Soft Matter
PAPER
11 Characteristics and behaviour of liposomes when incubated with natural bile salt extract: implications for their use as oral drug delivery systems
5 Laura G. Hermida, Manuel Sabe´s-Xaman´ı and Ramon Barnadas-Rodr´ıguez*
3 The use of liposomes for oral administration of drugs and for food applications is based on their ability to preserve
10 entrapped substances and to increase their bioavailability.
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ART C4SM00981A\_GRABS
Soft Matter
PAPER
1
1
Cite this: DOI: 10.1039/c4sm00981a
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Received 6th May 2014 Accepted 30th June 2014
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DOI: 10.1039/c4sm00981a www.rsc.org/softmatter
Characteristics and behaviour of liposomes when 1
incubated with natural bile salt extract:
implications for their use as oral drug delivery
5
systems
Laura G. Hermida,a Manuel Sabe´s-Xaman´ıb and Ramon Barnadas-Rodr´ıguez*b
The use of liposomes for oral administration of drugs and for food applications is based on their ability to 10 preserve entrapped substances and to increase their bioavailability. Bile salts are one of the agents that affect the liposome structure during intestinal digestion and the main reported studies on liposome/bile salt systems used only one bile salt. The aim of this work is to characterise the interaction of liposomes with a natural bile salt extract (BSE) at physiological pH and temperature. Three types of liposomes (fluid, 15 gel-state and liquid-ordered bilayers) were studied. Phase diagrams were obtained and a very different behaviour was found. Fluid bilayers were completely permeable to an entrapped dye with partial or complete disruption of vesicles (final size 10 nm). Gel-state bilayers released their content but BSE led to the formation of large mixed structures (2000 nm). Liquid-ordered bilayers formed mixed vesicles (1000 nm) and, surprisingly, retained a high percentage of their aqueous content (about 50%). As a 20 consequence, each type of liposome offers singular features to be used in oral applications due to their specific interaction with bile salts.
25 1 Introduction
with cell cultures, formulations are usually exposed to in vitro 25 digestion that mimics the gastrointestinal tract. The intestinal
Liposomes as (so) drug delivery systems are commonly used in step of the previous process usually requires sample incubation parenteral and topical administrations,1,2 but aer a period of with a bile salt extract (BSE) from a biological source.14–16
uncertainty the oral route potential has now emerged.3–7 Due to
Consequently, a detailed knowledge of the processes
30 their structure, liposomes can protect the entrapped substances involved in the interactions of liposomes and BSE would 30 from the environment of the gastrointestinal tract, and their in contribute to the improvement of the functionality of the oral vivo effectiveness depends on, among other factors, the changes formulations that contain these vesicles. In fact, the effect of
that they undergo when they interact with the bile salts present bile salts on phospholipid bilayers and monolayers is a particin the intestines. In this way, the macromolecular assembly of ular case of the well-known solubilisation process17–19 caused by 35 phospholipids can undergo several changes that can lead to the surfactants which involves three different stages: (a) during the 35
total disruption of the vesicles. The role of bile salts in the use of vesicular stage the liposome bilayer becomes progressively
liposomes and other colloids as drug carriers is not only limited enriched in the surfactant, (b) liposomes are gradually
to the digestion process, as it has been shown that the inclusion destroyed as the surfactant concentration increases and mixed
of some of them in the particle structure enhances their phar- micelles are formed and, (c) only (mixed) micelles exist in the
40 macological activity.8,9
bulk. Solubilisation curves can be obtained by monitoring 40
At the same time, the evaluation of oral formulations to absorbance/turbidity changes in different liposome suspen-
predict their bioavailability and mucoadhesion properties sions upon increasing the surfactant concentration. Some
greatly depends on the existence of in vitro models, such as the characteristic points that indicate the phase boundaries
45
Caco-2 cultures.10–13 In these cases, and prior to the incubation (usually the onset and full solubilisation points) can be observed from these curves. These points are then used to
45
obtain the phase diagram of the system. It is described in the
aCentre of Research and Development in Chemistry, National Institute of Industrial Technology (INTI), Av. Gral. Paz e/ Constituyentes y Albarellos San Mart´ın, Buenos Aires, Argentina
literature that, for a given surfactant, liposome solubilisation depends to a great extent on the size, lamellarity and compo-
50
bCentre d'Estudis en Biof´ısica, Unitat de Biof´ısica, Departament de Bioq´ımica i Biologia sition of the vesicles, as well as temperature and ionic strength 50
Molecular. Faculty of Medicine, Universitat Auto`noma de Barcelona, 08193 of the medium.20–22 Most of the liposome solubilisation studies
Cerdanyola, Catalonia, Spain. E-mail: ramon.barnadas@uab.cat; Fax: performed with bile salts have been made using pure molecules
+34 935811907; Tel: +34 935868476
This journal is © The Royal Society of Chemistry 2014
Soft Matter, 2014, xx, 1–9 | 1
Soft Matter
Paper
1 such as sodium cholate, sodium deoxycholate, or some articial ($99%) were purchased from Sigma-Aldrich (USA). Pyranine 1 mixtures.23–25 Even though the effect of natural BSE on lipo- was from Kodak (USA) and DPX (a pyranine quencher) was from
somes implies a closer approximation to the in vivo intestinal Molecular Probes (The Netherlands). All other reagents were of
digestion, and BSE is commonly used in in vitro liposome analytical grade.
