Concentration- and Temperature-Induced Effects of Incorporated

6336

J. Phys. Chem. B 2009, 113, 6336–6346

Concentration- and Temperature-Induced Effects of Incorporated Desmopressin on the Properties of Reverse Hexagonal Mesophase Dima Libster,†,§ Abraham Aserin,† Doron Yariv,† Gil Shoham,‡ and Nissim Garti*,† Casali Institute of Applied Chemistry, The Institute of Chemistry, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel, Department of Inorganic Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel ReceiVed: NoVember 24, 2008; ReVised Manuscript ReceiVed: February 23, 2009

In this paper we report on the solubilization of desmopressin, as a model for peptide drugs, into reverse hexagonal (HII) liquid crystals. Concentration- and temperature-induced interactions of desmopressin, as well as the conformation of the peptide, were studied using small-angle X-ray scattering, ATR-FTIR spectroscopy, SD-NMR, and rheological measurements. A considerable increase (up to 6 Å) in the lattice parameter of the mesophases was obtained upon incorporation of the peptide. According to the ATR-FTIR analysis, the chaotropic effect of peptide embedment was assigned to its interactions with hydroxyls of monoglyceride in the outer interface region. These interactions had only a minor influence on the conformation of the peptide; weakening or opening the γ-turns resulted in partial binding of the peptide’s free carbonyls to monoolein. Temperature-dependent SAXS measurements displayed a chaotropic destabilizing effect of desmopressin on the structure, shifting toward the lower temperature HII-L2 structural transition. Temperature increase resulted in an increase of the domain size in the presence of the peptide, in contrast to the trend observed in the empty mesophase. SD-NMR analysis enabled distinguishing between two factors impeding the diffusion of the peptide: the restriction of motion due to the geometrical constrain of diffusion within the water tubes, and the interactions of the guest molecule with monoglyceride. The onset of the critical behavior at 45 °C was found to be significant, indicating considerable weakening of the monoglyceride and desmopressin interactions and the destabilizing effect of the peptide on the mesophase above this temperature. Similar temperature-dependent behavior was revealed by rheological measurements displaying the same onset of the critical behavior. It was demonstrated by Franz diffusion cell measurements that hexagonal mesophases can potentially be used as delivery vehicles for sustained delivery of desmopressin. 1. Introduction Lyotropic liquid crystals (LLC) based on glycerol monooleate (GMO) have recently attracted much scientific attention, especially due to their interesting structural properties and various potential applications.1-5 LLC have a high degree of internal order and symmetry, combined with a large interfacial area and balanced hydrophobic and hydrophilic domains content. These properties make the LLC excellent universal drug carriers, with numerous advantages over most other systems currently used. The more common and relatively well-studied lyotropic liquid crystalline phases include the lamellar (La), hexagonal (normal, HI or inverted HII), and normal or inverted cubic (bicontinuous or micellar) structures.6-8 Among these LLC, the reverse hexagonal liquid crystals (HII) seem to be the most promising candidates as delivery vehicles for pharmaceuticals, mainly due to their unique structural properties. These mesophases can accommodate hydrophilic and amphiphilic compounds within their aqueous domains, which are composed of densely packed, infinitely long, and straight water-filled rods. They can also accommodate hydrophobic and * To whom correspondence should be addressed. Tel: +972-2-658-6574/ 5. Fax: +972-2-652-0262. E-mail; [email protected]. † The Institute of Chemistry, The Hebrew University of Jerusalem. ‡ Department of Inorganic Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem. § The results presented in this paper will appear in the Ph.D. dissertation of D.L. in partial fulfillment of the requirements for the degree of Doctor in Applied Chemistry, The Hebrew University of Jerusalem, Israel.

amphiphilic compounds by direct interactions within their lipid hydrophobic moieties, oriented radially outward from the centers of the water rods.9-11 These structures have lower viscosity compared to the relatively stiff cubic phase, an important property that makes practical applications much easier.12,13 In addition, the surface topology of the hexagonal mesophase was found to have fractal characteristics, indicating a discontinuous and disordered alignment of the corresponding internal water rods on the mesoscale.14 This can potentially enhance the drug delivery through the large surface area of these carriers, owing to the fractal nature of the surface. Due to the special properties described above, the HII mesophases can be specifically tailored to solubilize and transport therapeutic molecules for more efficient drug delivery. When delivered without a protecting carrier, peptides are prone to rapid cleavage by the various human proteases, thus leading to poor bioavailability.15,16 The entrapment potential of weakly water-soluble drugs and their release from the HII mesophases was recently demonstrated by both in vitro and in vivo experiments for several compounds, including vitamin K,17 cinnarizine,18 paclitaxel and irinotecan,19 and progesterone and cyclosporin A.20-22 Sustained release of hydrophilic drugs was demonstrated from cubic LLC in several cases.19,23,24 Desmopressin is a synthetic analogue of the antidiuretic hormone vasopressin, which is mainly used for treatment of enuresis in young children, central diabetes insipidus, hemophilia A, von Willebrand disease, and trauma-induced injuries (Figure

10.1021/jp810309d CCC: $40.75  2009 American Chemical Society Published on Web 04/09/2009

Solubilization of Desmopressin

Figure 1. Chemical structure of desmopressin.25

1).25,26 To get the more advanced analogue desmopressin (a linear nonapeptide of 1100 Da), the original drug, vasopressin, was modified in two positions, including deamination of the cysteine in position 1, resulting in 3-mercaptopropionic acid, and substitution of L-arginine with D-arginine at position 8.16 These alterations led to prolonged duration of the antidiuretic activity and reduced blood pressure effect. Desmopressin is usually administered in doses of 1-20 µg, perorally and intranasally, and by both routes it shows relatively low bioavailability of only 1% and 2-10%, respectively.27-29 The very high hydrophilicity of desmopressin and its enzymatic degradation in the gastrointestinal tract seem to be the major reasons for the poor bioavailability of the drug.30-32 In this respect, transdermal administration of desmopressin can therefore be a valuable alternative. Several studies evaluating transdermal delivery of desmopressin were recently conducted including microemulsion utilization,16 iontophoresis,33,34 and microneedle array technology35 to overcome the skin barrier. Nevertheless, only the pharmacokinetic aspects of the desmopressin delivery were addressed in these experiments. However, it is clear that a comprehensive study of desmopressin incorporation in LLC is needed in order to maximize the application potential of this alternative method of drug delivery. Macroscopic and molecular level characterization of the interactions between desmopressin and its carrier are required in order to fully understand its solubilization and release. The current study aims to address these specific aspects of the desmopressin/LLC system to facilitate rational improvement of its drug delivery. The structural properties of the hexagonal mesophases composed of GMO/tricaprylin/water were extensively studied in our group, and most of the results have been reported in our previous publications.9-11,13,14,36 In addition to the general structural characterization of the mesophase, we have demonstrated that a lipophilic peptide drug (cyclosporin A) was solubilized into the HII mesophase, and its solubilization effects on the phase and its conformational changes within the phase were studied on a macroscopic scale12 and a molecular level.37 In the current research we studied solubilization of desmopressin within the HII mesophase. Our aims were to define the exact location of desmopressin in the phase, its specific interactions with the carrier and follow their structural changes (if any) during the incorporation of the drug into the mesophase. In order to do that, several methodologies were used, including SAXS, FTIR, SD-NMR, and rheology. The efficiency of transdermal delivery of desmopressin using the hexagonal phase as a carrier was examined in vitro, using Franz diffusion cells. 2. Materials and Methods 2.1. Materials. Monoolein, GMO, distilled glycerol monooleate (min. 97 wt % monoglyceride), and 2.5 wt % diglyceride (acid value 1.2, iodine value 68.0, melting point 37.5 °C, and free glycerol 0.4 wt %) were obtained from Riken Vitamin Co. (Tokyo, Japan). Tricaprylin (TAG) (97-98 wt %) was purchased from Sigma Chemical Co. (St. Louis, MO). Desmopressin acetate (purity >98%) was purchased from ChemPep Inc. (Miami, FL). The water was double distilled. All ingredients were used without further purification. D2O (D,

