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Effect of three pluronic polymers on the transport of an organic cation across a POPG bilayer studied by Second Harmonic spectroscopy
Chemical Physics Letters 684 (2017) 267–272
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Research paper
Effect of three pluronic polymers on the transport of an organic cation across a POPG bilayer studied by Second Harmonic spectroscopy S.R. Kintali, G.K. Varshney, K. Das ⇑ a b
Photochem. & Photophys. Appl. Lab, Laser Bio-Medical Applications Section, Raja Ramanna Center for Advanced Technology, Indore 452013, M.P., India Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400094, India
a r t i c l e
i n f o
Article history: Received 20 March 2017 In final form 1 July 2017 Available online 3 July 2017
a b s t r a c t Pluronic polymer induced transport of an organic cation across a negatively charged POPG membrane bilayer were studied using interfacial selective Second Harmonic (SH) spectroscopic technique. The length of either hydrophilic (poly-ethylene oxide) or hydrophobic (poly-propylene oxide) unit in the polymer was varied to investigate their effect on membrane transport. Membrane transport was observed to depend critically on the length of the hydrophobic segment present in the polymer. Membrane transport studies using polymers which were either ‘incorporated’ or ‘incubated’ with the lipid bilayer suggested that bilayer packing plays a critical role in the insertion of polymers having a long hydrophilic chain. Ó 2017 Elsevier B.V. All rights reserved.
1. Introduction Several types of pluronic polymers are currently being investigated for a wide variety of bio-medical applications such as tumor therapy, chemo-sensitizing agents, drug delivery, DNA transfection, healing of injured cell membranes etc. [1]. These are amphiphilic tri-coblock polymers whose middle section consists of a hydrophobic poly-propylene oxide (PPO) unit and the two ends are hydrophilic poly-ethylene oxide (PEO) units having the general chemical structure: EOn-POm-EOn. A key prerequisite for such biomedical effects are specific interactions with biological membranes and therefore the interactions between Pluronic polymers and phospholipid membranes have been attracting increasing research interest in recent years [2–4]. Investigations employing various biophysical and microscopic techniques as well as theoretical studies have been performed to characterize the interactions between these polymers and bio-membranes [5–34]. It has been demonstrated by several studies using fluorescence spectroscopic techniques that Pluronic polymers affect membrane organization, especially membrane permeability [2,5–9,12–14,23,24,29]. A common conclusion of all the studies involving the interaction of Pluronic polymers with artificial and natural membranes is that the individual PPO and PEO units of the polymer plays a key role in ⇑ Corresponding author at: Photochem. & Photophys. Appl. Lab, Laser Bio-Medical Applications Section, Raja Ramanna Center for Advanced Technology, Indore 452013, M. P., India. E-mail addresses: [email protected], [email protected] (K. Das). http://dx.doi.org/10.1016/j.cplett.2017.07.002 0009-2614/Ó 2017 Elsevier B.V. All rights reserved.
deciding membrane permeability. The insertion and location of the hydrophobic part (PPO) of the polymer inside a membrane is another topic which has been studied exclusively by experimental [10,11,15,18–22] as well as theoretical [27,28,30–34] techniques. We report in this work a comparative study on the effect of three different pluronic polymers on the bilayer permeability of an organic cation across negatively charged POPG liposomes using the interfacial selective Second Harmonic (SH) spectroscopic technique. This (SH) spectroscopic technique has the ability to monitor the transport of certain molecules across a model membrane in real time provided the molecule of interest possesses a reasonable hyperpolarizability value at the excitation wavelength [35]. The principle of this technique lies on the fact that the SH field generated from the molecules adsorbed only on the outer surface of a membrane can add coherently to generate a measurable SH signal when the diameter of the membrane are of the order of the excitation wavelength. As the molecules transport from the outer surface to the inner surface of the membrane (membrane thickness 5 nm) the SH field generated from oppositely oriented molecules will cancel out because they are separated by a distance which is much less than the coherence length of this process. Therefore by monitoring the time dependent SH signal from the molecules, which is proportional to the population difference of the molecules adsorbed between the outer and inner surface of the membrane, its transport across the membrane can be monitored in real time. The capability of this technique has first been demonstrated using an organic cation Malachite Green [36] and later with other organic ions [37,38]. In this work we have studied how the transport
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properties of an organic cation (LDS-698: ([2-[4-[4-(dimethylamino)phenyl]-1,3-butadienyl]-1-ethylpyridinium monoperchlorate)) across a L-a Phosphatidyl DL-Glycerol (POPG) bilayer are affected in the presence of three different Pluronic polymers, F127 (EO100 PO65 EO100), L-61 (EO2 PO30 EO2) and F-68 (EO76 PO30 EO76). The objective of this study was to investigate the role of the PPO and PEO chain lengths on bilayer permeability and how the insertion of the hydrophobic PPO unit of the polymer depends on bilayer mobility. 2. Materials & methods LDS-698 (from Exciton), POPG (from Avanti) and the three Pluronic polymers: F-127, L-61 and F-68 (from Sigma) were used as received. Spherical liposomes from POPG lipid (details of preparation are provided in SI) were prepared in 10 mM phosphate buffer solution having a pH of 7.4. Their average size and zeta potential was 190 ± 10 nm and 80 ± 10 mV which was measured by a Brookhaven90Plus size and zeta potential analyzer. The liposome polymer complexes were prepared either by adding polymer to the liposome solution (polymer incubated liposomes) or by incorporating the polymer in the liposome. Polymer liposome complexes prepared by the first and second method are referred as ‘incubated’ and ‘incorporated’ throughout this work. The latter complex is prepared by adding a buffer solution containing the respective polymers to the dry lipid thin film and then preparing the liposomes. Addition of Pluronic polymers during the preparation of liposomes ensures that the hydrophobic PPO block of the polymer will span the bilayer. A one hour incubation time was given when these polymers were added to pre-formed liposome solution so as to ensure sufficient interaction time between the polymer and liposome [22,24]. Details of the SH experimental setup were similar to our previously published reports and therefore provided in the SI section. For temperature variation experiments, the sample temperature was controlled by a Neslab circulating water chiller having a temperature accuracy of ±1 °C. The electric field of the SH signal (E2x) were obtained from the observed SH signal (I2x) as [35]:
E2x ðtÞ ¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Ið2xÞdyeþliposome ðtÞ Ið2xÞbackground
ð1Þ
where I(2x)dye+liposome (t) is the SH signal detected at 2x at time t and I(2x)background represents the contributions from the SH signal generated by the buffer solution alone. The E2x field of LDS cation, before addition of the liposomes originates due to hyper-Rayleigh scattering which is confirmed in a previous study [38].