5 digestion models, to our knowledge there are no detailed
5
studies focused on the effects of natural BSE on these vesicles. 2.2 Liposome preparation
Moreover, no detailed studies providing phase diagrams for
SPC and HSPC were added into 10 mM TRIS, 145 mM sodium
mixtures of bile salt extracts with liposomes are available. If chloride, pH 6.5 buffer solutions (since pH changes during
liposomes are currently intended to be used for oral drug
10
intestinal digestion from, approximately, 5.7 to 7.4, this mean delivery, knowledge of their behaviour in the presence of a
10
value was selected and used throughout the work) and stirred 60
complex natural mixture of bile salts could provide important minutes at 40 C or 55 C respectively. The resulting multi-
information for the optimisation of the system. Our work endeavours to ll these gaps in the scientic
lamellar vesicles (33 mM) were further homogenized30 using a Microuidizer 110S at the previously indicated temperatures.
literature. On the one hand, it analyses and provides a
15
HSPC/CHOL liposomes were prepared at a lipid molar ratio of hypothesis, not previously described, about the formation of
15
3 : 2. Lipids were dissolved in chloroform : methanol (2 : 1 v/v)
mixed structures with BSE, especially in the cases of saturated
and solvent was eliminated by rotary-evaporation. The dry
and cholesterol containing liposomes. The phase diagrams of lm obtained was hydrated with the buffer solution (nal lipid
the studied systems are also obtained.
concentration 41 mM) and vortexed at 55–60 C before
We report and analyse the interactions of a natural BSE with
20
three types of liposomes that exhibit very different phase membrane properties at human body temperature (37 C): (a)
homogenization at the same temperature. When required, 2 mM pyranine (a water soluble uorescent dye) was added to the buffer.
20
soy phosphatidylcholine (SPC), which has a negative transition
temperature and, therefore, forms uid bilayers; (b) gel-state
25
membranes composed of hydrogenated SPC (HSPC), with a 2.3 Vesicle size determination
25
transition temperature higher than that of the human body, and; The particle size was measured by dynamic light scattering
(c) liquid-ordered bilayers of HSPC and cholesterol 3 : 2 mol (Ultrane Particle Analyzer UPA150, USA). Cell temperature was molÀ1 (HSPC/CHOL), which exhibit the characteristics of both controlled by an external bath, and the change of water viscosity
uid and gel phases, and have no transition temperature.26,27 with temperature was considered in the soware presets.
30 Consequently, a very different behaviour is expected when they Analyses were performed without sample dilution in order not 30 interact with a natural BSE. These lipids are puried by large to alter the phase equilibrium. Results are expressed as the
scale methods, are commercially available, and are usually used mean diameter of the volume distribution and SD (n $ 2).
by food and pharmaceutical industries. The BSE commercial
extract used has a bile salt composition that is very similar to 2.4 Solubilisation assays
35 that of human bile as previously determined.28,29 The present
35
paper specically describes the physical evolution of these Solubilisation curves of liposomes at several concentrations
liposomes at 37 C and pH 6.5 (with the absence of intestinal were obtained by monitoring sample absorbance (600 nm) on a
double beam spectrophotometer Varian CARY 3Bio. The wave-
enzymes) by measuring the turbidity changes of the sample and
length was chosen in order to minimize the interference of BSE
40
by dynamic light scattering. From these measurements, phase absorption. Appropriate dilutions of the BSE in TRIS buffer (pH diagrams were obtained and the temperature was eventually
40
6.5) were used as reference solutions. The required volumes of
raised to achieve full solubilisation. The capacity of the lipo-
concentrated BSE aliquots were added to the continuously
somes to maintain the entrapped aqueous medium was also stirred samples. Results are expressed as mean Æ SD (n $ 2).
evaluated by measuring the leakage of pyranine, a non-bilayer
45
permeable uorescent probe. We found that each type of The phase diagrams where obtained by calculating the characvesicle exhibits a very different behaviour not only with regard to teristic points of the solubilisation curves. At a given concen-
45
tration of lipid (each one of the curves), the corresponding BSE
the susceptibility to be fully solubilised by BSE, but also in the
concentrations were calculated from the break points of the
vesicular stage. The results set suggests the formation of three different molecular aggregates which, depending on the initially
curve, from it the rst derivative (0 value) and, in the case of
50
entrapped substances, may determine the effectiveness of each total liposome solubilisation, the BSE concentration that caused an absorbance value equal or smaller than 0.03 was also
50
type of liposome in nutritional formulations.
considered. Results of the phase diagrams are expressed as the
2 Experimental section
mean Æ SD (n $ 2).