J. Phys. Chem. B, Vol. 113, No. 18, 2009 6337 99.9%), NaOD solution (D, 99.5%), and DCl solution (D, 99.96%) were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). HCl solution and NaOH solution were purchased from Fluka (Switzerland). Phosphate-buffered saline (PBS) was purchased from Biological intustries (Kibbutz Beit Haemek, Israel). 2.2. Preparation of HII Mesophases. The starting composition of 74.7 wt % GMO and 8.3 wt % tricaprylin (9:1 weight ratio) at 17 wt % water content was chosen for the solubilization experiments. The GMO/tricaprylin/water hexagonal liquid crystals were prepared by mixing weighed quantities of GMO and tricaprylin while heating to 45 °C. This was done in sealed tubes under nitrogen atmosphere to avoid oxidation of the GMO. An appropriate quantity of preheated water at the same temperature was added, and the samples were stirred and cooled to 25 °C. Desmopressin was solubilized in the range of 1-10 wt %. The peptide was dissolved in water prior to its incorporation into the HII mesophases. D2O was used for FTIR and SD-NMR measurements, instead of water. The pH or pD were adjusted to 5 with appropriate quantities of NaOD, DCl, NaOH, or HCl. It should be noted that as a result of desmopressin solubilization, the concentrations of GMO and tricaprylin were decreased, keeping their weight ratio of GMO/tricaprylin (9:1) and water content of 17 wt % constant. 2.3. Small-Angle X-ray Scattering (SAXS). Scattering experiments were performed using Ni-filtered Cu KR radiation (0.154 nm) from an Elliott rotating anode X-ray generator that operated at a power rating of 1.2 kW. The X-ray radiation was further monochromated and collimated by a single Franks mirror and a series of slits and height limiters, and measured by a linear position-sensitive detector. The samples were held in 1.5 mm quartz X-ray capillaries inserted into a copper block sample holder. The temperature was maintained at T ( 0.5 °C with a recirculating water bath. The camera constants were calibrated using anhydrous cholesterol. The scattering patterns were desmeared using the Lake procedure implemented in homewritten software.38 In order to estimate a lower bound for the sizes of ordered domains (LH), the full width at half-height of the (10) diffraction peak was measured and this value inserted into the Scherrer formula.10 2.4. Attenuated Total Reflectance Fourier Transform Infrared (ATR FTIR) Measurements. An Alpha model spectrometer, equipped with a single reflection diamond ATR sampling module, manufactured by Bruker Optik GmbH (Ettlingen, Germany), was used to record the FT-IR spectra. The spectra were recorded with 50 scans, at 25 °C; a spectral resolution of 2 cm-1 was obtained. 2.5. ATR-FTIR Data Analysis. Multi-Gaussian fitting has been utilized to resolve individual bands in the spectra. The peaks were analyzed in terms of peak frequencies, width at halfheight, and area. In order to resolve the measured amide I′ band of desmopressin, the samples’ spectra were background subtracted against the appropriate hexagonal mesophase control spectra. Further, the amide I′ band was resolved by secondderivative Savitzky-Golay nine-point smoothing function. 2.6. Rheological Measurements. Rheological measurements were performed using a Rheoscope 1 rheometer (Thermo-Haake, Karlsruhe, Germany). A cone-plate sensor was used with a diameter of 35 mm, cone angle of 1°, and a gap of 0.024 mm. Temperature-dependent rheological measurements were conducted in the range of 20-85 °C at a constant frequency of 3 rad/s and stress of 75 Pa, determined according to the linear viscoelastic range (LVR) of the materials (data not shown). The viscoelasticity of the HII phases was characterized in terms of

6338

J. Phys. Chem. B, Vol. 113, No. 18, 2009

the elastic modulus G′, the loss modulus G′′ from which tan δ (G′′/G′) was calculated, the complex viscosity η*, and the longest relaxation time τmax. All the tests were triplicated and found to be reproducible. 2.7. SD-NMR Measurements. All NMR experiments were performed with a Bruker Avance II spectrometer equipped with GREAT 1/10 gradients. Isotropic liquid samples were analyzed using a 5 mm BBI probe with a shielded z-gradient with a maximum strength of 0.546 T m-1. Liquid crystal samples were analyzed at a spinning rate of 4000 Hz with a 4 mm HR-MAS probe with a shielded magic-angle gradient with a maximum strength of 0.639 T m-1. Diffusion was measured in an asymmetric bipolar LED39,40 experiment with an asymmetry factor of 20%, ramping the strongest gradient from 2% to 95% of the maximum strength in 32 steps. The spectrum was processed by a Fourier transform in the acquisition (t2) dimension and by a Levenberg-Marquardt fit41,42 to decaying Gaussians with the Bruker TOPSIN software, in the gradient ramp evolution (g) dimension. NMR spectra were recorded within the range of 25-60 °C. 2.7.1. In Vitro Skin Penetration Study. The permeability of desmopressin through porcine skin was determined in vitro with a Franz diffusion cell system (PermeGear, Inc., Hellertown, PA). The porcine skin was excised from ears of slaughtered white pigs, carefully dissected and dermatomized, stored at -20 °C, and used within a month. Before the experiments, the skin was thawed and mounted on a Franz cells (diffusion area of 0.635 cm2) with the stratum corneum facing the donor compartment. The receptor compartment was filled with PBS (pH 7.2). The receptor phase was kept under constant stirring at 37 ( 0.5 °C. 150 mg of the liquid crystalline formulations or water solution containing 1 wt % of desmopressin was applied to the surface of the stratum corneum. 2.7.2. Analytical Method. Desmopressin content in the samples was determined by high-performance liquid chromatography (HPLC) equipped with photodiode array detector (Waters, Milford, MA). Isocratic elution was carried out with 25% acetonitrile and 75% trifluoroacetic acid 0.1% (w/v).43 The wavelength for UV detection was 220 nm. The column used was Luna 5 µm, C18, 250 mm × 4.6 mm (Phenomenex, Torrance, CA). The experiments were performed at ambient temperature at a flow rate of 1 mL/min. The injection volume was 40 µL. The detection limit of desmopressin determination was 0.1 µg/ml. Retention time of the peptide was 7 min. 2.7.3. Calculation of the in Vitro Data. In the in vitro skin penetration trials using Franz cells, because of the sampling from the receiver solution and replacement with equal volumes of buffer, the receiver solution was constantly being diluted. Considering this, the cumulative drug permeation (Qt) was calculated from the following equation:44 t-1

Qt ) VrCt +

∑ VsCi i)0

where Ct is the drug concentration of the receiver solution at each sampling time, Ci the drug concentration of the ith sample, and Vr and Vs are the volumes of the receiver solution and the sample, respectively. The obtained data were expressed as the cumulative drug permeation per unit of skin surface area, Qt/S. The steady-state fluxes (Jss) were calculated by linear regression interpolation of the experimental data at a steady state:

Libster et al.