nificantly in the number of PO units. We have studied the average size, poly-dispersity and zeta potential of POPG liposomes incubated with the three pluronics at various lipid:pluronic molar ratio and also when they are incorporated within the POPG bilayer. The results are presented in Fig. S1 in SI which shows that no significant changes of these three parameters are occurring due to incubation/incorporation with the polymers. This is consistent with previous reports [6,15,18,20,12,23,28] where it was observed that liposome integrity is not compromised at polymer concentrations lying well below their CMC values. Fig. 1 describes the changes in the SH electric field of LDS cation (E2x) at 25 °C before and after addition of POPG liposomes (added at 50 s) for: (1) Only POPG liposomes (black curve). (2) POPG liposomes which were earlier incubated with 100 nano-molar F-127 pluronic polymer (red curve). (3) POPG liposomes which were earlier incubated with 3000 nano-molar L-61 pluronic polymer (green curve). (4) POPG liposomes which were earlier incubated with 10 000 nano-molar F-68 pluronic polymer (blue curve). Immediately after addition of liposomes, the SH signal increases instantaneously (<1 s) which is attributed to the electrostatic adsorption of the LDS cation on the outer surface of the POPG liposomes as demonstrated earlier [38]. After addition of POPG liposomes E2x of LDS decreases gradually due to transport of the LDS cation from the outer leaflet to the inner leaflet of the liposomes. It is obvious that presence of Pluronic polymers enhances the transport of the LDS cation across the membrane. The transport time constants (Tav) were obtained by exponential fitting of the curves from their maximum i.e. just after addition of the liposomes and their values are indicated in the legend in Fig. 1. The Tav values of LDS across the POPG membrane becomes 9 times faster in the presence of three polymers. However as indicated in Fig. 1, the concentrations of the individual polymers to induce such a change vary widely. This suggests that permeability of the POPG membrane against the LDS cation is critically dependent on the chemical architecture of the pluronic polymers. Fig. 2 describes how the number of polymer molecules per liposome affects the transport rate constant (kav i.e. 1/Tav) of the LDS cation. In order to capture
100
Tav ~ 110 s
+ 3000 nM L-61
Tav ~ 90 s
60
E2ω
The various physico-chemical properties of the three Pluronic polymers used in this study are listed in Table 1. While L-61 and F-68 have similar number of propylene oxide (PO) units but differ significantly in the number of ethylene oxide (EO) units, F-68 and F-127 have more-or-less similar number of EO units but differ sig-
Tav ~ 910 s
+ 100 nM F-127
+ 10000 nM F-68 Tav ~ 140 s
80
3. Results
Only LDS
40
20 Table 1 The various physico-chemical properties of the three Pluronic polymers used in this study.
a b
Pluronic polymer
Chemical formula
a
L-61 F-68 F-127
EO2PO30EO2 EO76PO30EO76 EO100PO65EO100
3 29 22
Ref. [6] Ref. [14]
HLB
a
b
1.1 104 4.8 104 2.8 106
0.24 ± 0.037 3.5 ± 0.53 N.A.
CMC
Log Kp
water/hexane
0 0
200
400
600
800
1000
1200
time (second) Fig. 1. Changes in the SH electric field of LDS cation with time after addition of POPG liposomes (added at 50 s) incubated with or without three different Pluronic polymers at 25 °C. The legends are colour matched with the data for clarity. The solid lines passing through the data points represent the exponential fits of the data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Transport rate constant of LDS cation (sec-1)
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F127: EO100 PO65 EO100 L61: EO2 PO30 EO2 F68: EO76 PO30 EO76
10-2
10-3 0.01
0.1
1
10
Polymer per POPG Liposome Fig. 2. Effect of three different pluronic polymer incubated POPG liposomes on the transport rates of LDS cation across the bilayer at 25 °C. Both axes are shown in logarithmic scale to capture the entire variation. The line connecting the data points are only meant for guidance. The legends are color matched with the data for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the entire variation in polymer concentration and kav both the axes are shown in logarithmic scale. Compared to L-61 and F-68, the transport of the LDS cation was observed to be severely affected by the presence of F-127 polymer. The experiments reported so far has been carried out with POPG liposomes incubated with the pluronic polymers. From Fig. 2, it can be safely speculated that the hydrophobic part of the polymer plays a crucial role in changing the membrane permeability. Since these experiments were carried out with polymer incubated liposomes, the question of polymer localization with respect to lipid bilayer remains an open issue. To address this, a comparison of the transport rates of the LDS cation using polymer incorporated and polymer incubated POPG liposomes (as detailed in the Expt. Section) were carried out. For polymer incorporated liposomes it is reasonable to assume that the PO unit of the polymer will span the entire bilayer. For the latter case the insertion of the hydrophobic PO unit in the bilayer will be accompanied by the translocation of the hydrophilic EO unit across the bilayer. This is expected to depend upon the bilayer rigidity which has been varied by changing the temperature of the system (5–45 °C). As evident from Fig. 2, it will be difficult to compare the effect of the three polymers using a similar concentration because their individual membrane permeability properties (and hence the transport of the LDS cation) will vary widely. Therefore we have decided to use those polymer concentrations where the magnitude of enhancement in the transport rate of the LDS cation across the polymer incubated POPG bilayer at room temperature (25 °C) were observed to be more or less similar (Fig. 2). Accordingly polymer ‘incorporated’ and polymer ‘incubated’ POPG liposomes were prepared with 500 nano-molar F-127, 1000 nano-molar L-61 and 5000 nano-molar F-68. It is pertinent to note that results of this experiment will provide an idea about how temperature will affect the insertion of the polymer in the bilayer for the ‘incubated’ case, by comparing with the ‘incorporated’ case. Fig. 3 describes how the preparation of the polymer-POPG complex affects the transport of the LDS cation at two different temperatures (10 and 25 °C). Only POPG liposomes were used as control for overall comparison. Following the addition of liposomes (at t = 100 s), the time dependent decrease in the E2x signal of the LDS cation (E2x(t)) were
