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Chitosan effect on the mesophase behavior of phosphatidylcholine supramolecular systems
Materials Science and Engineering C 29 (2009) 463–469
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Chitosan effect on the mesophase behavior of phosphatidylcholine supramolecular systems Omar Mertins ⁎, Maria I.Z. Lionzo, Yasmine M.S. Micheletto, Adriana R. Pohlmann, Nádya Pesce da Silveira ⁎ Instituto de Química, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, 91501-970, Caixa Postal 15003, Porto Alegre, RS, Brazil
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Article history: Received 29 April 2008 Received in revised form 20 August 2008 Accepted 29 August 2008 Available online 16 September 2008 Keywords: Soybean phosphatidylcholine Chitosan Liposome Phase transition temperature SAXS Light Scattering
a b s t r a c t Two distinct supramolecular self assemblies of phosphatidylcholine and chitosan, namely liposomes and their precursory organogel, have been investigated by means of SAXS, Light Scattering and Polarized Optical Microscopy. The main goal was the evaluation of the chitosan effect on the self assemblies phase transition behavior upon heating. A distinct smectic organization was observed for the organogel prepared in the presence of chitosan, if compared to that prepared only with phosphatidylcholine. In addition, the phosphatidylcholine–chitosan organogel showed unchanged optical properties upon heating and after 24 h, indicating increased stability when compared to the organogel prepared without chitosan. For the liposomes containing chitosan, the thermotropic behavior features a lamellar pattern that is preserved under heating, until at least 81 °C. A phase transition temperature has been determined around 64 °C, which was clearly higher than that observed for liposomes prepared without chitosan. The bilayer repeat distance typical of the liposomes increases slowly by increasing the temperature and stacking fluctuations of the bilayers are delayed due to enhancement of the membrane rigidity. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Phospholipid vesicles or liposomes have long been known as systems with great similarities to cellular membranes. Nowadays, liposomes are developed as nanometric drug carriers for specific treatments with controlled circulation time, reduced side effects and optimized drug action [1–8]. It is well known that the knowledge of physico-chemical stability is a crucial demand for applicability of liposomes. The bilayer organization, for instance, changes at well defined temperatures, called temperatures of phase transition (Tpht). At low temperature there is the crystalline lamellar phase Lc producing an ordered bilayer. Increasing the temperature, the first sub transition leads to the Lβ phase, where the lipid chains are extended and tilted with respect to the bilayer plane, keeping the molecular order on the bilayer. Following, a gel ripple phase Pβ′ can be sometimes observed, where the lipid bilayers become a periodic ripple. Afterwards, the main transition temperature forms the liquid crystalline lamellar phase Lα, where the apolar tail of the lipids in the bilayers are highly disordered. As a consequence, the bilayer repeat distances are changing according to the characteristic mesophase [9–11]. Values of Tpht in liposomes have been determined applying different methods like DSC [9,12,13], spectroscopy [14,15], X-ray
⁎ Corresponding authors. Tel.: +55 51 33087321; fax: +55 51 33087304. E-mail addresses: [email protected] (O. Mertins), [email protected] (N.P. Silveira). 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.08.038
diffraction [9,16] and FTIR [11,17]. Recently, Michel et al. [18] have determined the Tpht of lipids by Light Scattering during temperature variations. It was demonstrated that the changes in the measured scattered intensities during heating are representative of different molecular organizations in the liposomes. For the determination of Tpht in liposomes several parameters have to be considered. In these systems, Tpht is sensitive to the ionic strength of the dispersion buffer [9,19], the presence of foreign components, such as cholesterol, together with the phospholipids building the bilayer of the vesicles [20] and to products mixed in the dispersing solution [21], like polymers. Chitosan, a biocompatible polysaccharide [22] obtained from partial N-deacetylation of chitin [23–25], showed a high potential to be used as an extra cover for the improvement of liposomes stability in aggressive environments [26–29]. However, little is known about influence of chitosan on Tpht of liposomes. In recent works [30–32] we have applied the reverse phase evaporation method [33] for the production of liposomes containing chitosan. In this method, we first obtain an organogel, and after water addition, liposomes are formed. Considering that the introduction of a macromolecule may change the bilayer phase transition and, furthermore, the stability and properties of the whole system can also be modified [34], we investigate first the organogel by means of Polarized Optical Microscopy. Following, the liposomes have been studied by means of Static Light Scattering (SLS) and Small Angle X-ray Scattering (SAXS). The experiments have been carried out between 25 and 81 °C.
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2. Experimental 2.1. Materials Phosphatidylcholine (PC) from soybean (95% phosphatidylcholine, 5% lysophosphatidylcholine and phosphatidic acid) was a gift from Solae do Brasil S.A. The molecular composition of the soybean PC is around 75% distearoylphosphatidylcholine (DSPC), 12% dioleoylphosphatidylcholine (DOPC) and 8% dipalmitoylphosphatidylcholine (DPPC). The water content of 6.2% was obtained using Karl Fischer titration. Chitosan was obtained from the deacetylation of chitin as previously described [31]. The acetylation degree of 17% was determined by IR spectroscopy. The average molecular weight Mw = 160,000 g/mol and the gyration radius Rg = 43.5 nm from Static Light Scattering measurements [31] gave a critical concentration of c⁎ = 0.8 mg/ml in a buffer solution of 0.02 M acetate (0.02 M acetic acid and 0.02 M sodium acetate)/0.1 M NaCl (pH 4.5). All other reagents were of analytical grade. 2.2. Sample preparation The organogel and the liposomes were prepared by the reverse phase evaporation method [33]. Samples with or without chitosan were obtained: in 60 mg of soybean PC dissolved in 10 ml of ethyl acetate (Merck®), 200 μl of buffer (0.02 M acetate buffer/0.1 M NaCl (pH 4.5)) without polymer for samples free of chitosan and containing 1 mg/ml of chitosan (filtered 0.22 μm (Millipore®)) for samples with polymer (Fig. 1), were dropped in the solution to form a water in oil like emulsion which was sonicated (2 min) yielding a homogeneous opalescent dispersion of reverse micelles. The organic solvent was removed using a rotatory evaporator (Buchi) at 30–35 °C under vacuum, resulting in a high viscous organogel. The organogel was
reverted to liposomes with the addition of 10 ml of pure water (MilliQ®) under vigorous shaking. The final pH of the nanovesicle emulsions was 5.8 for both systems. The final chitosan concentration in the nanovesicles corresponds to 0.0033 mg per mg of PC. 2.3. Polarized Optical Microscopy The organogels were settled between two glass slides and examined under polarizing microscope (Olympus® BX-43). The texture images were captured using an Olympus® PM20 optical camera with exposure controller. Temperature was ranged from room temperature to 80 °C controlled by a Quasar MT 300 heating stage programmed at a similar range as for SLS and SAXS analyses, and the images acquisition was performed after each 4 °C increment. 2.4. Light Scattering Liposome sizes, polydispersity and diffusion coefficients were determined by Dynamic Light Scattering (DLS) as previously described [30,35]. Static Light Scattering (SLS) was performed on a Brookhaven Instruments standard setup with a He–Ne laser (λ = 632.8 nm) as light source. The samples were placed into dust free cells for Light Scattering (LS) measurements that were placed in the index-matching liquid decahydronaphthalene (Aldrich®) and heated from 25 to 87 °C in a rate of 1 °C/min with intervals of 10 min at each 2 °C when LS intensity (I) was measured 10 times with intervals of 0.1 min. The position of the detector was at 135° relative to the incident beam. The data analysis was carried out as previously described [31]. Experiments were repeated twice for each sample showing reproducibility and error bar was determined considering standard deviation of 10 measurements at each temperature.
