Interaction between amphipathic triblock copolymers and L-α-dipalmitoyl phosphatidylcholine large unilamellar vesicles

Colloids and Surfaces B: Biointerfaces 148 (2016) 30–40

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Interaction between amphipathic triblock copolymers and L-␣-dipalmitoyl phosphatidylcholine large unilamellar vesicles M.A. Palominos a , D. Vilches a , E. Bossel b , M.A. Soto-Arriaza (Dr.) a,c,∗ a Laboratorio de Biocoloides & Biointerfaces, Departamento de Química Física, Facultad de Química, Pontificia Universidad Católica de Chile, Av. Vicu˜ na Mackenna 4860, Casilla 306, Correo 22, C.P. 7820436 Santiago, Chile b Laboratorio de Polímeros, Departamento de Química Física, Facultad de Química, Pontificia Universidad Católica de Chile, Av. Vicu˜ na Mackenna 4860, Casilla 306, Correo 22, C.P. 7820436 Santiago, Chile c Centro de Investigación en Nanotecnología y Materiales Avanzados CIEN-UC, Pontificia Universidad Católica de Chile, Santiago, Chile

a r t i c l e

i n f o

Article history: Received 10 March 2016 Received in revised form 19 August 2016 Accepted 22 August 2016 Available online 27 August 2016

a b s t r a c t This study contributes to an understanding of how different polymeric structures, in special triblock copolymers can interact with the lipid bilayer. To study the phospholipid-copolymer vesicles system, we report the effect of two amphipathic triblock copolymers of the type BAB, i.e., hydrophobic-hydrophilic-hydrophobic triblock copolymers arranged as poly(␧-caprolactone)poly(ethylene oxide)-poly(␧-caprolactone) (PCLn -PEOm -PCLn ), where n = 12 and m = 45 for COP1 and n = 16 and m = 104 for COP2, on the dynamic and structural properties of dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles (LUVs). The interaction between the copolymers and DPPC LUVs was evaluated by means of several techniques: (a) Photographs of the dispersion for evaluation of colloidal stability; (b) Thermotropic behavior from generalized polarization of Laurdan and fluorescence anisotropy of DPH (c) Main phase transition temperature determination; (d) Order parameters and limiting anisotropy by time-resolved fluorescence anisotropy measurements; (e) Water outflow through the lipid bilayer and (f) Calcein release from DPPC LUVs. Steady-state fluorescence measurements as a function of temperature show a typical behavior. Laurdan and DPH are fluorescent probes that sense the interface and the inner part of the bilayer, respectively. Both copolymers increase the Tm value of DPPC LUVs sensed by DPH, i.e., in the inner part of the bilayer. On the contrary, only COP2 had an effect on increasing the Tm value at the interface of the bilayer. At low temperature, in the gel phase, the presence of the copolymers produced a slight decrease in generalized polarization of Laurdan sensed in the interface of the lipid bilayer, but in the liquid-crystalline phase it produced an increase. In contrast, the order parameters obtained from time-resolved fluorescence anisotropy of DPH show an increase in the presence of the copolymers in the gel phase, but a decrease in the liquid-crystalline phase. COP2 produces a greater effect than COP1 in decreasing the water outflow through DPPC LUVs in the same concentration range. Furthermore, calcein release was decreased to a minimum at low copolymer concentration; however, at high concentration, release factor percentage (RF%) increased slightly without reaching the values obtained in the absence of copolymers. Therefore, the copolymer concentrations studied decrease the calcein release from the liposome. © 2016 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: PCL, poly(␧-caprolactone); PEO, poly(ethylene COP1, PCL12 –PEO45 –PCL12 ; COP2, PCL16 –PEO104 –PCL16 ; DPH, oxide); 1,6-diphenyl-1,3,5-hexatriene; GP, Generalized Polarization; Laurdan, 2dimethylamino-6-lauroylnaphthalene; DMSO, dimethyl sulfoxide; LUVs, large unilamellar vesicles; DPPC, L-␣-dipalmitoyl-sn-glycero-3-phosphocholine; CMC, critical micelle concentration. ∗ Corresponding author at: Departamento de Química-Física, Facultad de Química, Pontificia Universidad Católica de Chile. E-mail address: [email protected] (M.A. Soto-Arriaza). http://dx.doi.org/10.1016/j.colsurfb.2016.08.038 0927-7765/© 2016 Elsevier B.V. All rights reserved.