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observed to depend on the temperature of the system and how the liposome-polymer complex were prepared. At 10 °C, the E2x(t) characteristics of the LDS cation after the addition of liposomes was observed to be similar for all polymerPOPG complex except F-68 and F-127 incorporated POPG liposomes where the changes in E2x(t) indicated that the transport of the LDS cation is significantly faster in these two cases. When the temperature is increased to 25 °C, while the observed changes in E2x(t) signal of the LDS cation suggested that the transport of the cation to be faster for the incubated complexes of L-61 and F-68 polymers compared to their incorporated complexes with POPG liposomes, data for F-127 polymer suggests that transport characteristics of the cation to be more or less similar for both incorporated and incubated complexes of the polymer with POPG liposomes. Finally Fig. 4 describes the effect of temperature on the kT values of the LDS cation across a pure POPG and different types of pluronic-POPG bilayers. The X-axis was deliberately shown in logarithmic scale so as to fully capture the wide variations of the transport rate constants. As the temperature is varied from 5 to 40 °C, the following observations can be summarized: (1) The kT values of LDS cation across POPG liposomes were observed to remain similar till 15 °C after which it increases sharply (10x) and remains more or less constant thereafter. (2) The kT values of LDS cation across POPG bilayer in presence of L-61 polymer (either incubated or incorporated) increases monotonically with temperature over the entire temperature range. (3) The kT values of LDS cation for F-127 or F-68 incorporated POPG liposomes was observed to be substantially higher compared to their incubated counter-part at lower temperature range (5–15 °C). (4) The kT values of LDS cation for F-127 or F-68 incubated POPG liposomes were observed to be similar to that of only POPG liposomes till 15 °C after which it increase sharply and somewhat starts to level off after 30 °C. 4. Discussions We have studied the role of three different pluronic polymers in altering the membrane permeability properties of a negatively charged POPG bilayer against an organic cation LDS by using the interface selective SH spectroscopic technique. Earlier, this have been studied by monitoring the trans-membrane transport of some fluorescent probes and also by monitoring the lipid flip-flop rates using fluorescently labelled lipids [2,6,8,9,13,14]. In one study it was observed that the leakage of a fluorescent dye (Carboxyfluorescein) from Egg-PC SUV’s increased by 100 times in presence of F-127 at a polymer:lipid (P/L) mole ratio of 0.10 [8]. A similar increase in the transport rate of the LDS cation was observed at a P/L ratio of 0.010 in this study (Fig. 4). In another study, it was observed that adsorption of 20 L-61 molecules to the surface of an Egg-PC vesicle results in a 3-fold increase in the permeation of Doxorubicin (an anti-cancer drug) and a 6-fold increase in the flip-flop rate of fluorescently labelled lipids [9]. In this study a 10-fold increase in the transport rate of LDS cation was observed at room temp when 5 molecules of L-61 were incubated per liposome (see later for polymer per liposome estimation). As expected, the SH spectroscopic technique, which probes exclusively the processes occurring at an interface, offers a better sensitivity to probe the bilayer permeability induced by the pluronic polymers. Result presented in Fig. 2 indicates that POPG membrane permeability against LDS cation depends more critically on the length of the hydrophobic (PO) segment in the polymer. The length of the hydrophobic PO unit is highest in F-127 (EO100 PO65 EO100) and
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1.0 0.8
Normalized E2ω (LDS cation)
Normalized E2ω (LDS cation)
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Control Incorporated Incubated
0.6 0.4
L-61: EO2PO30EO2 1.0 µM i.e. 5 per liposome
0.2 0.0
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0.0 0
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time (second)
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0.4 Control Incorporated Incubated
0.2
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Normalized E2ω (LDS cation)
Normalized E2ω (LDS cation)
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time (second)
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Normalized E2ω (LDS cation)
time (second)
0.0
1.0 F-127: EO100PO65EO100
0.5 µM i.e. 2.5 per liposome
0.8 0.6
Control Incorporated Incubated
0.4 0.2 0.0
0
500
1000
1500
2000
0
500
1000
1500
time (second)
time (second)
Temperature: 10°C
Temperature: 25°C
2000
Fig. 3. Time dependent changes in the SH electric field of LDS cation following addition of POPG liposomes (added at 100 s) with/without three different pluronic polymers. Two types of polymer-liposome complex are used: incorporated and incubated; the preparation details of which are discussed in the experimental section. The left and right panel describes experiments carried out at 10 and 25 °C respectively. The legends are colour matched with the data for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
lowest in both L-61 (EO2 PO30 EO2) and F-68 (EO76 PO30 EO76). While the length of the hydrophobic PO unit in L-61 and F-68 is of the order of membrane thickness ( 5 nm), in F-127 this is roughly twice of the order of membrane thickness. Therefore it appears that length of the PO unit present in these polymers is responsible for the enhanced permeability of the LDS cation. Comparing the effects of L-61 and F-68, where the lengths of the hydrophobic PO units are similar, it appears that membrane permeability increases as the length of the hydrophilic (EO) unit gets smaller, i.e. when the HLB values gets smaller. Indeed it has been
shown that the HLB (hydrophiliclipophilic balance) index (Table 1) determined by the empirical formula: HLB = 36n/(2 m + n) + 33 [39] determines the hydrophilic/lipophilic character of the polymer. At this point it is worthwhile to consider the number of polymer molecules present per liposome in our experimental conditions. The liposome concentration was calculated to be 350 nM (see SI for calculation) which indicates that 0.3 molecule of F-127 per liposome can reduce the transport time of the LDS cation by 9 times while for L-61 and F-68 this amounts to 9.0 and 29.0
Average transport rate (second-1)
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0.1
Control Incorporated complex Incubated complex
0.01
1E-3
L-61: EO2PO30EO2 1.0 µM i.e. 5 per liposome 1E-4 0
5
10
15
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25
30
35
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45
Average transport rate (second-1)
Temperature Control Incorporated complex Incubated complex
0.1
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F-68: EO76PO30EO76 5.0 µM i.e. 25 per liposome
1E-4 0
5
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20
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Average transport rate (second-1)
Temperature Control Incorporated complex Incubated complex
0.1
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1E-3
F-127: EO100PO65EO100 1E-4
0.5 µM i.e. 2.5 per liposome 0
5
10
15
20
25
30
35
40
45
Temperature Fig. 4. Temperature dependent changes in the transport rate constants of the LDS cation under different conditions: Top, middle and bottom panel represents POPG liposomes which were either incubated or incorporated with L-61, F-68 and F-127 polymers respectively. In all the panels the data for only POPG liposomes are also shown for comparison. The Y-axis is deliberately kept in logarithmic scale to capture the entire change. The legends are colour matched with the data for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