Jss )

∆Q ∆tS

Apparent permeability coefficients (Kp) were calculated according to the equation:

Kp )

Jss Cd

where Cd is the drug concentration in the donor compartment (1 × 104 µg/mL), while assuming that under sink conditions the drug content in the receiver was negligible compared with the drug in the donor. 3. Results and Discussion 3.1. Effect of Desmopressin Concentration on the Mesophase Structure. 3.1.1. SAXS Experiments. Small-angle X-ray scattering (SAXS) measurements were conducted to elucidate the effect of desmopressin solubilization on the lattice parameter of the hexagonal phase made of GMO/tricaprylin/ water mixtures. In general, the SAXS results indicated a noticeable increase in the lattice parameter of the mesophase, changing from 52.2 Å in the original (free) system to 58.5 Å in the same mixture with 10 wt % desmopressin content (Figure 2). It would be reasonable to assume that since the desmopressin molecule is of relatively high hydrophilicity, it would be intercalated within the water cylinders of the hexagonal structure. However, the nearly linear increase of the lattice parameter suggests an uneven peptide distribution between the bulk and the interfacial water. The tendency of the peptide to increase the lattice parameter can probably be attributed to its chaotropic effect. It is generally known that chaotropic solutes destabilize the structure of bulk water and therefore tend to accumulate at the water-surfactant interface.45 Consequently, the surfactant interface area is expanded in the presence of chaotropic solutes. The enlarged interface area resulting from such expansion may then lead to swelling of the water cylinders and thereby increase in the observed lattice parameter. Following this interpretation, the desmopressin guest molecule should be involved in a network of interactions with the polar moieties of monoglyceride molecules of the hosting system. Such interactions should take place to enable the effect described above, potentially affecting the overall conformation of the peptide. Assuming such interactions and conformational changes, the most suitable methodology to identify and follow them should be ATR-FTIR, as described in the following section. 3.1.2. ATR-FTIR Studies: Impact of Desmopressin on the Mesophase Structure. A number of ATR-FTIR studies recently conducted in our laboratory demonstrated the marked capability of this methodology for detailed analysis of specific molecular interactions within the HII mesophases.11,13,36,37 In the present study, we analyzed the investigated system in three distinct structural regions: the outer shell of the water-surfactant interface (region I; R-I), the more interior part of the water-surfactant interface (region II; R-II), and the area containing the lipophilic acyl-chains (region III; R-III). The absorption bands at 3200-3400 cm-1, which are usually attributed to the O-H stretching modes (νOH), were used to characterize the interactions of the O-H groups of the GMO headgroups and D2O molecules in the outer part of the interface (R-I) (Figure 3). In the inner part of the interface (R-II), for GMO two hydroxyl groups were identified in the observed spectra C-OH (β, ∼1117 cm-1), C-OH (γ, ∼1051 cm-1)),


J. Phys. Chem. B, Vol. 113, No. 18, 2009 6339

Figure 2. Lattice parameter (R) (0.6 Å of HII mesophases containing GMO:tricaprylin with weight ratio 9:1 and 17 wt % water as a function of desmopressin concentration, as measured by SAXS.

Figure 4. (a) Frequency (cm-1) as a function of desmopressin concentration (wt %) of the O-H absorption mode of GMO molecule. (b) Width in half-height (cm-1) as a function of desmopressin concentration (wt %) of the O-H absorption mode.

Figure 3. Representative FTIR spectrum obtained for a GMO: tricaprylin with weight ratio 9:1, 17 wt % water and 10 wt % desmopressin at 25 °C. The symbols marked on several absorption bands are explained in the text.

the “free” and the intramolecular hydrogen-bonded carbonyl groups, CdO at the R position, 1720-1740 cm-1, and the stretching of the bonds CO-O (ester at the R position, ∼1180 cm-1).11,13,36,37 Information about the conformational order of the acyl chains (R-III) was obtained from the specific IR peaks of the GMO methylene groups at 2853 cm-1 (symmetric stretching) and at ∼2918 cm-1 (antisymmetric stretching). Analysis of the specified parts of the observed IR spectra showed that no significant changes take place in the bands responsible for the R-II and R-III regions. On the contrary, the absorption bands at 3200-3400 cm-1, attributed to the O-H stretching modes (υOH) of GMO, demonstrated a visible shift to lower wavenumbers. In contrast to H2O, the D2O solvent used in the current experiments does not absorb in this IR region. We were, therefore, able to follow the effect of the desmopressin solubilization on the O-H stretching mode of the surfactant, without the interfering contribution of the water O-H stretching. The position of υOH was shifted toward lower frequencies (Figure 4a), changing from 3393 cm-1 in the original (free) mesophase to 3386 cm-1 in the system loaded with 10 wt % guest desmopressin, suggesting stronger hydrogen bonding between the monoolein O-H groups and the D2O. In addition, the downward shift of the υOH position was accompanied with enhancement of the half-width of this band (Figure 4b). It is assumed that hydrogen bonding with the guest peptide is the main reason for the enhancement of the half-width, suggesting that only the hydroxyls of GMO that interact with the peptide

via hydrogen bond are involved in the outer shell of the surfactant-water interface (R-I). Thus, the observed results indicate that neither the inner shell of the interface nor the acyl chains were affected by the embedded peptide. Desmopressin molecules probably did not penetrate deeply enough into the interface region of the mesophase and as a result could not interact with the inner parts of the host GMO molecules, including the carbonyl groups, the ester moiety, and the lipophilic parts. Hence, the increase in lattice parameter with the addition of desmopressin, as detected by the SAXS measurements, most likely originated from the GMO hydroxyl interactions with desmopressin, which take place only at the outer shell of the surfactant-water interface (R-I). 3.1.3. ATR-FTIR Studies: the Amide I′ Band Analysis. The amide I′ band of the IR spectra of desmopressin was analyzed in order to follow conformational changes (if any) resulted by its interactions with GMO. A number of studies were conducted by means of NMR spectroscopy and molecular dynamics simulations on vasopressin and its analogues.46-48 It was concluded that these peptides consist generally of two parts, a cyclic part of the polypeptide chain involving a β-turn or a γ-turn structure, and a highly flexible part consisting of the acyclic tail. In the most recent NMR study, conducted by Sikorska et al.49 on vasopressin analogues, it was demonstrated that β-turns generally occur at positions 2,3 and 7,8 of the peptide in H2O/ D2O solutions. Earlier studies indicated a β-turn structure at positions 4 and 5 of desmopressin in a trifluoroethanol solution.50 Desmopressin is considered to be a relatively flexible molecule. Molecular dynamic simulations of the conformation of argininevasopressin suggested the existence of a dynamic equilibrium with alternate β-turns around residues Phe3-Gln4 and around residues Gln4-Asn5.48 Several possibilities for γ-turn conformations were indicated in the desmopressin structures. In aqueous solution, an inverse γ-turn consisting of residues Phe3, Gln4, and Asn5 was detected by means of NMR and molecular dynamics in analogues of vasopressin.51-53 In addition, Sikorska et al.49 showed that a γ-turn or an inverse γ-turn at positions 5 and 9 of vasopressin analogues is stabilized by the appropriate intramolecular hydrogen bonds.