Lipid vesicles have been widely used us drug delivery systems because they can enclose hydrophilic compounds in the inner water pool or load hydrophobic compounds within the membrane bilayers. These properties have allowed their use as therapeutic agents for the delivery of different active molecules [1–3]. Although liposomes have numerous features that favor their use for drug delivery, there are several factors that complicate their use in vitro and in vivo systems, such as stability, undesirable size distribu-

M.A. Palominos et al. / Colloids and Surfaces B: Biointerfaces 148 (2016) 30–40

tion, low entrapment efficiency and short circulation half-lives. Multiple efforts have been made to obtain liposome formulations that stabilize their structure, for example, stealth liposomes or long circulating liposomes [4–7]. These liposomes are also known as sterically stabilized or PEG-ylated liposomes due to the incorporation of polyethylene glycol (PEG) on their surface [8]. Heldt and co-workers determined the effect of PEG attached to lipids in the membrane and found that the presence of PEG produces homogenous size liposomes, that is, PEG could help to regulate liposome size [9]. In this context, biocompatible and biodegradable copolymers are very interesting starting materials for the design of micelle-forming drug delivery vehicles. Different polymers have been used to promote liposome stabilization, such as, poly(ethylene oxide), PEO, and poly(N-vinyl pyrrolidone), PVP. These are appropriate polymers to be used as the hydrophilic block, while polyesters such as poly(lactide) and poly(␧-caprolactone) are appropriate as hydrophobic blocks due to their biocompatibility and can also undergo hydrolytic degradation [10]. PVP copolymer is a biocompatible polymer that has been extensively used in the pharmaceutical industry [11] and has been incorporated in different diblock [11,12] and triblock copolymers [13,14]. Furthermore, diblock copolymers have been studied in lipid vesicles constituted of different lipids. Previous reports have shown that the incorporation of diblock copolymers arranged as PEO-b-PCL in DPPC unilamelares vesicles forms nano-assemblies of smaller size and that these systems maintain their original physicochemical properties after two weeks. These qualities make that the diblock copolymer grafted DPPC vesicles behave like a nano-carrier for encapsulation of indomethacin at high molar ratio [15]. Furthermore, triblock copolymers, arranged as PEO-PPO-PEO, such as poloxamer 188 (P188, or Pluronic F68) prevent the acute necrosis of adult skeletal muscle cells due to their membrane sealing capability [16] and the increase in the structural stability of cell membrane, indicating that the membrane integrity can be a novel therapeutic target in the treatment of neuronal injury [17,18]. Different poloxamers have been incorporated into or adsorbed on lipid vesicles to study their potential as a stabilizing material for liposomes. In this context, several reports have shown the effect of different poloxamers in phosphatidylcholine (PC) liposomes [19–22]. These studies contribute to an understanding of the steric stabilization and mode of incorporation of triblock copolymers in PC liposomes. However, other authors have shown that the permeability of lipid bilayers can change significantly, increasing the leakage of water soluble molecules entrapped inside the liposomes when triblock copolymers are added [23,24]. Johnsson and co-workers found that triblock copolymers produce a significant morphological changes of the liposomes and promote the formation of bilayer discs and a size reduction of the aggregates formed from Egg-PC and DMPC [24]. Moreover, Wu and co-workers, have proven that the interaction between lipid bilayers and poloxamer depends on temperature and on the lipid bilayer phase, with formation of bilayer discs when the concentration of poloxamer is higher than the critical micelle concentration (CMC) and the temperature higher than the Tm in DMPC liposomes [25]. Triblock copolymers of PCL-PEOPCL have been studied as new drug carrier systems [26,27]. Ge and co-workers found that upon increasing the size of the hydrophobic block, the CMC decreases. The PEO/PCL copolymer ratio was determinant of micelle size, such that as the weight ratio of PCL increases the size increases [26]. Moreover, Quaglia and co-workers have shown that the copolymers self-assemble in aqueous media with a size less than 50 nm and a slightly negative surface potential [27]. In this context, our interest is focused on investigating the effect on L-␣-dipalmitoyl phosphatidylcholine vesicles of incorporation of amphipathic biodegradable and biocompatible triblock copolymers, that unlike poloxamer, are formed by a hydrophilic central block of poly(ethylene oxide, PEO) and two hydropho-