polymer molecules per liposome (Fig. 1). It is pertinent to note that number of polymer molecules adsorbed per liposome will depend upon the partition co-efficient (Log Kp water-hexane) of the individual
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polymers. Therefore the actual number of polymer molecules adsorbed on the surface of an individual liposome will be less than the number of polymer molecules added to the liposome solution. Further, from Table 1 it is clear that adsorption of L-61 on the liposome surface will be significantly higher compared to F-68 and F127. It is reasonable to expect that membrane permeability will depend on the mobility of the lipids constituting the membrane. Therefore in presence of pluronic polymers the enhanced mobility of the membrane components may originate from the global mobility of all the lipid constituents or by formation of small patches on the membrane surface in which the mobility of membrane components is increased drastically. It is difficult to assume that presence of 5 or less pluronic molecules per liposome (Fig. 2) would affect the global mobility of the lipids constituting the liposome, which is supported by the fact that the sizes of the liposomes do not alter at all the pluronic concentrations used in this study (Fig. 1, SI). Therefore, it seems likely that the mobility of the membrane surrounding the adsorbed polymer gets compromised. Since the length of the hydrophobic PO unit in F-127 is twice of the order of membrane thickness (5 nm) it appears that to accommodate this large hydrophobic unit the lipid packing around an adsorbed F-127 molecule is severely affected. We propose that formation of this localized ‘highly mobile’ areas are responsible for the enhanced permeability of the LDS cation. It might be noted that similar type of hypothesis was already proposed previously [9]. In addition it may be noted that several MD simulation studies [27,28,30–34] have been shown that under equilibrium conditions, the hydrophobic segment of the pluronic polymer in the bilayer adapts a folded conformation. In particular, it has been observed that for F-127 polymer, the hydrophobic PO unit remains in a folded conformation even when the polymer is in transmembrane state [31]. Due to this folded configuration, the ordered arrangement of the lipid molecules in the immediate vicinity is disturbed. At this point it is important to note that transport of ions across a bilayer is driven by the creation of a trans-membrane potential difference due to adsorption of oppositely charged ions on the outer bilayer surface. However, the transport rate will also depend upon the rigidity of the bilayer. Therefore if by any means the rigidity is compromised, the transport rate will increase. We propose that due to the folded conformation of the PO unit of F-127 inside the bilayer, the lipid molecules in the immediate vicinity will lose their ordering arrangement resulting in reduced rigidity which will result in an enhanced permeability of the LDS cation around this localized area. So far, we have discussed the effect of polymer present in the solution on the POPG bilayer permeability by analysing the transport characteristics of the LDS cation. The interaction of the polymer with the membrane consists of two parts; first there is adsorption of the polymer at the aqueous-membrane interface and then insertion of the hydrophobic part (PO unit) of the polymer inside the bilayer [11,15,18,22,24,27,30]. The insertion of the PO unit (whose length is of the order of bilayer thickness or more) in the bilayer can be of two types: bilayer spanning or localization in the outer leaflet of the membrane. Spanning the entire membrane requires the transport of the hydrophilic segment of the polymer across the hydrophobic bilayer which is an energetically costly process and was shown to be slow in time [22,24] at room temperature, however at elevated temperatures when the bilayer becomes sufficiently mobile this may be faster. A comparison of the kT values of the LDS cation across a POPG bilayer and POPG bilayer containing pluronic polymers which were present either in the incorporated or incubated state (Fig. 4) indicates that temperature plays a crucial role in controlling the interaction of the F-68 and F-127 polymers with the bilayer. The kT values of LDS cation across POPG bilayer shows an abrupt increase near the temperature range of 15–20 °C indicating that at this tem-
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perature the mobility of the POPG lipids increases significantly. This trend is also followed by F-68 and F-127 polymers (which have a long chain hydrophilic EO groups) when they are incubated with POPG liposomes which leads us to conclude that insertion of the PO chain in F-127 and F-68 polymers in the POPG bilayer starts when the mobility of the bilayer gets reduced abruptly around the 15–20 °C temperature range. Although the reason behind this sudden change in the POPG bilayer mobility is not clear at this point, it is relevant to note that liposomes made from egg PE (phosphatidyl ethanolamine) were observed to show a transition near 10 °C, which has been attributed to the lipid hydrocarbon chain orderdisorder conformational transition [40]. It is possible that a similar transition in the POPG liposomes occurs in the temperature range of 15–20 °C causing the above mentioned effect. Further experiments are needed to corroborate this possibility. Since above 20 °C the observed changes in the kT values of the LDS cation with temperature are more or less similar for both incubated or incorporated complexes of F-127 or F-68 polymers with POPG liposomes further leads to the conclusion that above this temperature polymer incorporation in the bilayer is similar for both incorporated and incubated polymer-lipid complexes and this is likely to be in a bilayer spanning state. The reduction of the lipid mobility is likely to assist the translocation of the bulky EO chains in these polymers across the bilayer. The temperature dependent changes in the kT values of the LDS cation in presence of POPG liposomes complexed with L-61 polymer is similar for both incubated and incorporated complex. This suggests that L-61 inserts itself in the bilayer when it is added externally. This is perhaps not surprising given the fact that among the three polymers used in this study, L-61, has the highest HLB value and therefore is expected to bind more strongly to the POPG bilayer. 5. Conclusions In conclusion it was observed that tri-block pluronic polymer induced permeability of POPG liposomes against an organic cation, LDS, depends on the individual sub-units of the polymer. Using three different polymers, F-127, F-68 and L-61 the effect of variation in the individual sub-units of the polymer, the hydrophobic PO chain and the hydrophilic EO chain, has been investigated. Results obtained indicate that compared to the EO chain, variation in the PO chain remarkably affects the transport of LDS cation across the bilayer which can be attributed to the significant alteration in the bilayer packing near the vicinity of the adsorbed polymer. This alteration results from accommodating a PO chain whose length is almost double to that of the bilayer thickness (F-127). The role of polymer structure and temperature (5–40 °C) on the insertion of the hydrophobic PO unit in the bilayer has also been investigated by monitoring the transport of the LDS cation. L-61, which is the most hydrophobic of the series, was observed to insert itself spontaneously into the bilayer. F-127 and F-68 was observed to insert their PO unit above 15–20 °C which was aided by an enhancement in the mobility of the POPG lipids at this