6340


The representative amide I′ IR spectra of the desmopressin within the HII mesophase are shown in Figure 5a. The measured amide I′ bands of the peptide were resolved by a secondderivative analysis, a well-established technique allowing the decomposition of the amide I′ contours into their contributing components.54 The relevant absorption peaks appeared between 1608 and 1700 cm-1, as shown at the representative spectra of a desmopressin solution in D2O (Figure 5b). It has been suggested that bands below 1645 cm-1 appear as a result of hydrogen-bonded carbonyl groups, while “free” carbonyls give rise to absorption bands above 1660 cm-1. Bands in the 1645-1660 cm-1 range usually correspond to relatively weak hydrogen-bonded carbonyl groups that belong to unordered structures.55 It is also well documented that in small and midsize cyclic peptides that cannot adopt R-helical or β-sheet conformations, the 1645-35 cm-1 IR band is an indication of the presence of H-bonded β-turns.55 Another region of the IR spectra, located around 1690-1660 cm-1, was also found to be extremely sensitive for β-turn characterization. According to FTIR spectroscopy, vasopressin derivatives adopt β-turns.47 Absorption bands characteristic of γ-turns are not always straightforward to determine. However, strong H-bonded γ-turns usually appear around 1635-1610 cm-1, while weak or less stable H-bonded γ-turns give rise to absorption bands, usually in the 1635-1625 cm-1 range. Bifurcated hydrogen bonds may induce a shift of the band toward lower wavenumbers in the range of 1615-1600 cm-1. Other bands that can be assigned to γ-turns, which usually appear above 1648 cm-1 (1690-1647 cm-1), originate from nonbonded or weakly hydrogen-bonded carbonyl groups of γ-turns.55-57 The second derivatives of amide I′ bands were assigned according to the general considerations outlined above. The ATR-FTIR spectrum of the desmopressin peptide solubilized in D2O is shown in Figure 5b. The presence of the lowwavenumber component bands at 1608 and 1618 cm-1 can be associated with strongly H-bonded γ-turns. The high intensity of the 1639 cm-1 band is characteristic of the presence of very dominant H-bonded β-turns. The band at 1648 cm-1 can be attributed either to a β-turn, or as weakly H-bonded or open γ-turn. The 1659 cm-1 component reflects relatively weak hydrogen-bonded carbonyl groups that belong to unordered structures. The 1668 and 1691 cm-1 bands were assigned to “free” carbonyls. The ATR-FTIR spectra of the peptide solubilized in the HII mesophases are presented in Figure 5c,d. Comparing these spectra with the peptide in D2O solution, it was noticed that 1639, 1648, 1659, and 1668 cm-1 bands did not undergo significant changes. The data presented above implies that the dominant β-turn elements, unordered structures of the peptide and part of its “free” backbone carbonyls remained practically unchanged after the desmopressin solubilization within the hexagonal mesophase. Despite the general conformational stability observed, several small conformational changes were monitored within the solubilized peptide structure. In the low concentration range of desmopressin within the mesophase (1-4 wt %), the lowwavenumber bands at 1608 and 1618 cm-1 were shifted to 1622 and 1628 cm-1, respectively, indicating weakening or opening of the γ-turns. Alternatively, this could indicate the disappearance of bifurcation of the peptide carbonyls, likely due to decreasing strength of the solvent as H-bond donor (Figure 5c). This behavior suggests a more hydrophobic environment around the peptide, compared to the water solution. Such modification could be a result of the interactions of amino acids, involved in γ-turns, with the hydroxyl groups of the monoolein. This

Libster et al.

Figure 5. (a) Amide I′ band of 10 wt % desmopressin solubilized in GMO:tricaprylin with weight ratio 9:1, 17 wt % water at 25 °C. Representative second-derivative FTIR spectra of desmopressin incorporated in (b) 10 wt % in D2O solution, (c) in relatively low concentration regime of 4 wt % in the HII system, and (d) in relatively high concentration regime of 10 wt % in the HII system.

Solubilization of Desmopressin interpretation was further confirmed with another band, which was shifted from 1691 cm-1 in water solution to 1679 cm-1 in the HII mesophase, suggesting that part of the “free” carbonyls of the peptide amide groups become more “bounded”. As the concentration of the peptide within the mesophase increased (up to 10 wt %), the 1622 cm-1 band completely disappeared, suggesting further weakening (or opening) of the original γ-turns (Figure 5d). Considering the described destabilization of the γ-turns and partial binding of the free carbonyls of the peptide, it seems very likely that, although the desmopressin molecule interacts with the hydroxyls of the GMO, this kind of interaction does not significantly influence the β-turn conformations at positions 2, 3 and/or 7, and 8, but rather the γ-turn structures alone. These results can be explained by a comparison with the active conformation of desmopressin. It was found that when the peptide is bound to the antidiuretic receptor, the residues at positions 1, 3, 5, and 8 are critical for binding and signal transduction, where especially Asn5 and Arg8 are the most crucial.46 The residues at positions 1, 3, and 5 were also found to be involved in the inverse γ-turn element. Hence, it could be speculated that the γ-turn, which consists of these residues, was significantly destabilized (or opened) by interactions with the hydroxyls of the GMO. It should be noted, however, that existence of a γ-turn or an inverse γ-turn at positions 5 and 9 was detected by Sikorska et al.49 Therefore, it is also possible that this γ-turn was affected by the interactions with GMO molecules. 3.1.4. Conclusions from the Concentration Variation Experiments. Taking into account the present FTIR results and the SAXS results, it could be deduced that desmopressin interacts with the hydroxyls of the GMO in the outer interface region (R-I). Such interactions led to a weaker solvent as a H-bond donor, compared to the strength of pure water. Consequently, weakening or opening of the γ-turns and partial binding of the peptide-free carbonyls is taking place, yet these interactions do not seem to influence the dominant β-turn elements, unordered structures of the peptide, and part of its “free” carbonyls. With this experimental evidence, it would be reasonable to infer that, analogous to the peptide active conformation,46 the overall peptide conformation did not undergo radical alterations as a result of its embedment into the water cylinders of the HII mesophase. Such a conclusion is consistent with the finding that the peptide did not penetrate to the inner layer of the lipid-water interface. 3.2. Effect of Temperature Variations. 3.2.1. SAXS Experiments. It is recognized that the behavior of both peptide and liquid crystalline phases is extremely temperature-dependent and therefore interactions between desmopressin and the hexagonal structures may be affected by temperature modulation. SAXS temperature-dependent measurements showed that upon increasing the temperature, the lattice parameter decreased in both empty and peptide-loaded structures. Between 20 and 60 °C, a decrease of 2 Å was detected in both systems (Figure 6a). The decrease in the lattice parameter with increasing temperature is an expected phenomenon that is attributed to either dehydration of the surfactant polar headgroups or an increase in the hydrocarbon chain mobility.58 Further heating caused melting of the HII mesophase (the empty system) and the formation of the micellar solution (L2) at elevated temperatures, ca. 70 °C. However, this structural transition (HII-L2) was shifted toward lower temperatures (ca. 60 °C) in the presence of 8 wt % desmopressin. The decrease in stability range of the HII mesophase is attributed to the fact that the interactions of desmopressin with hydroxyls of monoolein expand the


Figure 6. (a) Lattice parameter of (0) unloaded HII mesophases and (9) loaded with 8 wt % desmopressin as a function of temperature, as measured by SAXS. (b) Scherrer parameter values (LH) of unloaded HII mesophases (0) and loaded with 8 wt % desmopressin (9) as a function of temperature, as measured by SAXS.