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bic blocks of poly(␧-caprolactone, PCL) on either end. In aqueous medium, PCL-PEO-PCL triblock copolymers adopt a structure that mimics a phospholipid, favoring their interaction when the lipid and copolymer are co-solubilized for liposome preparation. PCL has a high hydrophobicity and low melting glass transition temperature, and PEO is hydrophilic and flexible and imparts steric stability to micelles and biomimetic properties. In this context, two copolymers with different sizes of the hydrophilic block (PEO) but similar size of the hydrophobic blocks (PCL) were investigated to elucidate their interaction with DPPC LUVs. 2. Materials and methods 2.1. Reagents L-␣-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was obtained from Sigma Chemical Co. (St. Louis, MO). Triblock copolymers PCL12 –PEO45 –PCL12 (COP1) (Mw: 5282 g/mol and Mw/Mn: 1.112 where PCL is 2750 g/mol) and PCL16 –PEO104 –PCL16 (COP2) (Mw 14375 g/mol and Mw/Mn: 1.742 where PCL is 3653 g/mol) were synthetized, fully characterized and kindly donated by the Polymer Laboratory, Chemistry Faculty of Pontificia Universidad Católica de Chile. Chloroform, methanol, dimethyl sulfoxide and ethanol were obtained from Merck (Darmstadt, Germany). Calcein was obtained from Aldrich Chemical Co. (Milwaukee, WI). The fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 2-dimethylamino-6-dodecanoylnaphthalene (Laurdan), were obtained from Molecular Probes (Oregon, USA). Milli-Q water was employed in all preparations with a resistivity of 18.2 M cm. All reagents were of the highest purity available and were used without further purification. The chemical structures of the lipid, copolymers and fluorescent probes used in this study are shown in Table S1 in the ESI. 2.2. Preparation of DPPC LUVs Multilamellar liposomes of DPPC and copolymer were prepared from a stock chloroform solution and co-dried under a nitrogen flux. The lipid films were kept under vacuum overnight at room temperature. Multilamellar liposomes were prepared by hydrating and vortexing the lipid films with 1 mM NaCl solutions. DPPC/copolymer LUVs dispersion were prepared by the extrusion method, after five freezing and thawing cycles [28–30]. Extrusion was performed through 400 nm nominal pore polycarbonate filters employing nitrogen as pressurizing gas. Laurdan and DPH were incorporated into the LUVs suspension by addition of a small aliquot of a concentrated solution in methanol and DMSO, respectively, and incubated at 10 ◦ C above the main lipid transition temperature of the bilayer for 60 min. 2.3. Determination of the critical micelle concentration of the copolymers (CMC) The CMC of the copolymers were determined using the ratio of intensities, I338/I333, of the fluorescence excitation spectrum of pyrene in water according to Wilhem without other modifications [31]. The fluorescence ␭em = 390 nm of pyrene was measured at varying polymer concentrations using a Perkin Elmer LS55 Luminescence Spectrometer. All determinations were carried out at room temperature, 25 ◦ C. 2.4. Determination of the colloidal stability of dispersions The DPPC/copolymers LUVs were prepared by hydrating the films with 15 mL of 1, 50 or 150 mM NaCl aqueous solutions and vortexing the dispersions at 70 ◦ C. The final lipid concentration

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was 2 mM. The colloidal stability was documented by taking photographs of the samples with Canon EOS 60D camera at time zero and every 24 h for three days. All samples were maintained in the same glass tube and at room temperature, 25 ◦ C. 2.5. Steady-state and time-resolved fluorescence measurements The fluorescence anisotropy measurements were carried out with a multifrequency phase and modulation spectrofluorimeter (K-2, ISS Inc., Champaign, Il, USA) equipped with Glan-Thompson polarizers. The exciting light was from a modulable ISS 375 nm LED. For DPH, the emission was measured through Schott KV-399 and WG-420 long bandpass filters. The fluorescence anisotropy of DPH defined by r = (I// − I⊥ )/(I// + 2I⊥ ) Eq. (1), where I// and I⊥ are the parallel and perpendicular emission intensities, respectively, was evaluated. The polarization measurements were performed in the “L” configuration using Glan-Thompson prism polarizers in both the excitation and emission beams. The Laurdan fluorescence spectral shifts were evaluated from the fluorescence intensities measured at 490 nm and 440 nm, and characterized by the value of the generalized polarization (GP), defined as: GP = (I440 − I490 )/(I440 + I490 ) Eq. (2) [32]. 2.6. Time-resolved fluorescence measurements The DPH fluorescence lifetime measurements were made with the polarizers oriented at the “magic angle” [33]. In the multifrequency phase and modulation technique, the intensity of the excitation light is modulated and the phase shift and relative modulation of the emitted light were determined. For the anisotropy decay determination, the differential phase angles and modulation ratios were obtained from parallel and perpendicular oriented sinusoidal polarized emission. The phase and modulation values were obtained as previously described [34,35]. DimethylPOPOP in ethanol was used as the reference for the intensity decay (␶ = 1.45 ns). Data were analyzed using the Global Unlimited software package (Laboratory for Fluorescence Dynamics, University of Illinois at Urbana–Champaign, Urbana, IL). The fitting function for the lifetime measurements was the sum of a continuously distributed Lorentzian component and a discrete component, which was fixed at 0.01 ns to account for scattered light [34]. The anisotropy decay data were fitted to a hindered rotator model of anisotropy decay according to the following equation: r(t) = (ro − r∞ )exp(−t/ 1 ) + r∞ Eq. (3), which is based on the “wobble-in-cone” model [36]. The model included a hindered rotation component, that is, two rotational correlation times, where the second rotational correlation time,  2 , was fixed at a large value (1 ms) relative to the lifetime. In the above equation, ro is the amplitude of the anisotropy decay at time zero,  1 is the fast rotational correlation time of the anisotropy decay and r∞ is the residual anisotropy at infinite time.  1 is related to the fractional amplitude f1 , that is, the fraction of molecules associated with  1 , where f1 = 1 − r∞ /ro Eq. (4). The fluorophore rotational rate, R1 , is related to  1 by R1 = 1/6 1 Eq. (5) and S, the mean second rank order parameter of the fluorescent probe in the bilayer, is related to the limiting and initial anisotropy by S = (r∞ /ro )1/2 Eq. (6) [37,38]. 2.7. Leakage assay for evaluation of the rate of water efflux through the bilayer Large Unilamellar Vesicle suspensions in Milli-Q water were exposed to a hypertonic shock by the addition of 1 mM NaCl solutions. The time-dependent light scattering change associated with the increase in sample turbidity was measured at 450 nm in a spectrophotometer. Values of t1/2 , corresponding to the time at which the change in light scattering equals 1/2(A)∞ , were obtained