Fig. 1. Reverse phase evaporation method with the addition of chitosan. 1. Phosphatidylcholine is dissolved in an organic solvent. 2. A portion of aqueous solution containing chitosan is added. 3. Under sonication an emulsion of reverse micelles containing chitosan is obtained. 4. The organic solvent is evaporated. 5. The organogel is obtained. 6. Vesicles containing chitosan are formed after water addition and shaking.
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2.5. Small Angle X-ray Scattering
3. Results and discussions
Synchrotron SAXS measurements were carried out on the bending magnetic beamline SAS of the Laboratório Nacional de Luz Synchrotron (LNLS, Campinas, Brazil). The samples were placed in a stainless steel sample holder [36] and thermostatized in the same range as for LS with accuracy of ± 1 °C. The wavelength of the incident beam was 1.605 Å and a linear detector (Princeton Instruments) was used at 43.5 cm from the sample. The exposure time of 10 min at each 2 °C interval was required for the spectral acquisitions. Data were normalized to constant beam intensity and corrected for the transmission, sample thickness, parasitic and background scattering according to standard procedure. Considering the size of vesicles larger than 50 nm, no deviation in the diffraction pattern can be expected from the infinite membrane approximation [37]. The bilayer thickness or characteristic length of the bilayer repeat distance (d) in the nanovesicles was determined through the Bragg relation:
3.1. Polarized Optical Microscopy
d ¼ 2π=ðnqÞ with q the wave vector related to the maximum peak intensity of the first Bragg reflection and n = 1, 2, 3,…, ∞. The fitting of the experimental curves was done applying Lorentzian functions with the Microcal Origin software [30,31,38].
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Szoka and Papahadjopoulos [33] described the phosphatidylcholine organogel as a viscous gel and a system with low stability. It undergoes spontaneously or with the application of some mechanical energy to the self assembly of lamellae that built the phospholipid bilayers of liposomes. The evolution of the molecular organization of supramolecular aggregates of phospholipids may consist of worm like micelles (hexagonal phase), cubic network (cubic phase), bicelles, ribbons and lamellae [39–41]. The transitions are related to water concentration and temperature representing respectively transitions of first and second order [41]. Herein the water concentration is kept constant, so that a second order transition is expected. Furthermore, the phospholipid used in this work is zwitterionic and temperature becomes an important parameter on phase transition. In this case, the obtained phases are known to be mostly lamellar. Domains of hexagonal and cubic phases may be found only at high temperatures [41]. In the present work, the microstructure evolution of the PC– organogel as a function of temperature range was investigated by Polarized Optical Microcopy (POM). Increasing the temperature, POM observations indicated the occurrence of two different phases for the PC–organogel. Indeed, as can be seen in Fig. 2 (left column) the phase
Fig. 2. POM images of the PC organogels (left column) and PC–chitosan organogels (right column) at different temperatures (26 (a, e), 62 (b, f) and 82 (c, g) °C), ranging over the phase transition temperatures and after overnight, at 26 °C (d and h) (scale bar = 100 nm).
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transition of the phospholipid modifies the optical properties of the PC–organogel. At 26 °C (Fig. 2a), we observe the presence of typical lamellar mesophase with mosaic texture (like Smectic C) [42,43]. Increasing the temperature up to 80 °C (Fig. 2c), the transition to a hexagonal mesophase with a fan-texture (Smectic A) is evidenced. Moreover, the texture of PC–organogel at 26 °C after 24 h (Fig. 2d) showed the presence of oily streaks (isotropic) and some “Maltese crosses” indicating the occurrence of a lamellar organization characteristic of a dispersion of multilamellar vesicles in an isotropic solution [39]. This texture is different from that visualized at the first condition (Fig. 2a) denoting a transition behavior for the supramolecular organization of PC–organogel. The images of the PC–chitosan organogel in Fig. 2 (right column) show a different pattern, which is similar to a mesophase with Schlieren texture (like Smectic C) [42] and some thread-like defects. Another difference is related to the birefringence of the PC–chitosan organogel that gives some evidence of a distinguished phospholipids organization due the presence of chitosan. Furthermore, since the PC is a lyotropic amphiphil, the presence of polymer may also lead to a reduction of the hydration water on the phospholipids. Thus, the water moieties displacement may modify the optical properties of the organogel. Moreover, the temperature effect on the PC–chitosan organogel was almost imperceptible. As we can see in Fig. 2e, f and g, after heating, the texture remains similar indicating that chitosan stabilizes the supramolecular organization. This stabilization was effective for at least 24 h at 26 °C (Fig. 2h). 3.2. Static Light Scattering PC liposomes free of chitosan and containing chitosan (1 mg/ml in the acetate buffer) feature respectively an average hydrodynamic radius of 173 ± 92 and 269 ± 87 nm, a zeta potential of −42 ± 3 and −34 ± 4 mV and a diffusion coefficient of 1.2 × 108 and 0.8 × 108 cm2/s [30]. The influence of temperature on the liposome sizes of particles was not considered, since phase transitions are not always related to changes in size and/or shape of such systems [18]. In LS experiments the discontinuity in the average number of photons detected per second (I) as the temperature is altered, corresponds to a change in the optical properties of the sample [18]. Indeed, a phase transition modifies the optical properties of the liposomes in water. Fig. 3 displays the averaged I upon heating, for both PC liposomes (a) and PC–chitosan liposomes (b). The abrupt change on I in Fig. 3a is related to the phase transition from gel to liquid crystal (Lα). For the system without chitosan the average I decreases between 53 and 65 °C. The decrease is not progressive, as expected for pure phospholipids [18]. As the PC from soybean contains mainly DSPC (75%), bearing 18 carbon atoms in both apolar chains of the molecule, the phase transition temperature for this phospholipid is expected to be at 55.6 °C [44]. However, the presence of DOPC (12%) and DPPC (8%) with shorter alkyl chains for the later (16 carbons) and some impurities (5%, see Experimental section) may influence the phase transition temperature. Since DSPC mainly determines the phase transition temperature, the other components enlarge the temperature range of the decrease of I. Hence, the phase transition is tuned by different molecules and the whole process is slowed down, resulting in an extended transition (Fig. 3a). A different behavior is observed when chitosan is present in the liposomes (Fig. 3b). Instead of a sharp decrease, a progressive reduction of scattered intensity I, covering the entire investigated range, is evidenced. It seems that chitosan dissipates the thermal energy furnished to the system leading to a very discrete phase transition, which occurs in moderated steps. In the presence of chitosan, the bilayers disorder is minimized somehow by a kinetic compensation that influences the molecular organization. Hence, the presence of
Fig. 3. Evolution of the mean count rate from Static Light Scattering Intensity (I) (squares) and bilayer repeat distances from SAXS (d ± 0.02 nm) (triangles) as temperature function for PC nanovesicles (a) and PC–chitosan nanovesicles (b).