surfactant interface area. The described interactions involving a relatively large molecule such as desmopressin (MW of about 1000 g/mol) led to a decrease in the critical packing parameter (CPP) values. This certainly destabilized the hexagonal mesophase that was reflected by 10 °C decrease of the stability range. However, the degree of order (as determined by diffraction peak broadening) of the hexagonal structures is a function of temperature, as demonstrated in different trends (Figure 6b). Upon temperature increase, a decrease in the domain size (LH) of ∼25% was detected in the empty hexagonal structure. We have recently shown a similar trend in the case of low water content hexagonal samples that became less ordered, with smaller effective crystallite size (lower LH values), and partially dehydrated head groups upon temperature increase.36 The hexagonal phase, which is strongly dependent on hydrogen bonding, gradually shrank to complete disruption, while the curvature and CPP values increased. The incorporation of desmopressin into the system reduced the LH values at room temperature (from 800 to ∼600 Å) due to the described destabilizing chaotropic effect (Figure 6b). Nevertheless, increasing the temperature induced a gradual increase of the domain size from 380 to ∼600 Å, contrary to the process observed in the empty mesophase. This could be explained if two competing processes took place with the temperature increase. In addition to the described process responsible for the decrease of the LH values in the empty system, the second competing process is due to the peptide and GMO temperature-dependent interactions. At higher temperatures, weakening of the monoglyceride hydroxyl interactions with the peptide is expected (will be discussed in SD-NMR section). As a result, part of the GMO-peptide bonds will be weakened and/or cleaved, leading to reconstitution of the initially low domain size (and accordingly degree order) caused by the chaotropic effect of desmopressin intercalation into the water channels.

6342


Figure 7. (a) Desmopressin diffusion coefficients in D2O solution as a function of temperature. (b) Arrhenius fitting of the diffusion coefficients of desmopressin in D2O solution (ln D vs 1/T) in the temperature range of 25-45 °C.

Upon heating, this process became more dominant and eventually, at higher temperatures, the domain size of both empty and loaded systems reached the same values of ∼550-600 Å. Similar LH values of both systems indicate very weak interactions of the peptide with GMO in the range of 45-55 °C. In order to more closely examine the phenomenological effect of peptide influence on the structures detected by SAXS measurements, SD-NMR analysis was utilized. 3.2.2. SD-NMR Experiments. It is well-known that, in SDNMR experiments, diffusion is measured over a distance of the order of micrometers and provides information about the components in systems possessing different translational mobility. The self-diffusion coefficient of a molecule confined within a closed aggregate, the cylindrical micelles in the present case, is expected be low, while the self-diffusion of the unassociated molecules will be higher, corresponding to molecular diffusion.59,60 The diffusion coefficients of desmopressin solution in D2O (D0) are reported in Figure 7a. As expected, with increasing temperature the diffusion coefficients increased. An increase in the diffusion coefficients from an order of magnitude of (1-3) × 10-10 at lower temperatures (25-45 °C) to an order of magnitude of (1-3) × 10-9 at higher temperatures (above 45 °C) was monitored upon heating. In the lower temperature range of 25-45 °C, the diffusion data showed the Arrhenius-like dependence on temperature (Figure 7b). The plot of water self-diffusion versus 1/T resulted in a straight line, giving activation energy

Libster et al.

Figure 8. (a) Desmopressin diffusion coefficients as a function of temperature in (4) 8 wt % in D2O solution (O) according to the Renkin model, (0) 8 wt % in HII mesophase as a function of temperature. (b) Obstruction factors calculated from the SD-NMR analysis as a function of temperature (O). The overall obstruction factor (λ); (9) β is the obstruction factor responsible for the physical restriction of the peptide in the hexagonal structure; (4) γ is the obstruction factor associated with the chemical interactions between the peptide and GMO.

(Ea) of about 38 kJ/mol. On the other hand, in the temperature range of 45-65 °C, the diffusion coefficients drastically increased and deviation from Arrhenius law dependence was monitored. Therefore, 45 °C seems to be the onset of temperature-activated critical behavior of the peptide in the water solution. Desmopressin mobility in the water cylinders of the HII mesophase (DHII) was compared to its diffusion in pure water. As displayed in Figure 8a, incorporation of the peptide into the mesophase domains significantly hindered its mobility, decreasing the diffusion coefficients by orders of magnitude. Between 25 and 45 °C, the measured DHIIwere in the range of 3.8 × 10-12 to 6.6 × 10-12; however, it should be noted that between 25 and 35 °C the DHII slightly decreased, exhibiting a minimum at ∼35 °C and then increasing up to 45 °C. Rapid increase in peptide mobility was observed above 45 °C, similar to the process noticed in the water solution. The diffusion of desmopressin in the hexagonal phase is hindered by two factors. The first is the physical restriction of the peptide motion due to the geometrical constrain, owing to its diffusion within the water cylinders. The second factor is the chemical interactions of the peptide with GMO hydrophilic moieties. These two effects can be separated and quantified by SD-NMR analysis. The restricted diffusion of spherical molecules within cylindrical pores, assuming no interactions between the molecules and the pores, as described by Renkin (eq 1),61 was successfully used to describe biomacromolecule diffusion in a restricted environment:62


( )


( ) ( ) ( )

DG RH RH 3 ) 1 - 2.1444 + 2.08877 D0 rp rp RH 5 RH 6 RH 8 RH 9 0.94813 - 1.372 + 3.87 - 4.19 rp rp rp rp (1)

( )

( )

where DG is the diffusion coefficient of the molecule in the pore, assuming no interactions; D0 is the diffusion coefficient of the molecule in the pure solvent, without the motion restriction; RH is the hydrodynamic radius of the molecule; and rp is the radius of the pore. We used this equation to calculate the theoretical diffusion coefficients of desmopressin within the channels of HII mesophase, assuming no interactions of the peptide with GMO and TAG. Considering the geometric structure of the hexagonal phase, and using the obtained lattice parameter R from the SAXS measurements, the radius of the water cylinder RW can be calculated according to eq 2:63

(

√3(1 - φlip) Rw ) R 2π

)

1/2

(2)

where φlip is the total lipid volume fraction for the ternary mixtures, which was calculated as eq 3 (Popescu et al.63):

φlip

ωTAG ωGMO + FGMO FTAG ) ωD2O ωTAG ωGMO + + FGMO FTAG FD2O

(3)

where ω is the weight fraction of the component and F is the density. The densities of the components were 0.942 for GMO, 0.954 for TAG, and 1.1056 for D2O g/cm3. Using the measured diffusion coefficients of desmopressin solution in D2O (D0), taking Rw as rp (∼12 Å), estimating RH from the structural NMR data (∼6 Å),46 the diffusion coefficient of the molecule in the water tubes of hexagonal mesophase DREN (which is DG in eq 1) was calculated (Figure 8a). The observed decrease in diffusion coefficient values of the peptide in the HII mesophase (DHII) can be explained in terms of obstruction factors. The obstruction factors allow quantifying the effects of both physical restriction and chemical interactions. The overall obstruction factor (λ) is defined by eq 4:

λ)

DHII D0

) βγ

(4)

where β is the obstruction factor responsible for the physical restriction and γ is the obstruction factor associated with the chemical interactions. Obstruction factor β can be defined by eq 5

β)

DREN D0

Obstruction factor γ is presented in eq 6

(5)

γ)

DHII DREN

)

DHII D0β

(6)