55 2.1 Chemicals and reagents
2.5 Fluorescence assays
55
SPC (minimum 95%) and HSPC (minimum 95%) were obtained Fluorescence assays were performed to study the effect of BSE on
from Degussa (Germany). BSE (hyocholic acid 5.3%, cholic acid the aqueous content of liposomes. Vesicles were obtained in
18.5%, deoxycholic acid 2.5%, glyco and taurocholic acid buffer containing pyranine 2 mM and subsequently puried
37.5%, glyco and taurodeoxycholic acid 21.6%) and CHOL by size exclusion chromatography (Sephadex G-25) to remove the
2 | Soft Matter, 2014, xx, 1–9
This journal is © The Royal Society of Chemistry 2014
Paper
Soft Matter
1
non-entrapped dye. Aer adjusting the lipid concentration, are formed (2 mM to approximately 6 mM of BSE) and; (III) only sample aliquots were incubated during 1 h at 37 C with micelles are present in the sample (BSE concentration > 6 mM).
1
increasing concentrations of BSE. The percentage of pyranine It can be observed that stage I (vesicular domain) exhibited
retention was calculated from the ratio of the corrected uo- particular absorbance changes. There was a clear initial
5 rescence measured before and aer the addition of 150 ml of DPX decrease of the absorbance (stage Ia) and a subsequent increase 5 200 mM to 3 ml of sample. As BSE exhibits intrinsic uores- (stage Ib). The uorescent changes shown in Fig. 1 indicate that,
cence, curves of BSE uorescence in the absence and presence of in one hour, the dye was released at BSE concentrations that did
DPX were acquired for data correction. Fluorescence was not cause bilayer disruption, that is, during stage I. This was
measured with a SLM Aminco 8100 Spectrouorometer using especially true during stage Ib, as the retention of the dye
10 417 nm and 511 nm as excitation and emission wavelengths drastically diminished (inset Fig. 1), and pyranine was almost 10
respectively. Results are expressed as mean Æ SD (n $ 2).
completely released before the onset of formation of mixed
micelles. Fig. 2 shows the absorbance variations of different
2.6 Differential scanning calorimetry (DSC)
concentrations of SPC liposomes aer the addition of
increasing amounts of BSE. From the similarity of the proles,
15 The main phase transition temperature of HSPC and HSPC/ in all cases, it can be assumed that equivalent morphological 15
CHOL liposomes was determined using a Microcal MC-2 DSC changes and processes took place. Several characteristic points
20
microcalorimeter (USA). Measurements were performed at a heating rate of 90 C hÀ1, from 25 to 80 C, and using TRIS
buffer as a blank.
can be obtained from the curves at different SPC concentrations: CIab, the BSE concentration that caused the change from stage Ia to stage Ib (minimum absorbance value of stage I); Csat, the BSE concentration that caused the saturation of the bilayer
20
3 Results
3.1 SPC liposomes
(limit between stages I and II); and Csol, the BSE concentration that caused full solubilisation of liposomes (limit between stages II and III). The inset of Fig. 2 was obtained by plotting the
25
Fig. 1 shows the behaviour of SPC vesicles when incubated with total BSE concentration vs. the total lipid concentration at the BSE for 1 h at 37 C (the absorbance remained constant aer previously obtained characteristic points. The results parallel
25
20 minutes of incubation at each BSE concentration, indicating the general behaviour of liposome/surfactant systems17–19,23 in
that all the mixtures reached a steady state). The results of which, from each family of characteristic points, a linear ansatz
absorbance reected the morphological changes of liposomes can be made using the following general equation:
30 caused by the interaction with BSE. The absorbance curve illustrates the well-known three-stage model for the interaction
[detergent]T ¼ [detergent]w + Re[Lip]T
30 (1)
between liposomes and surfactants:31 (I) bile salt molecules interact with the membranes without disrupting the vesicles (0– 2 mM of BSE); (II) aer saturation of the bilayers, vesicles are
where, when applied to the case of BSE, [BSE]T is the total BSE concentration, [BSE]w is the BSE concentration in the bulk,
35 progressively solubilised and, concomitantly, mixed micelles
35
40
40
45
45
50
50
55
55
Fig. 2 Solubilisation curves of SPC liposomes (circle: 3.33 mM; square:
Fig. 1 Absorbance (circles) and fluorescence (squares) changes of 2.33 mM; up triangle: 1.75 mM; down triangle: 0.88 mM; diamond:
suspensions of SPC liposomes (0.60 mM) in the presence of BSE after 0.60 mM) obtained in the presence of BSE after 20 min at 37 C. Inset:
1 h of incubation at 37 C. The inset corresponds to low concentrations the phase diagram of SPC/BSE mixtures obtained from the solubili-
of BSE.
sation curves. (*: Point rejected).