chitosan may lead to a non-homogeneous phase transition, e.g. parts of the liposomes may go trough the transition while others may delay the process. As proposed by Michel et al. (applying Boltzmann regression curves) [18], the phase transition temperatures were obtained from the middle of the slope of each curve, where the presence of microdomains allows the coexistence of two phases in the transition region [45]. In this work we differentiated the curves to obtain the points corresponding to the middle of the slopes. We have found Tpht = 57 °C for liposomes and Tpht = 64 °C for liposomes with chitosan. At this point, an important condition to be considered is the heating process applied. Since our temperature range (25–87 °C) was submitted to large intervals (10 min at each measured temperature, required for SAXS scattering diagrams acquisitions), we consider that these values of Tpht are approximated. Also, the addition of some NaCl in the buffer solution (0.1 M) to form the reverse micelles (Fig. 1) has been reported to lead to alterations in the Tpht of phosphatidylcholines [46]. Nevertheless, the result for liposomes in this work is close to the reported result of Tpht for DSPC (55.6 °C) [44]. After all, as our aim was to investigate the chitosan influence on the behavior of the system, the Tpht shows an increase of at least 7 °C for the system containing chitosan. 3.3. Small Angle X-ray Scattering One of the structural modifications upon heating which results from the phase transition in liposomes is the change in the bilayer
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repeat distance (d) [10]. Using SAXS, d can be obtained by analysis of the diffraction profiles of the scattered intensity [37]. Fig. 4 depicts the SAXS spectra obtained during the temperature increase (see the labeled temperatures for each scattering diagram) for the two samples. At the beginning, both systems display well defined first and second order Bragg reflections indicative of a lamellar phase. As we recently reported [30] such profile may also represent multilamellar structures. In multilamellae the bilayer stacks are less vulnerable to the fluctuations raised from the diffusional movements in water. While in unilamellae, the diffusional movements may constantly modify the format of vesicles changing the bilayer organization. As a result for the former case, the X-ray scattering furnishes a diffuse intensity in the background that provides broad curves in the spectra. In addition, Fig. 4 shows that for the system without chitosan the intensity decreases faster during temperature increase and the peaks broaden suggesting a phase transition [43]. In Fig. 4a, the first order peak can be seen until around 57 °C, despite of the slight intensity relative to the 35 °C profile. After 59 °C, only a small Bragg peak can be noticed superimposed to a broad halo. At 63 °C the peak disappears yielding a broad component, which persists until 81 °C (Fig. 4a). Again, such results may be interpreted as a phase transition that takes place around 57 to 59 °C, supporting the result obtained in SLS experiment (57 °C). This temperature range matches approximately with the region of I decrease in SLS (Fig. 3a). Analyzing the d parameter (Fig. 3a), a constant shift of the peak to smaller q values is detected, resulting in d increase with temperature.
Fig. 4. Small Angle X-ray Scattering Intensities (I) as wave vector function (q) at different temperatures (°C) as indicated for PC nanovesicles (a) and PC–chitosan nanovesicles (b). Spectra shift +0.001.