The lower the obstruction factors are, the stronger the described effects. As depicted in Figure 8b, between 25 and 45 °C both β and γ factors possess similar values. This suggests that the physical restriction and the chemical interactions contributed almost equally to the decrease in overall mobility of desmopressin within the HII mesophase, compared to the water solution. However, above 45 °C increased values of γ-factor suggest that significant weakening of the GMO and desmopressin interactions occurred. The high γ-factor value of 1.58 (γ > 1) at 55 °C even indicates repulsive interactions between the surfactant and the peptide. This may also contribute to the observed decreased thermal stability of the loaded mesophase. Thus, these results also show that the peptide dictates the phase behavior of the hexagonal structure, exhibiting the onset of the critical behavior at 45 °C. The diffusion of GMO was also examined in the empty and peptide-loaded structures (Figure 9a). In the empty mesophase, with increasing temperature the diffusion coefficients of the surfactants increased (Figure 9a) from an order of magnitude of 8 × 10-12 m2/s at lower temperatures (25 °C) to an order of magnitude of 3.5 × 10-11 m2/s at higher temperatures (above 60 °C). In the full temperature range of 20-65 °C, the diffusion data showed the Arrhenius-like dependence on temperature. Activation energy (Ea) of about 31 kJ/mol was obtained from the plot of water self-diffusion coefficients versus 1/T (Figure 9b). Addition of desmopressin altered the diffusion of the surfactant (Figure 9a). At a lower temperature range (25-45 °C) diffusion coefficients of GMO (DGMO) in the loaded system were slightly lower than in the empty one as a result of interactions with the peptide. Conversely, starting from 45 °C, the DGMO values increased considerably from being qualitatively similar to the peptide diffusion behavior. The same onset of critical behavior was monitored in the case of GMO diffusion and it deviated from Arrhenius law dependence. Thus, it could be inferred that the peptide interactions with GMO determine the diffusion of the surfactant and consequently the phase behavior of the liquid crystalline structure. In order to elucidate how the mentioned mutual interactions influenced the hexagonal structure on a macroscopic scale, rheological characterization of the mesophases was utilized. 3.2.3. Rheological Measurements. The thermally activated structural changes occurring in the mesophases were studied by a rheological temperature-sweep experiment on the blank and desmopressin-loaded mixtures. The evolution of G′, G′′, and tan δ with increasing temperature from 20 to 85 °C are presented in Figure 10. The transformations are consistent with previous SD-NMR measurements on the structural shifts with temperature. Three regions were clearly detected in the obtained rheological data of the unloaded system (Figure 10a). The first corresponds to the structural rearrangements of the randomly oriented macroscopic domains of the hexagonal liquid crystals to yield more oriented structures. This process in the temperature range of 20-30 °C is reflected by a significant increase in the elasticity of the sample (increase in G′), constant value of the loss modulus (G′′), and drastic decrease of the tan δ from 0.9 to 0.5. The elastic properties of the sample dominate (G′ > G′′) reveal viscoelastic behavior that can be referred to the onset of the flow region of the hexagonal phase. In one of our previous reports,14 using ESEM technique, it was shown that the mesoscopic organization of these systems

6344


Libster et al.

Figure 9. (a) Diffusion coefficients of GMO in (0) empty HII mesophase and (9) loaded with 8 wt % peptide as a function of temperature. (b) Arrhenius fitting of the diffusion coefficients of GMO in the empty HII mesophase (ln D vs 1/T) in the temperature range of 25-65 °C.

is based on an alignment of discontinuous and anisotropic polycrystalline domains possessing fractal characteristics, each of which is several dozen micrometers in size. In this temperature range the material reveals viscoelastic behavior and starts to flow. Consequently, the orientation of the initially disordered alignment of the nonoriented domains probably increased as a result of their alignment with the applied temperature and the direction of oscillation. At the temperature range of ca. 30-69 °C, a decrease in the slope of tan δ was detected, demonstrating a more moderate decrease in tan δ, compared to the first region. According to the SAXS measurements, the system possesses hexagonal symmetry in this temperature range, but both the lattice parameter and the domain size decreased. This relatively moderate decrease of tan δ can be explained in terms of the gradual dehydration of the surfactant headgroups. The enhanced flow ability of the liquid crystalline structure, induced by the additional temperature increase, contributed to the tan δ drop. However, the dehydration of the headgroups decreased the elasticity of the sample, hindering the decrease of tan δ, compared to the first region. Further temperature increase caused melting of the HII mesophase and the formation of the micellar solution at high temperatures, as shown by SAXS measurements. The onset of this structural transition (HII-L2) was clearly detected ca. 69 °C in the rheological thermogram. It was reflected by a drastic drop of the mechanical moduli (especially G′′) and tan δ. Hence, while the first transition ca. 30 °C was an indication of the structural rearrangements in the mesoscopic structure of the hexagonal mesophase, the second process (ca. 69 °C) showed order-disorder transition (ODT) of the system. The viscoelastic behavior of hexagonal structures was radically modified in the

Figure 10. Rheological thermograms of HII mesophases: (a) empty system, (b) system loaded with 4 wt % desmopressin, and (c) system loaded with 8 wt % desmopressin. (O) G′ (storage modulus) values, (4) G′′ (loss modulus values), and ([) tan δ values.

presence of 4 and 8 wt % desmopressin (Figure 10b,c). Three regions were also observed from the rheological thermograms of the loaded systems. The most significant changes were detected in the first region, which was expanded up to 45 °Csthe onset of the critical behavior of the peptide, which was demonstrated by SD-NMR. In contrast to the empty system, the solubilization of both 4 and 8 wt % desmopressin caused a decrease in the elasticity of the structure. This was reflected by similar values of G′ and G′′ in this region and consequently an almost constant value of tan δ (∼1), unlike the empty system, where the storage modulus was dominant and tan δ decreased from 0.9 to 0.5. Based on these results, it could be suggested that peptide-GMO molecular interactions, shown by SD-NMR analysis on the molecular scale, were responsible for more viscous behavior of the structure up to 45 °C on the macroscopic scale. Starting from 45 °C and up to 62 °C, a more drastic drop in tan δ of the loaded systems occurred, compared to the empty one. While in the 4 wt % loaded system the tan δ decreased from 0.94 to 0.36, the most pronounced effect was observed in the presence of 8 wt % desmopressin. Here the tan δ values dropped from 1 to 0.05, indicating increased flow ability of the loaded structures. This was induced by the high mobility of the peptide at elevated temperatures that dictated the monoolein behavior, increasing


Figure 11. Cumulative transdermal penetration of 1 wt % desmopressin (0) via water solution and (4) via HII mesophase.

its diffusion coefficients. Finally, as evident from the thermogram, the ODT to the L2 phase was promoted toward lower temperatures (62 to 70 °C) due to the described destabilizing effect of the peptide on the hexagonal structure. 3.3. Transdermal Delivery of Desmopressin. Franz diffusion cells were employed to test the applicability and efficiency of transdermal delivery of desmopressin using the HII mesophase as a carrier. Characteristic profiles of the cumulative drug permeation (Qt) per unit of skin surface area of desmopressin from the HII mesophase and water solutions are shown in Figure 11. As seen in Figure 11, the hexagonal mesophase actually slowed down the penetration of desmopressin through the porcine skin, compared with the penetration determined for the drug using plain aqueous solution. After a period of 24 h, the cumulative drug permeation Qt)24 was 12.4 µg/cm2 via the aqueous solution, and only 6 µg/cm2 via the hexagonal mesophase. The calculated steady-state flux (Jss) decreased from 0.46 µg cm-2 h-1 using the aqueous solution to 0.22 µg cm-2 h-1 using HII mesophase. Accordingly, the permeability coefficients through the skin (KP × 103) were decreased from 0.046 cm h-1 when using water solution to 0.022 cm h-1 when using LLC carrier. It is obvious from the presented results that the peptide molecules solubilized in water can penetrate more easily and freely through the skin, compared to the hexagonal phase vehicles. This behavior of desmopressin should hence enable a sustained release of the drug. Interestingly, it is possible that the mechanism by which hydrophilic drugs penetrate the skin is different when using hexagonal mesophase as a vehicle, compared to their delivery via water solution. In a very recent pioneering work by Bender et al.,64 the authors demonstrated a different penetration pathway of sulforhodamine B (SRB), a fluorescent hydrophilic model drug, when using liquid crystalline cubic phase, compared to water solution, as drug vehicles. Using two-photon microscopy (TPM), these investigators showed that when SRB was applied using the cubic phase (consisting of either monoolein or phytantriol), the drugs were found to accumulate in microfissures and in a three-dimensional network of thin threads. In contrast, for the water-treated sample, a more homogeneous fluorescence pattern was demonstrated, which is concentrated mainly in the intercellular matrix. Hence, it was suggested that the intercluster penetration pathway is preferable for delivery of hydrophilic compounds via elastic cubic LLC, in contrast to the intercellular pathway when using the water solution. In this work, according to the authors’ interpretation of the TPM micrographs, the elastic lipid cubic phases penetrate into the microfissures, which are approximately 5 µm wide, with an irregular and entangled structure. After such process, the hydrophilic drug was supposed