This journal is © The Royal Society of Chemistry 2014
Soft Matter, 2014, xx, 1–9 | 3
Soft Matter
1 [Lip]T is the total lipid concentration, and Re is the effective BSE to the lipid ratio, that is, the ratio of the total detergent concentration that is bound to the lipids in the different types of
mixed aggregates ([BSE]agg/[Lip]agg). Each one of the previous 5 parameters are used to establish the different phase boundaries
of the system. From the previous equation, it is also possible to
calculate the molar fraction of BSE in the mixed aggregates, xaBgSgE, that is:23,32,33
10
xBagSgE ¼
½BSEagg ½BSEagg þ ½Lipagg
¼
Re Re þ
1
(2)
Paper 1 5
10
The values of the parameters obtained for the relationships
are shown in Table 1 and the corresponding curves set the
15 limits of the four different stages mentioned previously (Ia, Ib, II
15
and III). As can be observed (inset Fig. 2), in the studied range Fig. 3 Effect of the BSE concentration on the mean diameter
the CIab points do not give a curve with a signicant slope and, (%volume distribution) after 1 hour of incubation of SPC (circle:
consequently, a mean value of 0.41 Æ 0.23 mM is obtained for 1.8 mM, 37 C), HSPC (dark square: 1.2 mM, 37 C; white square:
20
[BSE]Iwab. Due to the deviation from linearity, the absorbance at a
3.3 mM, 55 C), and HSPC/CHOL liposomes (dark up triangle: 1.1 mM, 37 C; white up triangle: 1 mM, 65 C). The mean diameter of HSPC/
20
lower lipid concentration was rejected for the calculation of the CHOL liposomes incubated with 56 mM BSE at 65 C is also expressed
saturation phase boundary. Fig. 3 shows the variation of the as number distribution (white down triangle).
mean diameter of SPC vesicles as a function of the total BSE
concentration. The results indicate a gradual decrease of the
25 vesicle size (initial diameter 539 Æ 150 nm) when BSE is added. leakage (90–95%) at a BSE concentration of 0.8 mM (although 25
By the end of the solubilisation process the diameter was no vesicle disruption took place). Similar absorbance changes
consistent with the size of mixed micelles (about 10 nm). As the were observed at different phospholipid concentrations (Fig. 5)
diameter is expressed in volume percentage, it can be inferred and full solubilisation of liposomes was not attained even at the
that all the aggregates detected corresponded to micelles, and highest BSE to the phospholipid ratio used. In regard to the size
30 no other kind of vesicles was present.
measurements performed at a phospholipid concentration of 30
1.2 mM (Fig. 3), it can be observed that their evolution was
3.2 HSPC Liposomes
concomitant to the absorbance changes at the same concen-
The absorbance variation of HSPC liposomes as a function of tration and no micelle size was achieved (initial diameter 800 Æ
35
the BSE concentration aer 1 h incubation at 37 C is shown in 240 nm; nal diameter 1800 Æ 260 nm).
Fig. 4. Results show a different behaviour than that of SPC
In order to achieve full vesicle solubilisation the temperature
35
liposomes. As can be observed, no stage Ia was detected and a high positive slope of the absorbance was obtained at low BSE concentrations. Subsequently, and in a similar way to SPC
of the sample had to be raised above the phase transition temperature of HSPC (52.2 Æ 0.1 C, n ¼ 4).
The absorbance results obtained at 55 C (Fig. 6) for the two
40
vesicles, a BSE concentration which corresponded to the onset higher phospholipid concentrations (3.3 and 2.1 mM) were of solubilisation was achieved (about 3.5 mM). A further quite similar to the curves of SPC liposomes at 37 C. They
40
increase in the BSE concentration caused a decrease of the showed an initial decrease of the absorbance and a subsequent
absorbance but, contrary to SPC liposomes, it did not lead to a increase before the onset of solubilisation. Then, in those cases
zero value of the absorbance. Instead, the absorbance decreased and as for SPC, the vesicular domain of the uid bilayers of
45 to a nal constant value (about 0.5) that was higher than the HSPC exhibited the stages Ia and Ib in the presence of BSE. But 45
initial value (0.2) obtained in the absence of BSE. In the uo- for lower phospholipid concentrations the shape of the curves
rescence assay (Fig. 4), the release of the dye took place at very was not totally maintained: The portion of the vesicular domain
low concentrations of BSE and there was an almost a complete corresponding to stage Ia gradually decreased with decreasing
50
50
Table 1 Solubilisation parameters of SPC, HSPC and HSPC/CHOL liposomes obtained by incubation with BSE
CIab
Csat
55
Temperature Inc.
Composition (C)
time
[BSE]wIab (mM)
RIeab
xaBgSgE,Iab
[BSE]swat (mM)
Rseat
Csol
[BSE]swol
xaBgSgE (mM)
Rseol
xaBgSgE,sol
55
SPC
37
HSPC
55
HSPC/CHOL 65
20 min 0.41 Æ 0.23 0 0
1h
3.1 Æ 1.3 0 0
1–2 h —
——
1.24 Æ 0.14 0.39 Æ 0.07 0.28 4.58 Æ 0.12 1.60 Æ 0.06 0.62 3.55 Æ 0.74 1.58 Æ 0.35 0.61 7.37 Æ 0.43 5.07 Æ 0.8 0.84 À1.1 Æ 2.3 22.9 Æ 3.6 0.96 1.24 Æ 1.46 43.0 Æ 2.6 0.98
4 | Soft Matter, 2014, xx, 1–9
This journal is © The Royal Society of Chemistry 2014
Paper 1
5
Soft Matter 1
5
10
10
15 Fig. 4 Absorbance (circle) and fluorescence (square) changes of
15
suspensions of HSPC liposomes (0.67 mM) in the presence of BSE after
1 h of incubation at 37 C.