from light scattering vs. time plots. The absorbance change after an ‘infinite’ time (A) ∞ was obtained by extrapolating the sample absorbance to ‘infinite’ time [39–41]. All measurements were carried out at room temperature.

2.8. Calcein leakage Leakage of entrapped fluorophore was measured via the fluorescence emission intensity of calcein in the outer solution at 520 nm on a fluorescence spectrometer. Previously, liposomal solutions with entrapped calcein were passed through a column packed with Sephadex G-25 to remove any free calcein. The maximal fluorescence (100%) was established by lysing the vesicles after adding 1% of Triton X-100. The extent of the release or dye leakage, RF(t) %, was determined as a percentage of the maximal fluorescence for dye leakage by RF(t) % = (F(t) − Fo )/(Fmax − Fo ) × 100% Eq. (7), where F(t) is the fluorescence intensity at time t, Fo is the initial residual fluorescence of the vesicles and Fmax is the fluorescence observed upon lysing the vesicles with 1% Triton X-100. Calcein release was performed at room temperature in the presence of 1 mM 1-octanol as a membrane fluidizing agent [42]. The permeability coefficient of calcein, Ps, was estimated from kinetic analysis of calcein fluorescence according to Sato and co-workers [43] as developed by Maherani and co-workers [44]. From the time-course release behavior of calcein, we obtained the permeability coefficient (Ps) values in the absence and in the presence of different copolymer concentrations according to Ps = (r/3) × k Eq. (8), where the radius of the liposomes, r(cm), was obtained from the data presented in Table 2. Apparent rate constants, k, were obtained from RF(t) % = RFmax %(1-exp(-kt)) Eq. (9), which represents the time-course release of calcein.

2.9. Size-average diameter and zeta-potential determination Size-average diameter (Dz) and the polydispersity in the presence of different quantities of copolymers were obtained from dynamic lights scattering (DLS) at 90◦ using a Nano-Zetasizer ZS90 Analyzer (Malvern Instruments Limited, Worcestershire, UK) equipped with a 632.8 nm laser. Only those samples with high opalescence were diluted with ultra-purified Milli-Q water before the size-average and polydispersity determinations. The mean diameters were obtained by fitting the data to log-normal size distributions. The fitting was performed with the apparatus software using the non-negatively constrained least squares (NNLS) algorithm, which always fits the size distribution as a multimodal one [45]. Zeta-potential was also measured in pure water, adjusting the conductivity (50 ␮S cm−1 ) with a solution of sodium chloride (0.1%). The zeta-potential was determined from the electrophoretic mobility using the Helmholtz-Smoluchowski equation. The processing was performed using the software included in the system. All samples were analyzed in polystyrene cuvettes at 25 ◦ C.