J. Phys. Chem. B, Vol. 113, No. 18, 2009 6345 to diffuse into the surrounding intercellular lipid from the microfissures matrix, acting like a source for sustained release. Based on the results and interpretation of Bender et al.,64 it seems reasonable that in the present case the hexagonal phase can deliver hydrophilic compounds by a slimilar mechanism. Boyd et al.19 showed that the HII phases released model hydrophilic and hydrophobic drugs slower than the GMO cubic phase matrix. It was demonstrated in the current work and in our earlier reports that the examined HII mesophases possess dominating elastic properties, as well as adequate microstructural (lattice parameter) and mesostructural parameters (alignment of discontinuous and anisotropic polycrystalline domains, possessing fractal characteristics),14 in order to allow penetration of the guest drug into the microfissures. In addition, the observed sustained release of the peptide can be assigned to the interactions of the peptide with the monoglyceride hydroxyls, denoting that the lowest self-diffusion coefficient of desmopressin was detected ca. 35 °C. Certainly, further research is required to determine the detailed mechanism of delivery of desmopressin and similar short hydrophilic peptides via HII mesophase carriers. 3.4. General Conclusions. In the present study we investigated the location and mutual interactions of desmopressin with the HII mesophase, conformation of the peptide, and its structural modifications caused by the solubilization into the carrier. The potential of using these structures as sustained delivery vehicles for desmopressin was shown. Concentration-dependent characterization of the system (up to 10 wt % desmopressin) demonstrated a significant increase (up to 6 Å) in the lattice parameter of the mesophases. From the obtained ATR-FTIR results it appears that such chaotropic effect of the peptide is attributed to its interactions with hydroxyls of GMO in the outer interface region. These interactions were reflected in the conformation of the peptide when weakening or opening of the γ-turns and partial binding of the peptide’s free carbonyls occurred, but this kind of interaction does not influence the dominant β-turn elements. Temperaturedependent SAXS measurements revealed a chaotropic destabilizing effect of the peptide on the structure, reflected by a 10 °C shift toward lower temperatures of the ODT structural transition (HII-L2). The solubilization of the guest molecule into the system reduced its domain size LH values at room temperature (from 800 to ∼600 Å), due to the described chaotropic influence. However, upon temperature increase, the LH values increased from 380 to ∼600 Å, contrary to the process observed in the empty mesophase. This result indicated that, upon heating, GMO interactions with the peptide weakened, leading to the reconstitution of the initially low domain order and size. Furthermore, temperature-dependent SD-NMR analysis was applied to allow distinguishing and quantifying two major factors hindering the diffusion of the peptide: the physical restriction of motion due to the geometrical constrain of diffusion within the water tubes, and the chemical interactions of the guest molecule with GMO. The obtained results indicated that between 25 and 45 °C the physical restriction and chemical interactions contributed almost equally to the decrease in overall mobility of desmopressin within the HII mesophase, compared to the water solution. Nevertheless, above 45 °C considerable weakening of the GMO and desmopressin interactions occurred, leading to repulsive interactions between the surfactant and the peptide and a destabilizing effect. It was inferred from the obtained results that the high mobility of the peptide at elevated temperatures dictated the monoolein behavior, increasing its diffusion coefficients, and therefore determined the phase behavior of the liquid crystalline structure. The obtained effects

6346


on the molecular and microscopic levels greatly influenced the macroscopic behavior of the mesophases, as illustrated by rheological measurements. The onset of the critical behavior of the peptide (45 °C), which was demonstrated by SD-NMR, was also detected by the rheological measurements. The solubilization of desmopressin caused a decrease in the elasticity of the structure up to 45 °C, due to the interactions with GMO. In the temperature range of 45-62 °C, increased flow ability of the loaded mesophases was detected, as a result of the high mobility of the peptide, which determined the monoolein behavior. The shift toward lower temperatures of the structural transition (HII-L2) in the loaded system was also detected by rheological measurements, in full agreement with the SAXS analysis. Finally, it was shown that the examined mesophases can potentially be used as drug delivery vehicles for desmopressin. The hexagonal structure slowed down the transdermal penetration of desmopressin in vitro, compared with the penetration determined via normal aqueous solution, enabling a sustained release of the drug. The results obtained in this study may advantageously be utilized to rationally tailor specific and more efficient peptide-loaded liquid crystalline drug delivery systems, considering both the strength of the interactions between the peptide with the carrier and the special transport characteristics of the drug. References and Notes (1) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 1999, 4, 449–456. (2) Clogston, J.; Caffrey, M. J. Controlled Release 2005, 107, 97– 111. (3) Shah, M. H.; Paradkar, A. Int. J. Pharm. 2005, 294, 161–171. (4) Farkas, E.; Kiss, D.; Zelko´, R. Int. J. Pharm. 2007, 340, 71–75. (5) Efrat, R.; Aserin, A.; Garti, N. J. Colloid Interface Sci. 2008, 321, 166–176. (6) Larsson, K. J. Phys. Chem. 1989, 93, 7304–7314. (7) Qiu, H.; Caffrey, M. Biomaterials 2000, 21, 223–234. (8) Yamashita, J.; Shiono, M.; Hato, M. J. Phys. Chem. B 2008, 112, 12286–12296. (9) Amar-Yuli, I.; Garti, N. Colloids Surf., B 2005, 43, 72–82. (10) Amar-Yuli, I.; Wachtel, E.; Ben-Shoshan, E.; Danino, D.; Aserin, A.; Garti, N. Langmuir 2007, 23, 3637–3645. (11) Amar-Yuli, I.; Aserin, A.; Garti, N. J. Phys. Chem. B 2008, 112, 10171–10180. (12) Libster, D.; Aserin, A.; Wachtel, E.; Shoham, G.; Garti, N. J. Colloid Interface Sci. 2007, 308, 514–524. (13) Amar-Yuli, I.; Wachtel, E.; Shalev, D. E.; Aserin, A.; Garti, N. J. Phys. Chem. B 2008, 112, 3971–3982. (14) Libster, D.; Ben Ishai, P.; Aserin, A.; Shoham, G.; Garti, N. Langmuir 2008, 24, 2118–2127. (15) Gabizon, A.; Shmeeda, H.; Barenholz, Y. Clin. Pharmacokinet. 2003, 42, 419–436. (16) Getie, M.; Wohlrab, J.; Neubert, R. H. H. J. Pharm. Pharmacol. 2005, 57, 423–427. (17) Lopes, L. B.; Speretta, F. F. F.; Bentley, M. V. L. B. Eur. J. Pharm. Sci. 2007, 32, 209–215. (18) Boyd, B. J.; Khoo, S.-M.; Whittaker, D. V.; Davey, G.; Porter, C. J. H. Int. J. Pharm. 2007, 340, 52–60. (19) Boyd, B. J.; Whittaker, D. V.; Khoo, S.-M.; Davey, G. Int. J. Pharm. 2006, 309, 218–226. (20) Swarnakar, N. K.; Jain, V.; Dubey, V.; Mishra, D.; Jain, N. K. Pharm. Res. 2007, 24, 2223–2230. (21) Lopes, L. B.; Lopes, J. L. C.; Oliveira, D. C. R.; Thomazini, J. A.; Garcia, M. T. J.; Fantini, M. C. A.; Collett, J. H.; Bently, M. V. L. B. Eur. J. Pharm. Biopharm. 2006, 63, 146–155. (22) Lopes, L. B.; Ferreira, D. A.; De Paula, D.; Garcia, M. T. J.; Thomazini, J. A.; Fantini, M. C. A.; Bently, V. L. B. Pharm. Res. 2006, 23, 1332–1342.