temperature range. Results obtained in this study are expected to provide further insight into the nature of pluronic polymer induced membrane permeability. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cplett.2017.07. 002. References [1] A.V. Kabanova, E.V. Batrakova, V.Yu. Alakhov, J. Contr. Rel. 82 (2002) 189–212. [2] A.A. Yaroslavov, N.S. Melik-Nubarov, F.M. Menger, Acc. Chem. Res. 39 (2006) 702. [3] W.H. Binder, Angew. Chem. Int. Ed. 47 (2008) 3092–3095. [4] E. Amado, J. Kressler, Curr. Op. Coll. & Interf. Sc. 16 (2011) 491. [5] N.S. Melik-Nubarova, O.O. Pomaza, T.Yu. Dorodnycha, G.A. Badunb, A.L. Ksenofontovc, O.B. Schemchukovad, S.A. Arzhakova, FEBS Lett. 446 (1999) 194. [6] M. Johnsson, M. Silvander, G. Karlsson, K. Edwards, Langmuir 15 (1999) 6314. [7] V.Yu. Erukova, O.O. Krylova, Y.N. Antonenko, N.S. Melik-Nubarov, Biochim. Biophys. Acta 1468 (2000) 73. [8] M. Johnsson, N. Bergstrand, K. Edwards, J.J.R. Stalgren, Langmuir 17 (2001) 3902. [9] O.O. Krylova, N.S. Melik-Nubarov, G.A. Badun, A.L. Ksenofontov, F.M. Menger, A.A. Yaroslavov, Chem. Eur. J. 9 (2003) 3930. [10] S.A. Maskarinec, K.Y.C. Lee, Langmuir 19 (2003) 1809. [11] M.A. Firestone, A.C. Wolf, S. Seifert, Biomacromolecules 4 (2003) 1539. [12] O.O. Krylova, P. Pohl, Biochemistry 43 (2004) 3696. [13] A.E. Zhirnov, T.V. Demina, O.O. Krylova, I.D. Grozdova, N.S. Melik-Nubarov, Biochim. Biophys. Acta 1720 (2005) 73. [14] T. Demina, I. Grozdova, O. Krylova, A. Zhirnov, V. Istratov, H. Frey, H. Kautz, N. Melik-Nubarov, Biochemistry 44 (2005) 4042. [15] X. Liang, G. Mao, K.Y. Simon Ng, J. Coll. Interf. Sci. 285 (2005). [16] L.-C. Chang, C.-Y. Lin, M.-W. Kuo, C.-S. Gau, J. Coll. Interf. Sci. 285 (2005) 640. [17] J. Gau-Racine, J. Lal, M. Zeghal, L. Auvray, J. Phys. Chem. B 111 (2007) 9900. [18] S.L. Frey, D. Zhang, M.A. Carignano, I. Szleifera, K.Y.C. Lee, J. Chem. Phys. 127 (2007) 114904. [19] S.L. Frey, K.Y.C. Lee, Langmuir 23 (2007) 2631. [20] G. Wu, H.A. Khant, W. Chiu, K.Y.C. Lee, Soft Matter 5 (2009) 1496. [21] G. Wu, K.Y.C. Lee, Langmuir 25 (2009) 2133. [22] G. Wu, K.Y.C. Lee, J. Phys. Chem. B 113 (2009) 15522. [23] Y.Y. Chieng, S.B. Chen, J. Phys. Chem. B 113 (2009) 14934. [24] J.-Y. Wang, J. Chin, J.D. Marks, K.Y.C. Lee, Langmuir 26 (2010) 12953. [25] J.-Y. Wang, J. Marks, K.Y.C. Lee, Biomacromolecules 13 (2012) 2616. [26] C.-Y. Cheng, J.-Y. Wang, R. Kausik, K.Y.C. Lee, S. Han, Biomacromolecules 13 (2012) 2624. [27] S. Hezaveh, S. Samanta, A. De Nicola, G. Milano, D. Roccatano, J. Phys. Chem. B 116 (2012) 14333. [28] S. Nawaz, M. Redhead, G. Mantovani, C. Alexander, C. Bosquillon, P. Carbone, Soft Matter 8 (2012) 6744. [29] A. Hadicke, A. Blume, J. Coll. Interf. Sci. 407 (2013) 327. [30] H. Rabbel, M. Werner, J.-U. Sommer, Macromolecules 48 (2015) 4724. [31] I. Wood, M.F. Martini, J.M.R. Albano, M.L. Cuestas, V.L. Mathet, M. Pickholz, J. Mol. Struct. 1109 (2016) 106. [32] A. Goliaei, E.Y. Lau, U. Adhikari, E. Schwegler, M.L. Berkowitz, J. Phys. Chem. B 120 (2016) 8631. [33] N.I. Ercan, P. Stroeve, J.W. Tringe, R. Faller, Langmuir 32 (2016) 10026. [34] U. Adhikari, A. Goliaei, L. Tsereteli, M.L. Berkowitz, J. Phys. Chem. B 120 (2016) 5823. [35] K.B. Eisenthal, Chem. Rev. 106 (2006) 1462. [36] A. Srivastava, K.B. Eisenthal, Chem. Phys. Lett. 292 (1998) 345. [37] R.K. Saini, A. Dube, P.K. Gupta, K. Das, J. Phys. Chem. B 116 (2012) 4199. [38] G.K. Varshney, R.K. Saini, P.K. Gupta, K. Das, Langmuir 29 (2013) 2912. [39] S. Fusco, J. Bioact. Compat. Polym. 21 (2006) 149. [40] G. Basaiez, J.L. Nieva, E. Rivas, A. Alonso, F.M. Goni, Biophys. J. 70 (1996) 2299.
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Research paper
Effect of three pluronic polymers on the transport of an organic cation across a POPG bilayer studied by Second Harmonic spectroscopy S.R. Kintali, G.K. Varshney, K. Das ⇑ a b
Photochem. & Photophys. Appl. Lab, Laser Bio-Medical Applications Section, Raja Ramanna Center for Advanced Technology, Indore 452013, M.P., India Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400094, India
a r t i c l e
i n f o
Article history: Received 20 March 2017 In final form 1 July 2017 Available online 3 July 2017
a b s t r a c t Pluronic polymer induced transport of an organic cation across a negatively charged POPG membrane bilayer were studied using interfacial selective Second Harmonic (SH) spectroscopic technique. The length of either hydrophilic (poly-ethylene oxide) or hydrophobic (poly-propylene oxide) unit in the polymer was varied to investigate their effect on membrane transport. Membrane transport was observed to depend critically on the length of the hydrophobic segment present in the polymer. Membrane transport studies using polymers which were either ‘incorporated’ or ‘incubated’ with the lipid bilayer suggested that bilayer packing plays a critical role in the insertion of polymers having a long hydrophilic chain. Ó 2017 Elsevier B.V. All rights reserved.
1. Introduction Several types of pluronic polymers are currently being investigated for a wide variety of bio-medical applications such as tumor therapy, chemo-sensitizing agents, drug delivery, DNA transfection, healing of injured cell membranes etc. [1]. These are amphiphilic tri-coblock polymers whose middle section consists of a hydrophobic poly-propylene oxide (PPO) unit and the two ends are hydrophilic poly-ethylene oxide (PEO) units having the general chemical structure: EOn-POm-EOn. A key prerequisite for such biomedical effects are specific interactions with biological membranes and therefore the interactions between Pluronic polymers and phospholipid membranes have been attracting increasing research interest in recent years [2–4]. Investigations employing various biophysical and microscopic techniques as well as theoretical studies have been performed to characterize the interactions between these polymers and bio-membranes [5–34]. It has been demonstrated by several studies using fluorescence spectroscopic techniques that Pluronic polymers affect membrane organization, especially membrane permeability [2,5–9,12–14,23,24,29]. A common conclusion of all the studies involving the interaction of Pluronic polymers with artificial and natural membranes is that the individual PPO and PEO units of the polymer plays a key role in ⇑ Corresponding author at: Photochem. & Photophys. Appl. Lab, Laser Bio-Medical Applications Section, Raja Ramanna Center for Advanced Technology, Indore 452013, M. P., India. E-mail addresses: [email protected], [email protected] (K. Das). http://dx.doi.org/10.1016/j.cplett.2017.07.002 0009-2614/Ó 2017 Elsevier B.V. All rights reserved.