2.10. Transmission electron microscopy DPPC vesicles were prepared in the presence and the absence of copolymers as described above. To obtain the images, 15 ␮L of 500 ␮M liposomes dispersion were placed on a copper grid covered with a Formvar polymer layer coated with carbon. After that, the drop of liposome dispersion was left standing for 5 min to promote the adsorption process and the excess dispersion was removed by blotting with filter paper. Subsequently, 15 ␮L of 2% aqueous uranyl acetate solution were added and left standing for 5 min. The excess was removed, and the grid was dried in a vacuum oven for 5 min at controlled temperature. The morphologies of DPPC vesicles in the absence and in the presence of copolymer aggregates were deter-

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Table 1 Size-average diameter (Dz), Polydispersity index (PDI) and zeta-potential (␰) of DPPC dispersions in the absence and in the presence of different copolymers concentrations. Data are show with standard deviation of three independent measurements. [Copolymers] (g/L)

Dz(nm)

COP1

110 ± 3 112 ± 3 118 ± 4 109 ± 2 134 ± 4 152 ± 6 172 ± 4 243 ± 5

Without 1 × 10−4 0.025 0.05 0.1 0.2 1.0 2.0

COP2

PDl

␰(mV)

Dz(nm)

PDl

␰(mV)

0.207 ± 0.004 0.180 ± 0.008 0.145 ± 0.009 0.186 ± 0.010 0.170 ± 0.010 0.132 ± 0.009 0.171 ± 0.011 0.173 ± 0.008

−15.60 ± 0.10 −9.96 ± 0.12 −7.45 ± 0.08 −4.15 ± 0.05 −3.05 ± 0.06 −2.66 ± 0.03 2.01 ± 0.05 6.87 ± 0.10

115 ± 2 113 ± 3 121 ± 3 115 ± 3 160 ± 5 240 ± 4 255 ± 4 ND

0.181 ± 0.007 0.170 ± 0.005 0.169 ± 0.005 0.132 ± 0.009 0.201 ± 0.012 0.167 ± 0.011 0.200 ± 0.015 ND

−15.60 ± 0.10 −3.17 ± 0.05 −3.25 ± 0.07 −3.78 ± 0.04 −4.35 ± 0.03 −4.61 ± 0.07 −4.92 ± 0.03 ND

Table 2 Time Resolved Fluorescence Anisotropy of DPH. Data are given as a function of the copolymer concentrations. Sample

[copolymer] (g/L)

DPPC

COP1

1 × 10−4

0.2

COP2

1 × 10−4

0.2

a b c d e f

T (◦ C)

␶a (ns)

␪b (ns)

rss c

ro d

r∞ e

Sf

20 40 50

13.74 13.20 10.70

0.651 0.502 0.319

0.296 0.282 0.116

0.400 0.400 0.400

0.307 0.165 0.047

0.876 0.829 0.343

20 40 50 20 40 50

13.53 13.11 10.50 12.42 12.74 10.40

0.204 0.562 0.924 0.316 0.598 0.950

0.306 0.303 0.116 0.328 0.347 0.126

0.400 0.400 0.400 0.400 0.400 0.400

0.313 0.275 0.037 0.331 0.288 0.039

0.885 0.829 0.305 0.910 0.849 0.312

20 40 50 20 40 50

12.51 12.31 10.60 12.27 12.09 10.50

0.221 0.517 1.010 0.302 0.665 0.965

0.306 0.292 0.119 0.330 0.309 0.120

0.400 0.400 0.400 0.400 0.400 0.400

0.306 0.270 0.028 0.316 0.272 0.046

0.875 0.822 0.266 0.889 0.825 0.339

Lifetime. Rotational Correlation time. Steady-State anisotropy. Anisotropy in the absence of rotational diffusion. Limiting anisotropy. Order parameter. All parameter were obtained from Eqs. (3) and (6) (see Materials and methods).

mined using a Philips Tecnai 12 BioTween transmission electron microscope operating at 80KV. 3. Results and discussion It has been well established that liposomes are systems that can be used for drug delivery. However, they have shown significant limitations for their use because they are cleared by the immune system. In this context, various efforts have been made to obtain stabilized liposome preparations that can effectively transport and deliver hydrophilic and hydrophobic drugs. In the present work, we have studied the influence of amphiphilic triblock copolymer arranged as PCL-PEO-PCL on DPPC Large Unilamellar Vesicles (LUVs) properties (see Table S1 in the ESI). We used triblock copolymers that differ in the size of the hydrophilic block (PEO) but for with PCL chain lengths are similar. Furthermore, we worked in a wide range of copolymer concentrations. 3.1. Colloidal stability, size, z-potential and morphology of DPPC LUVs-copolymer dispersions Colloidal stability measurements of DPPC LUVs in the presence of different COP1 and COP2 concentrations were carried out at three different ionic strengths. The results of the first 24 h of incubation can be observed in Fig. 1. According to our results, in the presence of the copolymers the initial turbidity measured in 50 mM or 150 mM NaCl solutions was higher than that obtained in 1 mM NaCl solutions and the same