Fig. 6 Solubilisation curves of HSPC liposomes (circle: 3.33 mM;
square: 2.13 mM; up triangle: 1.24 mM; down triangle: 0.67 mM; dia-
20
mond: 0.40 mM) obtained in the presence of BSE after 1 h at 55 C. 20
Inset: the phase diagram of HSPC/BSE mixtures obtained from the
solubilisation curves. (*: Point rejected).
25
measured in pure HSPC samples. This fact indicates that a 25
homogeneous liquid-ordered membrane was obtained. Fig. 7
corresponds to the incubation of HSPC/CHOL liposomes with
BSE. It can be observed that there was a sharp initial decrease of
the absorbance at low BSE concentrations, and a subsequent
30
constant value (0.22) at half the initial absorbance. There was a 30
concomitant decrease in the uorescence (Fig. 7), and the
minimum value (32% of the initial value) was reached at a BSE
concentration of about 2 mM. Consequently, and similar to SPC
35
and HSPC, the interaction of bile salts with HSPC/CHOL lipo- 35
somes at low BSE to lipid ratios increased the bilayer perme-
Fig. 5 Effect of the BSE concentration after 1 hour of incubation at 37 C on the absorbance of HSPC liposomes (circle: 3.33 mM; red
ability of the vesicles. Both the constant values of the
square: 2.13 mM; up triangle: 1.24 mM; down triangle: 0.67 mM; absorbance and the diameter evolution (Fig. 3) obtained at
diamond: 0.40 mM).
higher BSE concentrations reveal that liposomes were not
40
40
HSPC concentration and, consequently, it was not possible to
fully assess the behaviour in the vesicular stage. The boundary
phase parameters were calculated from the characteristic points
45 (Table 1) and the phase diagram obtained (Fig. 6). Due to its
45
negative deviation, the absorbance corresponding to a lipid
concentration of 0.4 mM was rejected. This change in the
expected value has been described by other authors34,35 at a very
low lipid concentration using pure bile salts, and taking into
50 account the nite size of mixed micelles and the end-cap effect
50
of cylindrical micelles. Size analysis (Fig. 3) also reects the complete solubilisation of HSPC liposomes at 55 C: A mean
diameter close to 10 nm was observed at the end of the process,
indicating the mere existence of mixed micelles.
55
55
3.3 HSPC/CHOL liposomes
DSC thermograms of HSPC/CHOL liposomes showed the abolition of the phase transition temperature (data not shown)
Fig. 7 Absorbance (circle) and fluorescence (square) changes of
suspensions of HSPC/CHOL liposomes (0.5 mM) in the presence of BSE after 1 h of incubation at 37 C.
This journal is © The Royal Society of Chemistry 2014
Soft Matter, 2014, xx, 1–9 | 5
Soft Matter
Paper
1 solubilised, as described for HSPC vesicles. Surprisingly, and
1
contrary to the incubations performed with SPC and HSPC
liposomes, a residual uorescence was maintained once the
absorbance reached a plateau (about 50% of the initial uo-
5 rescence). This remarkable resistance of HSPC/CHOL lipo-
5
somes to solubilisation is evident in Fig. 8: Not even diluted
liposomes (0.25 mM total lipid) at the highest BSE concentra-
tion (56 mM) were solubilised during the incubation, only
decreasing the absorbance to 50% of the initial value. The
10 curves corresponding to phospholipid concentrations of 0.76
10
and 1 mM (Fig. 8) clearly show the stage at which pyranine was
initially released. In this zone, there was an initial decrease of
the absorbance upon increasing the BSE concentration which
was concomitant with a vesicle size diminution (Fig. 3), and the
15 subsequent absorbance increase was also associated with a
15
diameter increase. This behaviour parallels that observed with
SPC liposomes, and could be indicative of similar mechanisms
of interaction of BSE with the two types of bilayers, that is, the
existence of Ia and Ib stages.