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The absolute d values are obtained from Lorentzian fits as previously reported [30,31] and shown in Fig. 5 (fitted straight lines). Plotting d from SAXS together with I from SLS (Fig. 3a), a constant increase of d is evidenced in the same region of the I decrease. Regarding the results for the system containing chitosan, a clear different pathway is found. The intensity of the Bragg reflections decreases upon heating, as well, however in a remarkable smaller degree (Fig. 4b). Afterwards, the first peak remains over the whole range of temperatures. Similar behavior appears for the broadening of the peak. A higher broadening starts only at 65 °C (see also Fig. 5), which is close to the 64 °C determined for the Tpht in SLS. Besides, at 75 °C a small shoulder on the right side of the peak can be noticed (Figs. 4b and 5). The same can be related to the formation of a coexistent fluid hexagonal HII phase [47] which complete transition must be delayed by the presence of chitosan. Until 81 °C, it was still possible to discern a reflection. Moreover, the d parameter also presents a distinct behavior. In Fig. 3b, d is slightly increased during the temperature increase, without any abrupt change. A relative faster change starts only after 61 °C. Comparing both systems (Fig. 3a and b), the data of the sample free of chitosan shows a continuous increase of d after 53 °C, whereas for the system with chitosan the same behavior takes place only after 61 °C. Defining d as the bilayer repeat distance means the thickness of the phospholipidic bilayer plus the water layer between the bilayers in a multilamellar vesicle. It is established that a temperature increase leads to an increase in the disorder of the apolar chains resulting in a reduction of their elongation in some mesophases [48]. In this way, the thickness of the phospholipidic bilayer is expected to be reduced, depending on the mesophase in which the system is found. Nevertheless, since the lamellar peaks in SAXS shift to smaller q values, our results show a global increase of d, suggesting that the water layer may also be increased as a result of molecular agitation under the increasing temperature. Semmler et al. [47] also suggested an increase of water between the bilayers as a reason for larger bilayer repeat distances. Another point to be evaluated is the peak broadening. Local fluctuations in the membrane curvature produce such phenomenon [30,37]. Free water between bilayers is necessary to facilitate a change of the interfacial curvature, which in turn controls the phase transition [43]. Assuming an increase of the water layer, membrane fluctuations can be understood considering a concomitant increase of water spatial volume upon heating. As molecular agitation of water increases, the membranes fluctuate. Furthermore, the head group area of phospholipids was also shown to increase as a consequence of the chain mobility in high temperatures [49]. This may also help towards d increase. After all, the lamellar structure can be preserved, but in such situation the bilayer order needs to be weak to result in the broad scattering band in the SAXS region [47]. Thus, the membrane fluctuations must indeed be important. Comparing SAXS data of the two systems, chitosan acts towards keeping a relative ordered bilayer, meaning higher molecular organization. A smaller lattice disorder must be energetically favorable. Hence, the effect of temperature is less important probably because chitosan dissipates the thermal energy, as already considered in SLS, and in this manner the presence of chitosan stabilizes the lamellar phase. Similar effect was also observed by Radlinska et al. in a surfactant plus random heteropolymer system [43]. Considering the moderated d increase, chitosan may also reduce the effect of water agitation between lamellae. Finally, all these contributions seem to move towards avoiding the Tpht. The results demonstrate an attenuated behavior for the decrease of I in SLS as well as for d increase and peak reduction and broadening in SAXS that may better be called as “transition regime”. Upon the increasing temperature, this attenuated behavior yielded by chitosan suggests not only the maintenance of a phospholipidic
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Fig. 5. Amplified SAXS profiles of PC nanovesicles (left) and PC–chitosan nanovesicles (right) at three different temperatures as indicated with applied Lorentzian fits (straight lines). Arrow indicates the appearance of a possible hexagonal HII phase.
bilayer with a higher molecular organization, but also infers rigidity to the bilayer since fluctuations in the membrane curvature are smaller and water layer increase, if it occurs, must be slower. Therefore, as a main result elapsing from the overall process, a higher thermal stability for the vesicles with chitosan is denoted. 4. Conclusions The present contribution analyzes structural characteristics provided on both supramolecular systems of phosphatidylcholine, organogel and nanovesicles, containing chitosan. Polarized Optical Microscopy of the organogel containing chitosan features a characteristic texture which remains unchanged over a large temperature range
and as well as after 24 h. Thus, a non-temperature-sensitive and relatively more stable organogel has been produced with the incorporation of chitosan. Light Scattering and Small Angle X-ray Scattering have shown to work as complementary tools in the investigation of the liposomes upon heating. The evolution of the scattered radiation as a function of temperature shows that addition of chitosan leads to enhancement of thermal stability of the liposomes. The liposomes containing chitosan bear relatively weaker influence of thermal energy on the bilayer fluctuations, if compared to the system without chitosan. Therefore, higher membrane rigidity is suggested in the presence of chitosan. Also, considering the molecular order of the liposomes bilayer as a fundamental aspect of their structural stability, results evidence that
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chitosan increases the stability providing less lattice disorder through the dissipation of thermal energy. It was possible to determine a characteristic phase transition temperature for the phospholipids in the liposomes around 57 °C. A higher phase transition temperature of 64 °C was determined for the liposomes containing chitosan, which represents mostly an intermediate stage of a moderated transitory process in this system. These impaired thermal differentiations produced by chitosan in the liposomes can be of biological and technological relevance. As a worthy applicability we could consider, for instance, the reduction of cytotoxicity of doxorubicin, which may be entrapped in liposomes [50] possessing high phase transition temperature. Acknowledgements The authors thank Solae do Brasil S.A. for the phosphatidylcholine and CNPq and Rede de Nanobiotecnologia (CNPq/MCT) for financial support. The Laboratório Nacional de Luz Synchrotron (LNLS), Campinas, Brazil, is acknowledged for the SAXS measurements. O.M. thanks CAPES and CNPq for a doctoral fellowship. References [1] M. Umrethia, P.K. Ghosh, R. Majithya, R.S.R. Murthy, Cancer Investig. 25 (2007) 117. [2] J.M. Koziara, P.R. Lockman, D.D. Allen, R.J. Mumper, J. Nanosci. Nanotechnol. 6 (2006) 2712. [3] E. Fattal, P. Couvreur, C. Dubernet, Adv. Drug Deliv. Rev. 56 (2004) 931. [4] M.H.B. Costa, O.A. Sant'Anna, P.S. Araujo, R.A. Sato, W. Quintilio, L.V.N. Silva, C.R.T. Matos, I. Raw, Appl. Biochem. Biotechnol. 73 (1998) 19. [5] D. Papahadjopoulos, D.B. Kirpotin, J.W. Park, K.L. Hong, Y. Shao, R. Shalaby, G. Colbern, C.C. Benz, J. Lipid Res. 8 (1998) 425. [6] D.D. Lasic, Nature 380 (1996) 561. [7] G. Gregoriadis, Trends Biotechnol. 13 (1995) 527. [8] D.D. Lasic, D. Papahadjopoulos, Science 267 (1995) 1275. [9] R.N.A.H. Lewis, D. Zweytick, G. Pabst, K. Lohner, R.N. McElhaney, Biophys. J. 92 (2007) 3166. [10] L. Dahbi, M. Arbel-Haddad, P. Lesieur, C. Bourgaux, M. Ollivon, Chem. Phys. Lipids 139 (2006) 43. [11] P.L.G. Chong, M. Zein, T.K. Khan, R. Winter, J. Phys. Chem., B 107 (2003) 8694. [12] R.L. Biltonen, D. Lichtenberg, Chem. Phys. Lipids 64 (1993) 129. [13] S.A. Simon, L.J. Lis, J.W. Kauffman, R.C. Macdonald, Biochim. Biophys. Acta 375 (1975) 317. [14] P.M. McDonald, V. Strashko, Langmuir 14 (1998) 4758. [15] G.T. Dimitrova, T.F. Tadros, P.F. Luckham, M.R. Kipps, Langmuir 12 (1996) 315.