deciding membrane permeability. The insertion and location of the hydrophobic part (PPO) of the polymer inside a membrane is another topic which has been studied exclusively by experimental [10,11,15,18–22] as well as theoretical [27,28,30–34] techniques. We report in this work a comparative study on the effect of three different pluronic polymers on the bilayer permeability of an organic cation across negatively charged POPG liposomes using the interfacial selective Second Harmonic (SH) spectroscopic technique. This (SH) spectroscopic technique has the ability to monitor the transport of certain molecules across a model membrane in real time provided the molecule of interest possesses a reasonable hyperpolarizability value at the excitation wavelength [35]. The principle of this technique lies on the fact that the SH field generated from the molecules adsorbed only on the outer surface of a membrane can add coherently to generate a measurable SH signal when the diameter of the membrane are of the order of the excitation wavelength. As the molecules transport from the outer surface to the inner surface of the membrane (membrane thickness 5 nm) the SH field generated from oppositely oriented molecules will cancel out because they are separated by a distance which is much less than the coherence length of this process. Therefore by monitoring the time dependent SH signal from the molecules, which is proportional to the population difference of the molecules adsorbed between the outer and inner surface of the membrane, its transport across the membrane can be monitored in real time. The capability of this technique has first been demonstrated using an organic cation Malachite Green [36] and later with other organic ions [37,38]. In this work we have studied how the transport
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properties of an organic cation (LDS-698: ([2-[4-[4-(dimethylamino)phenyl]-1,3-butadienyl]-1-ethylpyridinium monoperchlorate)) across a L-a Phosphatidyl DL-Glycerol (POPG) bilayer are affected in the presence of three different Pluronic polymers, F127 (EO100 PO65 EO100), L-61 (EO2 PO30 EO2) and F-68 (EO76 PO30 EO76). The objective of this study was to investigate the role of the PPO and PEO chain lengths on bilayer permeability and how the insertion of the hydrophobic PPO unit of the polymer depends on bilayer mobility. 2. Materials & methods LDS-698 (from Exciton), POPG (from Avanti) and the three Pluronic polymers: F-127, L-61 and F-68 (from Sigma) were used as received. Spherical liposomes from POPG lipid (details of preparation are provided in SI) were prepared in 10 mM phosphate buffer solution having a pH of 7.4. Their average size and zeta potential was 190 ± 10 nm and 80 ± 10 mV which was measured by a Brookhaven90Plus size and zeta potential analyzer. The liposome polymer complexes were prepared either by adding polymer to the liposome solution (polymer incubated liposomes) or by incorporating the polymer in the liposome. Polymer liposome complexes prepared by the first and second method are referred as ‘incubated’ and ‘incorporated’ throughout this work. The latter complex is prepared by adding a buffer solution containing the respective polymers to the dry lipid thin film and then preparing the liposomes. Addition of Pluronic polymers during the preparation of liposomes ensures that the hydrophobic PPO block of the polymer will span the bilayer. A one hour incubation time was given when these polymers were added to pre-formed liposome solution so as to ensure sufficient interaction time between the polymer and liposome [22,24]. Details of the SH experimental setup were similar to our previously published reports and therefore provided in the SI section. For temperature variation experiments, the sample temperature was controlled by a Neslab circulating water chiller having a temperature accuracy of ±1 °C. The electric field of the SH signal (E2x) were obtained from the observed SH signal (I2x) as [35]:
E2x ðtÞ ¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Ið2xÞdyeþliposome ðtÞ Ið2xÞbackground
ð1Þ
where I(2x)dye+liposome (t) is the SH signal detected at 2x at time t and I(2x)background represents the contributions from the SH signal generated by the buffer solution alone. The E2x field of LDS cation, before addition of the liposomes originates due to hyper-Rayleigh scattering which is confirmed in a previous study [38].
nificantly in the number of PO units. We have studied the average size, poly-dispersity and zeta potential of POPG liposomes incubated with the three pluronics at various lipid:pluronic molar ratio and also when they are incorporated within the POPG bilayer. The results are presented in Fig. S1 in SI which shows that no significant changes of these three parameters are occurring due to incubation/incorporation with the polymers. This is consistent with previous reports [6,15,18,20,12,23,28] where it was observed that liposome integrity is not compromised at polymer concentrations lying well below their CMC values. Fig. 1 describes the changes in the SH electric field of LDS cation (E2x) at 25 °C before and after addition of POPG liposomes (added at 50 s) for: (1) Only POPG liposomes (black curve). (2) POPG liposomes which were earlier incubated with 100 nano-molar F-127 pluronic polymer (red curve). (3) POPG liposomes which were earlier incubated with 3000 nano-molar L-61 pluronic polymer (green curve). (4) POPG liposomes which were earlier incubated with 10 000 nano-molar F-68 pluronic polymer (blue curve). Immediately after addition of liposomes, the SH signal increases instantaneously (<1 s) which is attributed to the electrostatic adsorption of the LDS cation on the outer surface of the POPG liposomes as demonstrated earlier [38]. After addition of POPG liposomes E2x of LDS decreases gradually due to transport of the LDS cation from the outer leaflet to the inner leaflet of the liposomes. It is obvious that presence of Pluronic polymers enhances the transport of the LDS cation across the membrane. The transport time constants (Tav) were obtained by exponential fitting of the curves from their maximum i.e. just after addition of the liposomes and their values are indicated in the legend in Fig. 1. The Tav values of LDS across the POPG membrane becomes 9 times faster in the presence of three polymers. However as indicated in Fig. 1, the concentrations of the individual polymers to induce such a change vary widely. This suggests that permeability of the POPG membrane against the LDS cation is critically dependent on the chemical architecture of the pluronic polymers. Fig. 2 describes how the number of polymer molecules per liposome affects the transport rate constant (kav i.e. 1/Tav) of the LDS cation. In order to capture
100
Tav ~ 110 s
+ 3000 nM L-61
Tav ~ 90 s
60
E2ω
The various physico-chemical properties of the three Pluronic polymers used in this study are listed in Table 1. While L-61 and F-68 have similar number of propylene oxide (PO) units but differ significantly in the number of ethylene oxide (EO) units, F-68 and F-127 have more-or-less similar number of EO units but differ sig-
Tav ~ 910 s
+ 100 nM F-127
+ 10000 nM F-68 Tav ~ 140 s
80
3. Results
Only LDS
40
20 Table 1 The various physico-chemical properties of the three Pluronic polymers used in this study.
a b
Pluronic polymer
Chemical formula
a
L-61 F-68 F-127
EO2PO30EO2 EO76PO30EO76 EO100PO65EO100
3 29 22
Ref. [6] Ref. [14]
HLB
a
b
1.1 104 4.8 104 2.8 106
0.24 ± 0.037 3.5 ± 0.53 N.A.