behavior was observed after 24 h. All DPPC/polymer preparations showed similar behavior after 48 and 72 h of incubation (Figs. S1 and S2 in the ESI), i.e., all samples decanted during the incubation time, but could easily be resuspended with gentle shaking. Flocculation or precipitation of the dispersion of lipid/copolymers was not observed until after 72 h of incubation of the samples at room temperature. The CMC of the copolymer is a parameter that describes the physical properties of the micelles and is related to their thermodynamic stability [46,47]. Both copolymers have similar CMC values, 2.5 × 10−2 and 1.59 × 10−2 mg/mL for COP1 and COP2, respectively. These low CMC values indicate that the block copolymers have a similar tendency to aggregate in aqueous dispersion [48,49]. Since both copolymers have a similar length of hydrophobic block (PCL), but COP2 has hydrophilic block (PEO) twice as long as that of COP1, the PEO/PCL ratio for COP1 is smaller and it should have a greater hydrophobicity than COP2. It is noteworthy that both copolymers, COP1 and COP2 can form aggregates themselves in aqueous dispersions, a process accompanied by a slight turbidity, which is characteristic of these micellar dispersions [50]. For COP1 and COP2 copolymers, the PCL block forms a hydrophobic core while the PEO block formed a hydrophilic crown surrounding the core after both copolymers are dispersed in aqueous media, giving steric stability to the colloidal system [50]. When both copolymers and phospholipid are dispersed together in aqueous media, the hydrophobic block of the copolymers (PCL) interacts with the acyl chains of the phospholipid and the hydrophilic block of the copolymers (PEO) with the interface of the lipid bilayer. In this context, is important to determine whether COP1 and COP2

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Fig. 1. Turbidity at 450 nm of large unilamellar vesicles of DPPC in the presence of amphipathic triblock copolymers (a) COP1 and (b) COP2. Data were obtained at 25 ◦ C at 0 h (closed symbols) and 24 h (open symbols) at 1 mM NaCl (-䊏-,-䊐-), 50 mM NaCl (-䊉-,--) and 150 mM NaCl (--, −-). Both copolymers and DPPC were co-solubilized and re-suspended in Milli-Q water according to Section 2.2 of Materials and Methods. Inset: Images of DPPC LUVs dispersions in the presence of copolymers in (1) 1 mM, (2) 50 mM and (3) 150 mM NaCl. All images were taken at 25 ◦ C. Data were obtained at 0 h and 24 h.

have an influence on the distribution of net charge on the liposome surface. Even though the DPPC molecules are zwitterionic and have no formal net charge, electrostatic double-layer forces may play an important role in the stability of DPPC colloidal dispersions because the vesicles can require a net charge in solution. Several authors have used zeta-potential measurements to test the electrostatic effects at short or long distances on the stability of lipid vesicles in aqueous phases with different counterions or salt concentrations and different kinds of phospholipid molecules [51,52]. Wong and Thompson determined several conditions under which DPPC vesicles may coagulate and then coalesce or fuse [53]. For this purpose we determined the zeta-potential () for DPPC LUVs in the absence and in the presence of different quantities of the two copolymers, where the sign and magnitude of the zeta-potential is determined by the net charge accumulated on the liposome surface. The results can be seen in Table 1. In the absence of the copolymers, the zeta-potential,  was −15.6 mV and increased to +6.87 mV in the presence of COP1, an increase that was dependent on the amount of COP1 added. On the other hand,  increased slightly to −4.92 mV when COP2 was added up to 1 g/L. The zeta-potential of DPPC vesicles in the absence of copolymers obtained in the present work correlates well with those obtained by Park [54]. For the same conditions 1 mM NaCl, they determined a surface charge density () of 1.0 × 10−3 C/m2 and vesicle surface potential (z) of −60 mV and explain this result by preferential adsorption of Cl− and Na+ ions from the solution [54]. As can be observed, COP1 and COP2 increase the zeta-potential values of DPPC liposomes at 25 ◦ C and the effect was greater for COP1. Considering the recent work of Chibowski and Szcze´s [55] for the zeta-potential obtained of DPPC vesicles dispersed in 1 mM NaCl at different temperatures, the observed change in the zeta-potential can be explained by different orientations of the phospholipid switterionic head group. The range of zeta-potential values found for DPPC vesicles at 37 ◦ C in 1 mM NaCl can be explained on the basis of the model proposed by Makino and co-worker [56] in which the polar heads of phospholipids oriented in different ways at the surface, can result in negative, positive or even zero zeta-potential; in the same manner, polar head groups can reorient as a function of ionic strength. According to the interpretation given by these authors, the negative zeta-potential at low ionic strength is determined by the phosphate groups, whereas the positive zetapotential at a high ionic strength is determined by the choline groups of the phospholipid [55]. Considering our results, we can infer that the copolymers COP1 and COP2 favor a change in the