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Chitosan effect on the mesophase behavior of phosphatidylcholine supramolecular systems Omar Mertins ⁎, Maria I.Z. Lionzo, Yasmine M.S. Micheletto, Adriana R. Pohlmann, Nádya Pesce da Silveira ⁎ Instituto de Química, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, 91501-970, Caixa Postal 15003, Porto Alegre, RS, Brazil
a r t i c l e
i n f o
Article history: Received 29 April 2008 Received in revised form 20 August 2008 Accepted 29 August 2008 Available online 16 September 2008 Keywords: Soybean phosphatidylcholine Chitosan Liposome Phase transition temperature SAXS Light Scattering
a b s t r a c t Two distinct supramolecular self assemblies of phosphatidylcholine and chitosan, namely liposomes and their precursory organogel, have been investigated by means of SAXS, Light Scattering and Polarized Optical Microscopy. The main goal was the evaluation of the chitosan effect on the self assemblies phase transition behavior upon heating. A distinct smectic organization was observed for the organogel prepared in the presence of chitosan, if compared to that prepared only with phosphatidylcholine. In addition, the phosphatidylcholine–chitosan organogel showed unchanged optical properties upon heating and after 24 h, indicating increased stability when compared to the organogel prepared without chitosan. For the liposomes containing chitosan, the thermotropic behavior features a lamellar pattern that is preserved under heating, until at least 81 °C. A phase transition temperature has been determined around 64 °C, which was clearly higher than that observed for liposomes prepared without chitosan. The bilayer repeat distance typical of the liposomes increases slowly by increasing the temperature and stacking fluctuations of the bilayers are delayed due to enhancement of the membrane rigidity. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Phospholipid vesicles or liposomes have long been known as systems with great similarities to cellular membranes. Nowadays, liposomes are developed as nanometric drug carriers for specific treatments with controlled circulation time, reduced side effects and optimized drug action [1–8]. It is well known that the knowledge of physico-chemical stability is a crucial demand for applicability of liposomes. The bilayer organization, for instance, changes at well defined temperatures, called temperatures of phase transition (Tpht). At low temperature there is the crystalline lamellar phase Lc producing an ordered bilayer. Increasing the temperature, the first sub transition leads to the Lβ phase, where the lipid chains are extended and tilted with respect to the bilayer plane, keeping the molecular order on the bilayer. Following, a gel ripple phase Pβ′ can be sometimes observed, where the lipid bilayers become a periodic ripple. Afterwards, the main transition temperature forms the liquid crystalline lamellar phase Lα, where the apolar tail of the lipids in the bilayers are highly disordered. As a consequence, the bilayer repeat distances are changing according to the characteristic mesophase [9–11]. Values of Tpht in liposomes have been determined applying different methods like DSC [9,12,13], spectroscopy [14,15], X-ray
⁎ Corresponding authors. Tel.: +55 51 33087321; fax: +55 51 33087304. E-mail addresses: [email protected] (O. Mertins), [email protected] (N.P. Silveira). 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.08.038
diffraction [9,16] and FTIR [11,17]. Recently, Michel et al. [18] have determined the Tpht of lipids by Light Scattering during temperature variations. It was demonstrated that the changes in the measured scattered intensities during heating are representative of different molecular organizations in the liposomes. For the determination of Tpht in liposomes several parameters have to be considered. In these systems, Tpht is sensitive to the ionic strength of the dispersion buffer [9,19], the presence of foreign components, such as cholesterol, together with the phospholipids building the bilayer of the vesicles [20] and to products mixed in the dispersing solution [21], like polymers. Chitosan, a biocompatible polysaccharide [22] obtained from partial N-deacetylation of chitin [23–25], showed a high potential to be used as an extra cover for the improvement of liposomes stability in aggressive environments [26–29]. However, little is known about influence of chitosan on Tpht of liposomes. In recent works [30–32] we have applied the reverse phase evaporation method [33] for the production of liposomes containing chitosan. In this method, we first obtain an organogel, and after water addition, liposomes are formed. Considering that the introduction of a macromolecule may change the bilayer phase transition and, furthermore, the stability and properties of the whole system can also be modified [34], we investigate first the organogel by means of Polarized Optical Microscopy. Following, the liposomes have been studied by means of Static Light Scattering (SLS) and Small Angle X-ray Scattering (SAXS). The experiments have been carried out between 25 and 81 °C.
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2. Experimental 2.1. Materials Phosphatidylcholine (PC) from soybean (95% phosphatidylcholine, 5% lysophosphatidylcholine and phosphatidic acid) was a gift from Solae do Brasil S.A. The molecular composition of the soybean PC is around 75% distearoylphosphatidylcholine (DSPC), 12% dioleoylphosphatidylcholine (DOPC) and 8% dipalmitoylphosphatidylcholine (DPPC). The water content of 6.2% was obtained using Karl Fischer titration. Chitosan was obtained from the deacetylation of chitin as previously described [31]. The acetylation degree of 17% was determined by IR spectroscopy. The average molecular weight Mw = 160,000 g/mol and the gyration radius Rg = 43.5 nm from Static Light Scattering measurements [31] gave a critical concentration of c⁎ = 0.8 mg/ml in a buffer solution of 0.02 M acetate (0.02 M acetic acid and 0.02 M sodium acetate)/0.1 M NaCl (pH 4.5). All other reagents were of analytical grade. 2.2. Sample preparation The organogel and the liposomes were prepared by the reverse phase evaporation method [33]. Samples with or without chitosan were obtained: in 60 mg of soybean PC dissolved in 10 ml of ethyl acetate (Merck®), 200 μl of buffer (0.02 M acetate buffer/0.1 M NaCl (pH 4.5)) without polymer for samples free of chitosan and containing 1 mg/ml of chitosan (filtered 0.22 μm (Millipore®)) for samples with polymer (Fig. 1), were dropped in the solution to form a water in oil like emulsion which was sonicated (2 min) yielding a homogeneous opalescent dispersion of reverse micelles. The organic solvent was removed using a rotatory evaporator (Buchi) at 30–35 °C under vacuum, resulting in a high viscous organogel. The organogel was
reverted to liposomes with the addition of 10 ml of pure water (MilliQ®) under vigorous shaking. The final pH of the nanovesicle emulsions was 5.8 for both systems. The final chitosan concentration in the nanovesicles corresponds to 0.0033 mg per mg of PC. 2.3. Polarized Optical Microscopy The organogels were settled between two glass slides and examined under polarizing microscope (Olympus® BX-43). The texture images were captured using an Olympus® PM20 optical camera with exposure controller. Temperature was ranged from room temperature to 80 °C controlled by a Quasar MT 300 heating stage programmed at a similar range as for SLS and SAXS analyses, and the images acquisition was performed after each 4 °C increment. 2.4. Light Scattering Liposome sizes, polydispersity and diffusion coefficients were determined by Dynamic Light Scattering (DLS) as previously described [30,35]. Static Light Scattering (SLS) was performed on a Brookhaven Instruments standard setup with a He–Ne laser (λ = 632.8 nm) as light source. The samples were placed into dust free cells for Light Scattering (LS) measurements that were placed in the index-matching liquid decahydronaphthalene (Aldrich®) and heated from 25 to 87 °C in a rate of 1 °C/min with intervals of 10 min at each 2 °C when LS intensity (I) was measured 10 times with intervals of 0.1 min. The position of the detector was at 135° relative to the incident beam. The data analysis was carried out as previously described [31]. Experiments were repeated twice for each sample showing reproducibility and error bar was determined considering standard deviation of 10 measurements at each temperature.