CMC
Log Kp
water/hexane
0 0
200
400
600
800
1000
1200
time (second) Fig. 1. Changes in the SH electric field of LDS cation with time after addition of POPG liposomes (added at 50 s) incubated with or without three different Pluronic polymers at 25 °C. The legends are colour matched with the data for clarity. The solid lines passing through the data points represent the exponential fits of the data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Transport rate constant of LDS cation (sec-1)
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F127: EO100 PO65 EO100 L61: EO2 PO30 EO2 F68: EO76 PO30 EO76
10-2
10-3 0.01
0.1
1
10
Polymer per POPG Liposome Fig. 2. Effect of three different pluronic polymer incubated POPG liposomes on the transport rates of LDS cation across the bilayer at 25 °C. Both axes are shown in logarithmic scale to capture the entire variation. The line connecting the data points are only meant for guidance. The legends are color matched with the data for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the entire variation in polymer concentration and kav both the axes are shown in logarithmic scale. Compared to L-61 and F-68, the transport of the LDS cation was observed to be severely affected by the presence of F-127 polymer. The experiments reported so far has been carried out with POPG liposomes incubated with the pluronic polymers. From Fig. 2, it can be safely speculated that the hydrophobic part of the polymer plays a crucial role in changing the membrane permeability. Since these experiments were carried out with polymer incubated liposomes, the question of polymer localization with respect to lipid bilayer remains an open issue. To address this, a comparison of the transport rates of the LDS cation using polymer incorporated and polymer incubated POPG liposomes (as detailed in the Expt. Section) were carried out. For polymer incorporated liposomes it is reasonable to assume that the PO unit of the polymer will span the entire bilayer. For the latter case the insertion of the hydrophobic PO unit in the bilayer will be accompanied by the translocation of the hydrophilic EO unit across the bilayer. This is expected to depend upon the bilayer rigidity which has been varied by changing the temperature of the system (5–45 °C). As evident from Fig. 2, it will be difficult to compare the effect of the three polymers using a similar concentration because their individual membrane permeability properties (and hence the transport of the LDS cation) will vary widely. Therefore we have decided to use those polymer concentrations where the magnitude of enhancement in the transport rate of the LDS cation across the polymer incubated POPG bilayer at room temperature (25 °C) were observed to be more or less similar (Fig. 2). Accordingly polymer ‘incorporated’ and polymer ‘incubated’ POPG liposomes were prepared with 500 nano-molar F-127, 1000 nano-molar L-61 and 5000 nano-molar F-68. It is pertinent to note that results of this experiment will provide an idea about how temperature will affect the insertion of the polymer in the bilayer for the ‘incubated’ case, by comparing with the ‘incorporated’ case. Fig. 3 describes how the preparation of the polymer-POPG complex affects the transport of the LDS cation at two different temperatures (10 and 25 °C). Only POPG liposomes were used as control for overall comparison. Following the addition of liposomes (at t = 100 s), the time dependent decrease in the E2x signal of the LDS cation (E2x(t)) were
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observed to depend on the temperature of the system and how the liposome-polymer complex were prepared. At 10 °C, the E2x(t) characteristics of the LDS cation after the addition of liposomes was observed to be similar for all polymerPOPG complex except F-68 and F-127 incorporated POPG liposomes where the changes in E2x(t) indicated that the transport of the LDS cation is significantly faster in these two cases. When the temperature is increased to 25 °C, while the observed changes in E2x(t) signal of the LDS cation suggested that the transport of the cation to be faster for the incubated complexes of L-61 and F-68 polymers compared to their incorporated complexes with POPG liposomes, data for F-127 polymer suggests that transport characteristics of the cation to be more or less similar for both incorporated and incubated complexes of the polymer with POPG liposomes. Finally Fig. 4 describes the effect of temperature on the kT values of the LDS cation across a pure POPG and different types of pluronic-POPG bilayers. The X-axis was deliberately shown in logarithmic scale so as to fully capture the wide variations of the transport rate constants. As the temperature is varied from 5 to 40 °C, the following observations can be summarized: (1) The kT values of LDS cation across POPG liposomes were observed to remain similar till 15 °C after which it increases sharply (10x) and remains more or less constant thereafter. (2) The kT values of LDS cation across POPG bilayer in presence of L-61 polymer (either incubated or incorporated) increases monotonically with temperature over the entire temperature range. (3) The kT values of LDS cation for F-127 or F-68 incorporated POPG liposomes was observed to be substantially higher compared to their incubated counter-part at lower temperature range (5–15 °C). (4) The kT values of LDS cation for F-127 or F-68 incubated POPG liposomes were observed to be similar to that of only POPG liposomes till 15 °C after which it increase sharply and somewhat starts to level off after 30 °C. 4. Discussions We have studied the role of three different pluronic polymers in altering the membrane permeability properties of a negatively charged POPG bilayer against an organic cation LDS by using the interface selective SH spectroscopic technique. Earlier, this have been studied by monitoring the trans-membrane transport of some fluorescent probes and also by monitoring the lipid flip-flop rates using fluorescently labelled lipids [2,6,8,9,13,14]. In one study it was observed that the leakage of a fluorescent dye (Carboxyfluorescein) from Egg-PC SUV’s increased by 100 times in presence of F-127 at a polymer:lipid (P/L) mole ratio of 0.10 [8]. A similar increase in the transport rate of the LDS cation was observed at a P/L ratio of 0.010 in this study (Fig. 4). In another study, it was observed that adsorption of 20 L-61 molecules to the surface of an Egg-PC vesicle results in a 3-fold increase in the permeation of Doxorubicin (an anti-cancer drug) and a 6-fold increase in the flip-flop rate of fluorescently labelled lipids [9]. In this study a 10-fold increase in the transport rate of LDS cation was observed at room temp when 5 molecules of L-61 were incubated per liposome (see later for polymer per liposome estimation). As expected, the SH spectroscopic technique, which probes exclusively the processes occurring at an interface, offers a better sensitivity to probe the bilayer permeability induced by the pluronic polymers. Result presented in Fig. 2 indicates that POPG membrane permeability against LDS cation depends more critically on the length of the hydrophobic (PO) segment in the polymer. The length of the hydrophobic PO unit is highest in F-127 (EO100 PO65 EO100) and
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1.0 0.8
Normalized E2ω (LDS cation)
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Control Incorporated Incubated
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time (second)
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time (second)
time (second)
Temperature: 10°C
Temperature: 25°C
2000
Fig. 3. Time dependent changes in the SH electric field of LDS cation following addition of POPG liposomes (added at 100 s) with/without three different pluronic polymers. Two types of polymer-liposome complex are used: incorporated and incubated; the preparation details of which are discussed in the experimental section. The left and right panel describes experiments carried out at 10 and 25 °C respectively. The legends are colour matched with the data for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
lowest in both L-61 (EO2 PO30 EO2) and F-68 (EO76 PO30 EO76). While the length of the hydrophobic PO unit in L-61 and F-68 is of the order of membrane thickness ( 5 nm), in F-127 this is roughly twice of the order of membrane thickness. Therefore it appears that length of the PO unit present in these polymers is responsible for the enhanced permeability of the LDS cation. Comparing the effects of L-61 and F-68, where the lengths of the hydrophobic PO units are similar, it appears that membrane permeability increases as the length of the hydrophilic (EO) unit gets smaller, i.e. when the HLB values gets smaller. Indeed it has been