Libster et al. (23) Lynch, M. L.; Ofori-Boateng, A.; Hippe, A.; Kochvar, K.; Spicer, P. T. J. Colloid Interface Sci. 2003, 260, 404–413. (24) Chang, C. M.; Bodmeier, R. J. Pharm. Sci. 1997, 86, 747–752. (25) Pedersen, T. B.; Frokjaer, S.; Mouritsen, O. G.; Jørgensen, K. Int. J. Pharm. 2002, 233, 199–206. (26) Law, S. L.; Huang, K. J.; Chou, H. Y. J. Controlled Release 2001, 70, 375–382. (27) Harris, A. S.; Nilsson, I. M.; Wagner, Z. G.; Alkner, U. J. Pharm. Sci. 1986, 75, 1085–1088. (28) Vilhardt, H.; Lundin, S. Gen. Pharmacol. 1986, 17, 481–483. (29) Ko¨hler, M.; Harris, A. Eur. J. Clin. Pharmacol. 1988, 35, 281– 285. (30) Lundin, S.; Artursson, P. Int. J. Pharm. 1990, 64, 181–186. (31) Fjellestad-Paulsen, A.; So¨derberg-Ahlm, C.; Lundin, S. Peptides 1995, 16, 1141–1147. (32) Fredholt, K.; Lstergaard, J.; Savolainen, J.; Friis, G. J. Int. J. Pharm. 1999, 178, 223–229. (33) Nakakura, M.; Kato, Y.; Ito, K. Biol. Pharm. Bull. 1998, 21, 268– 271. (34) Nakakura, M.; Kato, Y.; Ito, K. Biol. Pharm. Bull. 1997, 20, 537– 540. (35) Cormier, M.; Johnson, B.; Ameri, M.; Nyam, K.; Libiran, L.; Zhang, D. D.; Daddona, P. J. Controlled Release 2004, 97, 503–511. (36) Amar-Yuli, I.; Wachtel, E.; Shalev, D. E.; Moshe, H.; Aserin, A.; Garti, N. J. Phys. Chem. B 2007, 111, 13544–13553. (37) Libster, D.; Ben Ishai, P.; Aserin, A.; Shoham, G.; Garti, N. Int. J. Pharm. 2009, 367, 115-126. (38) Lake, J. A. Acta Crystallogr. 1967, 23, 191–194. (39) Wu, D. H.; Chen, A. D.; Johnson, C. S. J. Magn. Reson. A 1995, 115, 260–264. (40) Pelta, M. D.; Morris, G. A.; Stchedroff, M. J.; Hammond, S. J. Magn. Reson. Chem. 2002, 40, S147-S152. (41) Levenberg, K. J. Appl. Math. 1944, 2, 164–168. (42) Marquardt, D. W. J. Appl. Math. 1963, 11, 431–441. (43) Polnok, A.; Verhoef, J. C.; Borchard, G.; Sarisuta, N.; Junginger, H. E. Int. J. Pharm. 2004, 269, 303–310. (44) Sintov, A. C.; Botner, S. Int. J. Pharm. 2006, 311, 55–62. (45) Koynova, R.; Brankov, J.; Tenchov, B. Eur. Biophys. J. 1997, 25, 261–274. (46) Walse, B.; Kihlberg, J.; Drakenberg, T. Eur. J. Biochem. 1998, 252, 428–440. (47) Szendrei, G. I.; Fabian, H.; Mantsch, H. H.; Lovas, S.; Nye´ki, O.; Scho¨n, L. O., Jr. Eur. J. Biochem. 1994, 226, 917–924. (48) Liwo, A.; Tempczyk, A.; Oldziej, S.; Shenderovich, M. D.; Hruby, V. J.; Talluri, S.; Ciarkowski, J.; Kasprzykowski, F.; Lankiewicz, L.; Grzonka, Z. Biopolymers 1996, 38, 157–175. (49) Sikorska, E.; S´lusarz, M. J.; S´lusarz, R.; Kowalczyk, W.; Lammek, B. J. Peptide Sci. 2006, 12, 13–24. (50) Wang, J.; Hodges, R. S.; Sykes, B. D. J. Am. Chem. Soc. 1995, 117, 8627–8634. (51) Shenderovich, M. D.; Kover, K. E.; Wilke, S.; Collins, N.; Hruby, V. J. J. Am. Chem. Soc. 1997, 119, 5833–5846. (52) Yu, C.; Yang, T. H.; Yeh, C. J.; Chuang, L. C. Can. J. Chem. 1992, 70, 1950–1955. (53) Rodziewicz-Motowidło, S.; Zhukov, I.; Kasprzykowski, F.; Grzonka, Z.; Ciarkowski, J.; Wo´jcik, J. J. Pept. Sci. 2002, 8, 347–364. (54) Zhang, J.; Yan, Y.-B. Anal. Biochem. 2005, 340, 89–98. (55) Vass, E.; Hollosi, M.; Besson, F.; Buchet, R. Chem. ReV. 2003, 103, 1917–1954. (56) Shaw, R. A.; Perczel, A.; Mantsch, H. H.; Fasman, G. D. J. Mol. Struct. 1994, 324, 143. (57) Vass, E.; Kurz, M.; Konat, R. K.; Hollo´si, M. Spectrochim. Acta, Part A 1998, 54, 773. (58) Mezzenga, R.; Meyer, C.; Servais, C.; Romoscanu, A. I.; Sagalowicz, L.; Hayward, R. C. Langmuir 2005, 21, 3322–3333. (59) Coppola, L.; Oliviero, C.; Pogliani, L.; Ranieri, G. A.; Terenzi, M. Colloid Polym. Sci. 2000, 278, 434–442. (60) Coppola, L.; Oliviero, C.; Olsson, U.; Ranieri, G. A. Langmuir 2000, 16, 4180–4184. (61) Renkin, E. M. J. Gen. Physiol. 1954, 38, 225–243. (62) Pluen, A.; Netti, P. A.; Jain, R. K.; Berk, D. A. Biophys. J. 1999, 77, 542–552. (63) Popescu, G.; Barauskas, J.; Nylander, T.; Tiberg, F. Langmuir 2007, 23, 496–503. (64) Bender, J.; Simonsson, C.; Smedh, M.; Engstro¨m, S.; Ericson, M. B. J. Controlled Release 2008, 129, 163–169.

JP810309D

Concentration- and Temperature-Induced Effects of Incorporated

Recommend Documents