Fig. 1. Reverse phase evaporation method with the addition of chitosan. 1. Phosphatidylcholine is dissolved in an organic solvent. 2. A portion of aqueous solution containing chitosan is added. 3. Under sonication an emulsion of reverse micelles containing chitosan is obtained. 4. The organic solvent is evaporated. 5. The organogel is obtained. 6. Vesicles containing chitosan are formed after water addition and shaking.
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2.5. Small Angle X-ray Scattering
3. Results and discussions
Synchrotron SAXS measurements were carried out on the bending magnetic beamline SAS of the Laboratório Nacional de Luz Synchrotron (LNLS, Campinas, Brazil). The samples were placed in a stainless steel sample holder [36] and thermostatized in the same range as for LS with accuracy of ± 1 °C. The wavelength of the incident beam was 1.605 Å and a linear detector (Princeton Instruments) was used at 43.5 cm from the sample. The exposure time of 10 min at each 2 °C interval was required for the spectral acquisitions. Data were normalized to constant beam intensity and corrected for the transmission, sample thickness, parasitic and background scattering according to standard procedure. Considering the size of vesicles larger than 50 nm, no deviation in the diffraction pattern can be expected from the infinite membrane approximation [37]. The bilayer thickness or characteristic length of the bilayer repeat distance (d) in the nanovesicles was determined through the Bragg relation:
3.1. Polarized Optical Microscopy
d ¼ 2π=ðnqÞ with q the wave vector related to the maximum peak intensity of the first Bragg reflection and n = 1, 2, 3,…, ∞. The fitting of the experimental curves was done applying Lorentzian functions with the Microcal Origin software [30,31,38].
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Szoka and Papahadjopoulos [33] described the phosphatidylcholine organogel as a viscous gel and a system with low stability. It undergoes spontaneously or with the application of some mechanical energy to the self assembly of lamellae that built the phospholipid bilayers of liposomes. The evolution of the molecular organization of supramolecular aggregates of phospholipids may consist of worm like micelles (hexagonal phase), cubic network (cubic phase), bicelles, ribbons and lamellae [39–41]. The transitions are related to water concentration and temperature representing respectively transitions of first and second order [41]. Herein the water concentration is kept constant, so that a second order transition is expected. Furthermore, the phospholipid used in this work is zwitterionic and temperature becomes an important parameter on phase transition. In this case, the obtained phases are known to be mostly lamellar. Domains of hexagonal and cubic phases may be found only at high temperatures [41]. In the present work, the microstructure evolution of the PC– organogel as a function of temperature range was investigated by Polarized Optical Microcopy (POM). Increasing the temperature, POM observations indicated the occurrence of two different phases for the PC–organogel. Indeed, as can be seen in Fig. 2 (left column) the phase
Fig. 2. POM images of the PC organogels (left column) and PC–chitosan organogels (right column) at different temperatures (26 (a, e), 62 (b, f) and 82 (c, g) °C), ranging over the phase transition temperatures and after overnight, at 26 °C (d and h) (scale bar = 100 nm).
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transition of the phospholipid modifies the optical properties of the PC–organogel. At 26 °C (Fig. 2a), we observe the presence of typical lamellar mesophase with mosaic texture (like Smectic C) [42,43]. Increasing the temperature up to 80 °C (Fig. 2c), the transition to a hexagonal mesophase with a fan-texture (Smectic A) is evidenced. Moreover, the texture of PC–organogel at 26 °C after 24 h (Fig. 2d) showed the presence of oily streaks (isotropic) and some “Maltese crosses” indicating the occurrence of a lamellar organization characteristic of a dispersion of multilamellar vesicles in an isotropic solution [39]. This texture is different from that visualized at the first condition (Fig. 2a) denoting a transition behavior for the supramolecular organization of PC–organogel. The images of the PC–chitosan organogel in Fig. 2 (right column) show a different pattern, which is similar to a mesophase with Schlieren texture (like Smectic C) [42] and some thread-like defects. Another difference is related to the birefringence of the PC–chitosan organogel that gives some evidence of a distinguished phospholipids organization due the presence of chitosan. Furthermore, since the PC is a lyotropic amphiphil, the presence of polymer may also lead to a reduction of the hydration water on the phospholipids. Thus, the water moieties displacement may modify the optical properties of the organogel. Moreover, the temperature effect on the PC–chitosan organogel was almost imperceptible. As we can see in Fig. 2e, f and g, after heating, the texture remains similar indicating that chitosan stabilizes the supramolecular organization. This stabilization was effective for at least 24 h at 26 °C (Fig. 2h). 3.2. Static Light Scattering PC liposomes free of chitosan and containing chitosan (1 mg/ml in the acetate buffer) feature respectively an average hydrodynamic radius of 173 ± 92 and 269 ± 87 nm, a zeta potential of −42 ± 3 and −34 ± 4 mV and a diffusion coefficient of 1.2 × 108 and 0.8 × 108 cm2/s [30]. The influence of temperature on the liposome sizes of particles was not considered, since phase transitions are not always related to changes in size and/or shape of such systems [18]. In LS experiments the discontinuity in the average number of photons detected per second (I) as the temperature is altered, corresponds to a change in the optical properties of the sample [18]. Indeed, a phase transition modifies the optical properties of the liposomes in water. Fig. 3 displays the averaged I upon heating, for both PC liposomes (a) and PC–chitosan liposomes (b). The abrupt change on I in Fig. 3a is related to the phase transition from gel to liquid crystal (Lα). For the system without chitosan the average I decreases between 53 and 65 °C. The decrease is not progressive, as expected for pure phospholipids [18]. As the PC from soybean contains mainly DSPC (75%), bearing 18 carbon atoms in both apolar chains of the molecule, the phase transition temperature for this phospholipid is expected to be at 55.6 °C [44]. However, the presence of DOPC (12%) and DPPC (8%) with shorter alkyl chains for the later (16 carbons) and some impurities (5%, see Experimental section) may influence the phase transition temperature. Since DSPC mainly determines the phase transition temperature, the other components enlarge the temperature range of the decrease of I. Hence, the phase transition is tuned by different molecules and the whole process is slowed down, resulting in an extended transition (Fig. 3a). A different behavior is observed when chitosan is present in the liposomes (Fig. 3b). Instead of a sharp decrease, a progressive reduction of scattered intensity I, covering the entire investigated range, is evidenced. It seems that chitosan dissipates the thermal energy furnished to the system leading to a very discrete phase transition, which occurs in moderated steps. In the presence of chitosan, the bilayers disorder is minimized somehow by a kinetic compensation that influences the molecular organization. Hence, the presence of
Fig. 3. Evolution of the mean count rate from Static Light Scattering Intensity (I) (squares) and bilayer repeat distances from SAXS (d ± 0.02 nm) (triangles) as temperature function for PC nanovesicles (a) and PC–chitosan nanovesicles (b).