Fig. 9 Solubilisation curves of HSPC/CHOL liposomes (circle:
20
1.04 mM; square: 0.76 mM; up triangle: 0.5 mM; down triangle: In order to achieve full solubilisation of HSPC/CHOL lipo- 0.36 mM; diamond: 0.25 mM) obtained in the presence of BSE after 1 h
20
somes in 1–2 hours the temperature had to be raised to 65 C. at 65 C. Inset: the phase diagram of HSPC/CHOL/BSE mixtures
The results (Fig. 9) show the complex behaviour of the system, obtained from the solubilisation curves. (*: Points rejected).
with some consecutive absorbance peaks before the onset of
25 solubilisation. Consequently, unlike that which occurs with SPC
25
and HSPC, more than two vesicular sub-domains can be corresponding to the stages II and III which are complemented
considered for HSPC/CHOL liposomes when they interact with in the inset of Fig. 9 with some other points of the vesicular
BSE if all the break points of the absorbance plots are taken into domain. Note that, as for the cases of SPC and HSPC, some account. Unfortunately, the proles of the curves were similar characteristics points corresponding to vesicular stages at low
30 only for the higher lipid concentrations used, thus it was not phospholipid concentrations showed deviations from linearity 30 possible to obtain the equivalent characteristic points for all the and were excluded in the linear regression t. With regard to the
lipid concentrations.
vesicle size analysis, the mean diameter determined aer full
This fact conditioned the calculation of the boundary phases solubilisation was about 200 nm (% volume average). However, and explains the existence of a negative value of [BSE]wsat (Table the measured mean diameter expressed as % number average 35 1) which is, obviously, a consequence of the experimental error was 20 nm. These results provide evidence of the heterogeneity 35
of the extrapolation. For this reason Table 1 shows the data of the sample at this stage, although both the absorbance value
and the diameter obtained from the number distribution
indicate that a large quantity of the initial liposomes was
solubilised.
40
40
4 Discussion
The experimental design used in the present work combines, on
45
the one hand, absorbance and size measurements that can 45
explain the changes that take place during the interaction of
BSE with liposomes. On the other hand, the uorescence
experiments provide information on how the membrane
permeability/integrity is affected during this process. As it is
50
known, the in vivo effectiveness of the liposomes depends on 50
the interaction with bile salts, on lipid digestion, and also on
the solubilisation of the incorporated drug, and possible
enhancement of permeability. Consequently, our study
provides specic information which could partially explain the
55
in vivo effectiveness of liposomes as oral drug delivery systems. 55
Fig. 8 Effect of the BSE concentration on the absorbance of HSPC/ CHOL liposomes (black circle: 1.04 mM; square: 0.76 mM; up triangle:
0.5 mM; down triangle: 0.36 mM; diamond: 0.25 mM) after 1 hour of incubation at 37 C.
From the results obtained by the incubation of SPC vesicles with a natural BSE for 1 h at 37 C, three primary conclusions may be reached: (a) SPC liposomes can be efficiently solubilised
under these conditions; (b) their aqueous content can be
6 | Soft Matter, 2014, xx, 1–9
This journal is © The Royal Society of Chemistry 2014
Paper
Soft Matter
1 completely released to the bulk, even if no destruction of the phosphatidylcholine (PC) liposomes in saline medium are 1.4 1 molecular structure of the vesicle has taken place and; (c) at very mM for deoxycholate (DC) and 6 mM for C (large unilamellar
low BSE to lipid ratios (stage Ia) the aqueous content is main- liposomes, 30 C),37 and 3 mM for TC (small unilamellar lipo-
tained, although an interaction between BSE and liposomes is somes, 25 C).23 In our experiments, a smaller value was
5 detected.
obtained (1.24 Æ 0.14 mM), and as [detergent]swat increases with 5
The observed effect of BSE on phosphatidylcholine lipo- temperature,37 it can be shown that at 37 C BSE mixed micelles
somes (Fig. 1–3) gave similar absorbance results to that are formed at a lower concentration than the mentioned pure obtained by Andrieux et al.23 and Elsayed and Cevc21 using pure bile salts. The Resat, value obtained with BSE (0.39 Æ 0.07) is
bile salts. These authors performed a continuous slow addition similar to that achieved with DC (0.35), C (0.33) and TC (0.29) by
10 of taurocholate (TC) or worked under equilibrium conditions the same previous authors.23,37 Thus, at a given lipid concen- 10
using cholate (C), respectively. In our case, before the onset of tration, the maximum quantity of BSE, DC, C and TC in the
solubilisation (that is, in the vesicular domain) BSE rst causes liposome membranes before the formation of mixed micelles is
a strong decrease of the absorbance and then a small increase. approximately the same. As expected, and instead of being This behaviour parallels the previously mentioned studies and, equal, [BSE]swol >[BSE]wsat as usually occurs with bile salts due to 15 therefore, a similar explanation of the vesicular changes could intermicellar interactions and its value (4.58 Æ 0.12 mM) is 15
be proposed. Elsayed and Cevc21 explained these absorbance results by a vesicle-apparent shrinkage caused by bilayer uctuations induced by the surfactant (which would produce a
located between that of TC (4 mM),23 C (from 5 to 8 mM)21,37 and DC (2 mM).37 With reference to Rseol, the value obtained for BSE (1.60 Æ 0.06) is higher than that obtained with the previous bile
20
decrease in the absorbance) and an expansion of the membrane salts. This fact means that the minimum molar fraction of BSE and an increase of the vesicle size caused, merely, by its inser- necessary to transfer all the PC into mixed micelles (0.62) about
20
tion into the membrane (which would cause an increase in the 30% higher than that of C (0.47),21 which, in turn, is higher than
absorbance). Consequently, as they said, there is a counter-play that of DC and TC.
between both phenomena, which would explain the negative
The observed effect of BSE on PC liposomes is not conclusive
25 and positive slopes of the absorbances observed at stages Ia and when compared to in vitro digestions carried out with simulated 25
Ib, respectively.
complex intestinal uids instead of pure bile salts. Literature
If we now consider our uorescence results, it can be shows contradictory results14,38 which could be caused by the
demonstrated that SPC bilayers remain stable while they different grade of purity of the PC used, as well as the different
interact with the BSE during stage Ia (more than 80% of the lipid digestion models.