average orientation of the polar heads of the phospholipids. In our previous work,  values were not reported for DPPC dispersions in 1 mM NaCl because they were quite small and would not be comparable because the DPPC dispersions were prepared by a different methodology [57]. Size-average diameter (Dz) of the DPPC LUVs dispersions in the absence and in the presence of different copolymer concentrations can be observed in Table 1. According to our results, for DPPC liposomes in the presence of COP1 or COP2, the size-average diameter increases to twice the value of liposomes in the absence of copolymers. These results correlate well with TEM images of the different samples. Fig. 2 shows TEM images for DPPC in the absence and in the presence of COP1 triblock copolymer aggregates (Fig. 2a and b, respectively) and for the DPPC dispersions in the presence of COP2 (Fig. S3c in the ESI). As shown in Fig. 2a, the TEM images of DPPC in the absence of copolymer show differences in morphology, size and bilayer properties relative to the DPPC/COP1 dispersion. As shown in Fig. 2b, clear changes in the thickness, contrast of the bilayer and size are observed when DPPC was dispersed in the presence of COP1 triblock copolymer. TEM images of COP1, COP2 copolymers and DPPC/COP2 dispersion can be seen in Fig. S3 in the ESI. The DPPC/COP2 dispersion shows the same morphology. The images show a population of heterogeneous liposomes in which we emphasize the presence of closed bilayer structures with free internal structure. Nevertheless, the shape of liposomes appeared to be distorted, although this technique ensures the complete structural analysis of thin transparent samples. Therefore, the distortion or crumpled structures of the liposomes may be due to the presence of copolymer micelles, since images were obtained at high copolymer concentration (2 g/L). On the other hand, the shaded parts might also correspond to the negative staining methods, i.e., the interaction between the sample and the negative stain, or eventual alterations induced during the drying step. The interaction of triblock copolymers with the lipid membrane depends on the arrangement of the lipophilic and hydrophilic chain blocks. In our case, we worked with two types of copolymers whose arrangement of block is different from to that reported by Cheng [58] and Wang and collaborators [59,60]. These authors studied the effect of different copolymers arranged as hydrophilic–hydrophobic–hydrophilic triblock copolymers composed of PEO-PPO-PEO in giant unilamellar vesicles. They proposed a mechanism of copolymer-lipid interaction that involves, in a first stage, an adsorption process and, in a second stage, insertion of

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3.2. Effects of COP1 and COP2 on the physical properties of DPPC LUVs dispersion

Fig. 2. TEM images of (a) DPPC large unilamellar vesicles and (b) DPPC/COP1 adjusted to the same scale. TEM images of other suspensions are provided in Fig. S3 of the ESI. Lipid concentration (DPPC) was 500 ␮M and COP1 2 g/L.

the copolymer in the lipid bilayers. They found that in the first stage (adsorption process) the copolymer protects the lipid membrane due to the loss of integrity, while in the insertion process the copolymer disrupts the packing of the lipid bilayer, thus improving membrane permeability [59]. Furthermore, theoretical studies carried out by Rabbel and co-workers [61], have shown that triblock copolymers of this type may have two conformational states that can interact with the lipid membrane, hairpin and transmembrane states, where the relative stability of the two states can be favored by slight changes of relative hydrophobicity and length of the central block. Triblock copolymers with intermediate chain lengths of the central block lead to stable transmembrane conformations, while those triblock copolymers with central chain longer or shorter than the thickness of the lipid bilayer produce a metastability of the transmembrane state [61]. Studies performed in LUVs of POPC-PLPC-POPG as a model membrane show that the adsorption of hydrophilic poloxamers at the surface of the lipid bilayer suppresses lipid peroxidation, an effect that is not observed when poloxamers are inserted into the bilayer [58,60]. These studies are important to understand how copolymers can interact with lipid membranes. In our case, both triblock copolymers were arranged as hydrophobic–hydrophilic–hydrophobic blocks with similar hydrophobic PCL chains that mimicked the thickness of the bilayer, but with a different size of the hydrophilic block (PEO). According to our methodology of preparing LUVs of DPPC in the presence of copolymers (coinstrusion, see Material and Methods) and the TEM images obtained, we believe that the triblock copolymers are predominantly inserted into the lipid bilayers rather than being merely adsorbed on the surface of the liposomes. This assumption is also consistent with the changes in the thickness of the lipid bilayer observed by TEM.