chitosan may lead to a non-homogeneous phase transition, e.g. parts of the liposomes may go trough the transition while others may delay the process. As proposed by Michel et al. (applying Boltzmann regression curves) [18], the phase transition temperatures were obtained from the middle of the slope of each curve, where the presence of microdomains allows the coexistence of two phases in the transition region [45]. In this work we differentiated the curves to obtain the points corresponding to the middle of the slopes. We have found Tpht = 57 °C for liposomes and Tpht = 64 °C for liposomes with chitosan. At this point, an important condition to be considered is the heating process applied. Since our temperature range (25–87 °C) was submitted to large intervals (10 min at each measured temperature, required for SAXS scattering diagrams acquisitions), we consider that these values of Tpht are approximated. Also, the addition of some NaCl in the buffer solution (0.1 M) to form the reverse micelles (Fig. 1) has been reported to lead to alterations in the Tpht of phosphatidylcholines [46]. Nevertheless, the result for liposomes in this work is close to the reported result of Tpht for DSPC (55.6 °C) [44]. After all, as our aim was to investigate the chitosan influence on the behavior of the system, the Tpht shows an increase of at least 7 °C for the system containing chitosan. 3.3. Small Angle X-ray Scattering One of the structural modifications upon heating which results from the phase transition in liposomes is the change in the bilayer
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repeat distance (d) [10]. Using SAXS, d can be obtained by analysis of the diffraction profiles of the scattered intensity [37]. Fig. 4 depicts the SAXS spectra obtained during the temperature increase (see the labeled temperatures for each scattering diagram) for the two samples. At the beginning, both systems display well defined first and second order Bragg reflections indicative of a lamellar phase. As we recently reported [30] such profile may also represent multilamellar structures. In multilamellae the bilayer stacks are less vulnerable to the fluctuations raised from the diffusional movements in water. While in unilamellae, the diffusional movements may constantly modify the format of vesicles changing the bilayer organization. As a result for the former case, the X-ray scattering furnishes a diffuse intensity in the background that provides broad curves in the spectra. In addition, Fig. 4 shows that for the system without chitosan the intensity decreases faster during temperature increase and the peaks broaden suggesting a phase transition [43]. In Fig. 4a, the first order peak can be seen until around 57 °C, despite of the slight intensity relative to the 35 °C profile. After 59 °C, only a small Bragg peak can be noticed superimposed to a broad halo. At 63 °C the peak disappears yielding a broad component, which persists until 81 °C (Fig. 4a). Again, such results may be interpreted as a phase transition that takes place around 57 to 59 °C, supporting the result obtained in SLS experiment (57 °C). This temperature range matches approximately with the region of I decrease in SLS (Fig. 3a). Analyzing the d parameter (Fig. 3a), a constant shift of the peak to smaller q values is detected, resulting in d increase with temperature.
Fig. 4. Small Angle X-ray Scattering Intensities (I) as wave vector function (q) at different temperatures (°C) as indicated for PC nanovesicles (a) and PC–chitosan nanovesicles (b). Spectra shift +0.001.