30 uorescence is retained). As the molar fraction xBagSgE,Iab is 0 mM,
Taking into account the previous facts, it can be concluded 30
it can be concluded that in stage Ia there is no insertion of BSE that PC liposomes, characterised by their uid state membrane,
into the bilayers. This scenario is compatible with the hypoth- are greatly susceptible to release all their entrapped aqueous
esis of Andrieux et al.23 who proposed that, in the rst range of content during intestinal digestion due to the effect of bile salts.
the vesicular domain, TC monomers are located at the phos- The very small range of stage Ia hardly ensures the maintenance
35 pholipid–water interface with no interaction with the hydro- of the water soluble entrapped molecules. By contrast, stage Ib 35
phobic core of the membrane. The results observed in our work and (obviously) partial and full liposome solubilisation lead to
suggest that this arrangement can be also applied to all the BSE the total release of the aqueous core to the medium. This
components in the Ia stage: the non-existence of surfactant behaviour can be an advantage if lipophilic substances are
insertion during the incubation ensures the retention of the incorporated into the liposome bilayer. In these cases, if the 40 probe by preserving the permeability of the liposomal membrane uidity is not altered, vesicles will be solubilised 40
membrane.
into mixed micelles which would increase the intestinal uptake.
However, it is obvious that the changes in absorbance and The behaviour of HSPC liposomes upon incubation is totally
the mean diameter measured in the entire vesicular domain different from that of SPC vesicles. The results shown in Fig. 3–5
45
provide evidence that there are variations of the liposome shape indicate that stage Ib is the main one in the vesicular domain and/or size. Such variations only affect membrane permeability and that full solubilisation of liposomes is not achieved under
45
during stage Ib (pyranine is released), that is, when the bilayer the reported conditions. Consequently, large mixed structures
expansion caused by the insertion of the bile salts causes the are formed instead of micelles, even at high BSE to HSPC ratios.
positive slope of the absorbance. In a recent work, Niu et al.36 The uorescence and absorbance results are compatible with
50 evaluated the effectiveness of insulin-loaded liposomes that those observed by Andrieux et al.20 in DSC experiments per- 50 contained several bile salts using a phospholipid (SPC) to bile formed with dipalmitoylphosphatidylcholine (DPPC) liposomes
salt molar ratio of 4 : 1. The increased transport of insulin under the continuous addition of TC. They detected a decrease
observed by the authors in Caco-2 cell cultures is compatible in the fusion enthalpy of membranes as a consequence of TC
with the existence of liposomes in the Ib stage, that is, the insertion in the vesicular domain, and concluded that under
55 vesicular structure is maintained, and at the same time they those conditions (before the phase transition temperature) the 55
gradually release their content into the medium.
DPPC gel-phase exhibits a disordered state when TC is present
As can be seen in Table 1, BSE exhibits an increased effec- in the bilayer. In our case, this proposed pre-transition disor-
tiveness of solubilisation compared with some pure bile salts. dered state, caused by a rapid insertion of bile salts into The [detergent]swat values obtained by other authors using the membranes, is consistent with both the dramatic
This journal is © The Royal Society of Chemistry 2014
Soft Matter, 2014, xx, 1–9 | 7
Soft Matter
Paper
1 morphological changes evidenced by the absorbance changes stage, where practically no release of HTPS nor insertion of the 1 and the intense pyranine leakage. In contrast, when the surfactants into the bilayers were detected. This fact suggests
concentration of TC is increased, Andrieux et al.20 achieve a full that, during the vesicle-apparent shrinkage of the HSPC/CHOL
liposome solubilisation at 37 C, that is, below the phase tran- liposomes, a surfactant insertion into the liquid-ordered bila-
5 sition temperature of DPPC (Tm ¼ 41 C), a phenomenon that is yers takes place. At higher BSE concentrations, when a subse- 5 not observed in our HSPC liposomes. This different behaviour quent increase of the vesicle size is produced (stage Ib), the loss
could be caused by the transition temperature of HSPC (Tm ¼ of the uorescent dye ends, just the opposite of that which 51.8 Æ 0.2 C, n ¼ 4). This hypothesis is compatible with the occurs with SPC and HSPC liposomes. Therefore, there is a
results obtained by Kokkona et al.39 They performed incubation critical inserted-surfactant to the lipid ratio that leads to a 10 experiments (1 h, 37 C) with 10 mM C or TC and 2.5 mM DPPC strong stabilisation of the HSPC/CHOL membrane (Fig. 8) and 10
or distearoylphosphatidylcholine (DSPC, Tm ¼ 55 C) liposomes prevents the loss of the entrapped aqueous medium. Accordand found that around 15% and 20% of the entrapped dye ingly, HSPC/CHOL liposomes were denitely the most resistant