shown that the HLB (hydrophiliclipophilic balance) index (Table 1) determined by the empirical formula: HLB = 36n/(2 m + n) + 33 [39] determines the hydrophilic/lipophilic character of the polymer. At this point it is worthwhile to consider the number of polymer molecules present per liposome in our experimental conditions. The liposome concentration was calculated to be 350 nM (see SI for calculation) which indicates that 0.3 molecule of F-127 per liposome can reduce the transport time of the LDS cation by 9 times while for L-61 and F-68 this amounts to 9.0 and 29.0
Average transport rate (second-1)
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0.1
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0.5 µM i.e. 2.5 per liposome 0
5
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20
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30
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45
Temperature Fig. 4. Temperature dependent changes in the transport rate constants of the LDS cation under different conditions: Top, middle and bottom panel represents POPG liposomes which were either incubated or incorporated with L-61, F-68 and F-127 polymers respectively. In all the panels the data for only POPG liposomes are also shown for comparison. The Y-axis is deliberately kept in logarithmic scale to capture the entire change. The legends are colour matched with the data for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
polymer molecules per liposome (Fig. 1). It is pertinent to note that number of polymer molecules adsorbed per liposome will depend upon the partition co-efficient (Log Kp water-hexane) of the individual
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polymers. Therefore the actual number of polymer molecules adsorbed on the surface of an individual liposome will be less than the number of polymer molecules added to the liposome solution. Further, from Table 1 it is clear that adsorption of L-61 on the liposome surface will be significantly higher compared to F-68 and F127. It is reasonable to expect that membrane permeability will depend on the mobility of the lipids constituting the membrane. Therefore in presence of pluronic polymers the enhanced mobility of the membrane components may originate from the global mobility of all the lipid constituents or by formation of small patches on the membrane surface in which the mobility of membrane components is increased drastically. It is difficult to assume that presence of 5 or less pluronic molecules per liposome (Fig. 2) would affect the global mobility of the lipids constituting the liposome, which is supported by the fact that the sizes of the liposomes do not alter at all the pluronic concentrations used in this study (Fig. 1, SI). Therefore, it seems likely that the mobility of the membrane surrounding the adsorbed polymer gets compromised. Since the length of the hydrophobic PO unit in F-127 is twice of the order of membrane thickness (5 nm) it appears that to accommodate this large hydrophobic unit the lipid packing around an adsorbed F-127 molecule is severely affected. We propose that formation of this localized ‘highly mobile’ areas are responsible for the enhanced permeability of the LDS cation. It might be noted that similar type of hypothesis was already proposed previously [9]. In addition it may be noted that several MD simulation studies [27,28,30–34] have been shown that under equilibrium conditions, the hydrophobic segment of the pluronic polymer in the bilayer adapts a folded conformation. In particular, it has been observed that for F-127 polymer, the hydrophobic PO unit remains in a folded conformation even when the polymer is in transmembrane state [31]. Due to this folded configuration, the ordered arrangement of the lipid molecules in the immediate vicinity is disturbed. At this point it is important to note that transport of ions across a bilayer is driven by the creation of a trans-membrane potential difference due to adsorption of oppositely charged ions on the outer bilayer surface. However, the transport rate will also depend upon the rigidity of the bilayer. Therefore if by any means the rigidity is compromised, the transport rate will increase. We propose that due to the folded conformation of the PO unit of F-127 inside the bilayer, the lipid molecules in the immediate vicinity will lose their ordering arrangement resulting in reduced rigidity which will result in an enhanced permeability of the LDS cation around this localized area. So far, we have discussed the effect of polymer present in the solution on the POPG bilayer permeability by analysing the transport characteristics of the LDS cation. The interaction of the polymer with the membrane consists of two parts; first there is adsorption of the polymer at the aqueous-membrane interface and then insertion of the hydrophobic part (PO unit) of the polymer inside the bilayer [11,15,18,22,24,27,30]. The insertion of the PO unit (whose length is of the order of bilayer thickness or more) in the bilayer can be of two types: bilayer spanning or localization in the outer leaflet of the membrane. Spanning the entire membrane requires the transport of the hydrophilic segment of the polymer across the hydrophobic bilayer which is an energetically costly process and was shown to be slow in time [22,24] at room temperature, however at elevated temperatures when the bilayer becomes sufficiently mobile this may be faster. A comparison of the kT values of the LDS cation across a POPG bilayer and POPG bilayer containing pluronic polymers which were present either in the incorporated or incubated state (Fig. 4) indicates that temperature plays a crucial role in controlling the interaction of the F-68 and F-127 polymers with the bilayer. The kT values of LDS cation across POPG bilayer shows an abrupt increase near the temperature range of 15–20 °C indicating that at this tem-
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perature the mobility of the POPG lipids increases significantly. This trend is also followed by F-68 and F-127 polymers (which have a long chain hydrophilic EO groups) when they are incubated with POPG liposomes which leads us to conclude that insertion of the PO chain in F-127 and F-68 polymers in the POPG bilayer starts when the mobility of the bilayer gets reduced abruptly around the 15–20 °C temperature range. Although the reason behind this sudden change in the POPG bilayer mobility is not clear at this point, it is relevant to note that liposomes made from egg PE (phosphatidyl ethanolamine) were observed to show a transition near 10 °C, which has been attributed to the lipid hydrocarbon chain orderdisorder conformational transition [40]. It is possible that a similar transition in the POPG liposomes occurs in the temperature range of 15–20 °C causing the above mentioned effect. Further experiments are needed to corroborate this possibility. Since above 20 °C the observed changes in the kT values of the LDS cation with temperature are more or less similar for both incubated or incorporated complexes of F-127 or F-68 polymers with POPG liposomes further leads to the conclusion that above this temperature polymer incorporation in the bilayer is similar for both incorporated and incubated polymer-lipid complexes and this is likely to be in a bilayer spanning state. The reduction of the lipid mobility is likely to assist the translocation of the bulky EO chains in these polymers across the bilayer. The temperature dependent changes in the kT values of the LDS cation in presence of POPG liposomes complexed with L-61 polymer is similar for both incubated and incorporated complex. This suggests that L-61 inserts itself in the bilayer when it is added externally. This is perhaps not surprising given the fact that among the three polymers used in this study, L-61, has the highest HLB value and therefore is expected to bind more strongly to the POPG bilayer. 5. Conclusions In conclusion it was observed that tri-block pluronic polymer induced permeability of POPG liposomes against an organic cation, LDS, depends on the individual sub-units of the polymer. Using three different polymers, F-127, F-68 and L-61 the effect of variation in the individual sub-units of the polymer, the hydrophobic PO chain and the hydrophilic EO chain, has been investigated. Results obtained indicate that compared to the EO chain, variation in the PO chain remarkably affects the transport of LDS cation across the bilayer which can be attributed to the significant alteration in the bilayer packing near the vicinity of the adsorbed polymer. This alteration results from accommodating a PO chain whose length is almost double to that of the bilayer thickness (F-127). The role of polymer structure and temperature (5–40 °C) on the insertion of the hydrophobic PO unit in the bilayer has also been investigated by monitoring the transport of the LDS cation. L-61, which is the most hydrophobic of the series, was observed to insert itself spontaneously into the bilayer. F-127 and F-68 was observed to insert their PO unit above 15–20 °C which was aided by an enhancement in the mobility of the POPG lipids at this
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