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The absolute d values are obtained from Lorentzian fits as previously reported [30,31] and shown in Fig. 5 (fitted straight lines). Plotting d from SAXS together with I from SLS (Fig. 3a), a constant increase of d is evidenced in the same region of the I decrease. Regarding the results for the system containing chitosan, a clear different pathway is found. The intensity of the Bragg reflections decreases upon heating, as well, however in a remarkable smaller degree (Fig. 4b). Afterwards, the first peak remains over the whole range of temperatures. Similar behavior appears for the broadening of the peak. A higher broadening starts only at 65 °C (see also Fig. 5), which is close to the 64 °C determined for the Tpht in SLS. Besides, at 75 °C a small shoulder on the right side of the peak can be noticed (Figs. 4b and 5). The same can be related to the formation of a coexistent fluid hexagonal HII phase [47] which complete transition must be delayed by the presence of chitosan. Until 81 °C, it was still possible to discern a reflection. Moreover, the d parameter also presents a distinct behavior. In Fig. 3b, d is slightly increased during the temperature increase, without any abrupt change. A relative faster change starts only after 61 °C. Comparing both systems (Fig. 3a and b), the data of the sample free of chitosan shows a continuous increase of d after 53 °C, whereas for the system with chitosan the same behavior takes place only after 61 °C. Defining d as the bilayer repeat distance means the thickness of the phospholipidic bilayer plus the water layer between the bilayers in a multilamellar vesicle. It is established that a temperature increase leads to an increase in the disorder of the apolar chains resulting in a reduction of their elongation in some mesophases [48]. In this way, the thickness of the phospholipidic bilayer is expected to be reduced, depending on the mesophase in which the system is found. Nevertheless, since the lamellar peaks in SAXS shift to smaller q values, our results show a global increase of d, suggesting that the water layer may also be increased as a result of molecular agitation under the increasing temperature. Semmler et al. [47] also suggested an increase of water between the bilayers as a reason for larger bilayer repeat distances. Another point to be evaluated is the peak broadening. Local fluctuations in the membrane curvature produce such phenomenon [30,37]. Free water between bilayers is necessary to facilitate a change of the interfacial curvature, which in turn controls the phase transition [43]. Assuming an increase of the water layer, membrane fluctuations can be understood considering a concomitant increase of water spatial volume upon heating. As molecular agitation of water increases, the membranes fluctuate. Furthermore, the head group area of phospholipids was also shown to increase as a consequence of the chain mobility in high temperatures [49]. This may also help towards d increase. After all, the lamellar structure can be preserved, but in such situation the bilayer order needs to be weak to result in the broad scattering band in the SAXS region [47]. Thus, the membrane fluctuations must indeed be important. Comparing SAXS data of the two systems, chitosan acts towards keeping a relative ordered bilayer, meaning higher molecular organization. A smaller lattice disorder must be energetically favorable. Hence, the effect of temperature is less important probably because chitosan dissipates the thermal energy, as already considered in SLS, and in this manner the presence of chitosan stabilizes the lamellar phase. Similar effect was also observed by Radlinska et al. in a surfactant plus random heteropolymer system [43]. Considering the moderated d increase, chitosan may also reduce the effect of water agitation between lamellae. Finally, all these contributions seem to move towards avoiding the Tpht. The results demonstrate an attenuated behavior for the decrease of I in SLS as well as for d increase and peak reduction and broadening in SAXS that may better be called as “transition regime”. Upon the increasing temperature, this attenuated behavior yielded by chitosan suggests not only the maintenance of a phospholipidic
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Fig. 5. Amplified SAXS profiles of PC nanovesicles (left) and PC–chitosan nanovesicles (right) at three different temperatures as indicated with applied Lorentzian fits (straight lines). Arrow indicates the appearance of a possible hexagonal HII phase.
bilayer with a higher molecular organization, but also infers rigidity to the bilayer since fluctuations in the membrane curvature are smaller and water layer increase, if it occurs, must be slower. Therefore, as a main result elapsing from the overall process, a higher thermal stability for the vesicles with chitosan is denoted. 4. Conclusions The present contribution analyzes structural characteristics provided on both supramolecular systems of phosphatidylcholine, organogel and nanovesicles, containing chitosan. Polarized Optical Microscopy of the organogel containing chitosan features a characteristic texture which remains unchanged over a large temperature range
and as well as after 24 h. Thus, a non-temperature-sensitive and relatively more stable organogel has been produced with the incorporation of chitosan. Light Scattering and Small Angle X-ray Scattering have shown to work as complementary tools in the investigation of the liposomes upon heating. The evolution of the scattered radiation as a function of temperature shows that addition of chitosan leads to enhancement of thermal stability of the liposomes. The liposomes containing chitosan bear relatively weaker influence of thermal energy on the bilayer fluctuations, if compared to the system without chitosan. Therefore, higher membrane rigidity is suggested in the presence of chitosan. Also, considering the molecular order of the liposomes bilayer as a fundamental aspect of their structural stability, results evidence that
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chitosan increases the stability providing less lattice disorder through the dissipation of thermal energy. It was possible to determine a characteristic phase transition temperature for the phospholipids in the liposomes around 57 °C. A higher phase transition temperature of 64 °C was determined for the liposomes containing chitosan, which represents mostly an intermediate stage of a moderated transitory process in this system. These impaired thermal differentiations produced by chitosan in the liposomes can be of biological and technological relevance. As a worthy applicability we could consider, for instance, the reduction of cytotoxicity of doxorubicin, which may be entrapped in liposomes [50] possessing high phase transition temperature. Acknowledgements The authors thank Solae do Brasil S.A. for the phosphatidylcholine and CNPq and Rede de Nanobiotecnologia (CNPq/MCT) for financial support. The Laboratório Nacional de Luz Synchrotron (LNLS), Campinas, Brazil, is acknowledged for the SAXS measurements. O.M. thanks CAPES and CNPq for a doctoral fellowship. References [1] M. Umrethia, P.K. Ghosh, R. Majithya, R.S.R. Murthy, Cancer Investig. 25 (2007) 117. [2] J.M. Koziara, P.R. Lockman, D.D. Allen, R.J. Mumper, J. Nanosci. Nanotechnol. 6 (2006) 2712. [3] E. Fattal, P. Couvreur, C. Dubernet, Adv. Drug Deliv. Rev. 56 (2004) 931. [4] M.H.B. Costa, O.A. Sant'Anna, P.S. Araujo, R.A. Sato, W. Quintilio, L.V.N. Silva, C.R.T. Matos, I. Raw, Appl. Biochem. Biotechnol. 73 (1998) 19. [5] D. Papahadjopoulos, D.B. Kirpotin, J.W. Park, K.L. Hong, Y. Shao, R. Shalaby, G. Colbern, C.C. Benz, J. Lipid Res. 8 (1998) 425. [6] D.D. Lasic, Nature 380 (1996) 561. [7] G. Gregoriadis, Trends Biotechnol. 13 (1995) 527. [8] D.D. Lasic, D. Papahadjopoulos, Science 267 (1995) 1275. [9] R.N.A.H. Lewis, D. Zweytick, G. Pabst, K. Lohner, R.N. McElhaney, Biophys. J. 92 (2007) 3166. [10] L. Dahbi, M. Arbel-Haddad, P. Lesieur, C. Bourgaux, M. Ollivon, Chem. Phys. Lipids 139 (2006) 43. [11] P.L.G. Chong, M. Zein, T.K. Khan, R. Winter, J. Phys. Chem., B 107 (2003) 8694. [12] R.L. Biltonen, D. Lichtenberg, Chem. Phys. Lipids 64 (1993) 129. [13] S.A. Simon, L.J. Lis, J.W. Kauffman, R.C. Macdonald, Biochim. Biophys. Acta 375 (1975) 317. [14] P.M. McDonald, V. Strashko, Langmuir 14 (1998) 4758. [15] G.T. Dimitrova, T.F. Tadros, P.F. Luckham, M.R. Kipps, Langmuir 12 (1996) 315.
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