remained in DPPC and DSPC vesicles, respectively. Therefore, C vesicles to a complex natural mixture of bile salts, as demon-
and TC highly modied bilayer permeability but did not solu- strated by the fact that they were only solubilised at high bile 15 bilise the liposomes. In our case, the effect of BSE on bilayer extract to lipid ratios and elevated temperatures (Table 1 and 15
permeability is higher than that caused by TC and C alone, due Fig. 3 and 9). As can be observed in the inset of Fig. 9, at low
to the fact that the dye is completely released to the bulk. But lipid concentrations there were large deviations from linearity,
the high transition temperature of HSPC liposomes, similar to and this fact prevented the precise determination of the vesic-
20
that of DSPC, could explain their resistance to solubilisation. ular sub-domains. Solubilisation of HSPC liposomes by bile salts was found to Consequently, if HSPC/CHOL liposomes are used in oral
20
be dependent on the temperature. Full vesicle solubilisation applications, it is expected that during the intestinal digestion can be achieved at 55 C (Fig. 3 and 6), that is, when they would maintain a part of their entrapped aqueous material
the membrane is in the uid state. Under these conditions and would preserve, partially, their vesicular structure. This 25 [BSE]wsat (3.55 Æ 0.74 mM) and [BSE]wsol (7.37 Æ 0.43 mM) are particular behaviour has to be due to their liquid-ordered 25
similar to that obtained by Hildebrand et al.34 with DPPC lipo- molecular organisation, induced by cholesterol, and, theresomes, in saline, with C and at 60 C (5.6 mM and 6.6 mM fore, could be also caused by other lipophilic molecules that, at
respectively). On the other hand, the slopes of BSE (Table 1) are the same time, could be of pharmacological interest. This could
one order of magnitude higher than that of C. This fact could be the case, for example, of phytosterols, which have a very
30 be a consequence of the transition temperature of the phos- similar structure to cholesterol. In this sense, it has been shown 30
pholipid. It is known that solubilisation of dimyr-
istoylphosphatidylcholine (Tm ¼ 24 C) liposomes with C at 30 C shows slopes 10 times smaller than that of DPPC.34
that, in mixtures with DPPC, some of these vegetable sterols cause a similar phase behaviour to that caused by cholesterol, and also induced, in different degrees, an increase of the bilayer
In light of what has been previously said, HSPC liposomes thickness.40
35 used in oral formulations would release all the entrapped water-
35
5 soluble substances during their interaction with bile salts. In Conclusions
the presence of BSE, and although they cannot be solubilised by
it, the organisation of the gel-state bilayers is drastically altered, The present work shows that each one of the three liposome
resulting in an increased permeability and, nally, mixed nano/ preparations assayed show different types of interaction with 40 microstructures. For this reason, if lipophilic substances were natural BSE at the human physiological concentration range, 40
included in the bilayers, they would not be as efficiently trans- that is, from 4 mM to 11 mM, pH 6.5 and 37 C. As all the
ferred to mixed micelles, as in the case of SPC liposomes.
vesicles contained a phospholipid with a common headgroup
From the results obtained with HSPC/CHOL liposomes (choline) the different behaviour can be attributed mainly to the
45
(Fig. 3, 7 and 8) it can be inferred that the incubations per- difference in their membrane status (uid, gel-state and liquidformed at 37 C and with high BSE concentrations resulted in ordered bilayers). These particular responses to physiological
45
mixed vesicles, which retain part of their initial entrapped bile salts should be taken into account when designing lipo-
aqueous volume. Despite the signicant morphological some formulations as drug carriers. They also indicate that each
changes, they retained a high percentage of the entrapped type of vesicle offers singular features that can be useful in oral
50 uorescent dye. This behaviour markedly contrasts with that of delivery systems. Thus, our ndings may be useful for investi- 50 uid (SPC) and gel-state (HSPC) liposomes at 37 C and provides gators developing liposomal delivery systems in the oral form,
evidence that the liquid-ordered bilayer of HSPC/CHOL vesicles which is our main objective.
can undergo a very particular interaction with the BSE bile salts
during the intestinal digestion.
Acknowledgements
55
In this regard, the morphological changes of HSPC/CHOL
55
liposomes that cause a decrease in the absorbance (Fig. 8) This research was supported (PTR95-0480-OP) by the
and mean diameter (Fig. 3) (characteristic events of the Ia stage) Ministry of Science and Technology of Spain. The authors
are correlated with a leakage of the entrapped dye (Fig. 7). Note would like to thank Dr Josep Cladera for helpful comments
that this was not the behaviour of SPC liposomes during the Ia and suggestions.
8 | Soft Matter, 2014, xx, 1–9
This journal is © The Royal Society of Chemistry 2014
Paper
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