The interaction of COP1 and COP2 copolymers with the lipid bilayers of DPPC liposomes can be followed by the generalized polarization function (GP) of Laurdan. When the lipids are in a gelphase, the emission maximum of Laurdan is centered at 440 nm and when the lipids are in a liquid-crystalline phase the emission maximum is centered at 490 nm. This red spectral shift is the result of the dipolar relaxation of Laurdan in the lipidic environment. The origin of this dipolar relaxation has been attributed to a few water molecules present in the bilayer at the level of the glycerol backbone, where the Laurdan naphthalene moiety resides. The concentration and molecular dynamics of these water molecules is a function of the phospholipid phase state, where water reorientation along the probe excited-state dipole occurs only in the liquid-crystalline phase [32]. The spectral shift of Laurdan was measured in DPPC LUVs in the absence and in the presence of the copolymers and the resulting GP function has a typical behavior clearly showing the phase change from gel to liquid-crystalline (Fig. 3a). A main phase transition temperature (Tm) around 41 ◦ C was obtained from the minimum value of the first derivative of the measured GP with respect to the temperature (Fig. 3a, Inset). Our results show that, while COP1 does not affect Tm, i.e., does not affect the interface of the bilayer, COP2 produce a small increase around of 5 ◦ C at 1 g/L of copolymer, sensed by Laurdan in the interface of the bilayer. Both copolymers produce a diminution in the difference between the GP values obtained in the gel phase with respect to the GP values obtained in the liquidcrystalline phase at high concentrations. On the other hand, at the inner part of the bilayer sensed by DPH, both copolymers increase of 5 ◦ C at a high copolymer concentration (Figs. 3 b and S4b in the ESI). The observation of a smaller difference between gel and liquid-crystalline phases observed and a broader main transition temperature range at high copolymer concentrations than at low copolymer concentrations suggests that, at high concentrations, the copolymers homogenize the lipid bilayer. As shown above, the zeta-potential of DPPC LUVs dispersion was also affected by COP1 and COP2 in all the concentration range. For DPPC LUVs dispersions, the presence of COP2 produces an increase in the zeta-potential consistent with the increase of about 5 ◦ C in Tm. According to the results shown in Fig. 3, the effect of copolymer concentration on DPPC LUVs has a different behavior depending on the temperature. The presence of amphipathic copolymers co-solubilized with DPPC LUVs increases the GP values in the liquid-crystalline phase for almost all copolymers concentrations, whereas in the gel phase neither copolymer has a marked effect. This behavior suggests that both COP1 and COP2 reduce the quantity or mobility of water molecules in the interface of the lipid bilayer in the liquid-crystalline phase. In contrast, in the deeper region of the bilayer, steady-state fluorescence anisotropy of DPH, which senses the mobility of the acyl chains, shows a typical behavior as a function of temperature in the absence and in the presence of COP1 and COP2 (Figs. 3 b; S4b in the ESI). Both copolymers increase the Tm sensed by DPH from 41 ◦ C to 45 ◦ C for COP1 and from 41 ◦ C to 43.5 ◦ C for COP2. The Tm of liposomes formed by pure lipids or mixtures thereof determined by the present methodology shows a strong correlation with determinations made by differential scanning calorimetry. For a system consisting of a pure lipid, the change of the fluorescent property (GP or r) is very sharp and the accuracy and precision of the technique is favored by increasing the number of measurements (data) around the Tm. In contrast, COP1 and COP2 increase the range of the phase transition temperature at high concentrations, an effect that was also observed in the interface of the bilayer sensed by Laurdan (Fig. S4a in the ESI). In view of these

36

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0,6

(a)

(b)

0,00

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0,3 -0,10

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15

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0,1 DPPC -4 1E g/L 0,025 g/L 0,05 g/L 0,10 g/L 0,20 g/L 1,00 g/L

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0,15 DPPC -4 1*10 g/L 0,025 g/L 0,10 g/L 0,20 g/L 0,50 g/L 1,00 g/L

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50

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Temperature (°C)

Fig. 3. Thermotropic behavior of (a) generalized polarization (GP) of Laurdan and (b) fluorescence anisotropy (r) of DPH in the presence of different concentrations of COP2. Inset in each graph: first derivative of generalized polarization of Laurdan and fluorescence anisotropy of DPH. The minimum represents the main phase transition temperature (Tm). Thermotropic behavior of COP1 can be seen in Fig. S4 in the ESI.

0,6

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Fig. 4. Amphipathic triblock copolymer effect on (a) Generalized Polarization of Laurdan and (b) fluorescence anisotropy of DPH in DPPC LUVs. COP1 (-䊐-,-䊏-) and COP2 (--,-䊉-) in the gel phase at 25 ◦ C (open symbols) and the liquid-crystalline phase at 55 ◦ C (closed symbols).

results, the fluorescence anisotropy data obtained at 25 ◦ C and 55 ◦ C were plotted as a function of copolymer concentration, as shown in Fig. 4. Plainly, COP1 and COP2 show their main effect in the gel phase at 25 ◦ C, producing a decrease of the fluorescence anisotropy at high copolymer concentrations. Therefore, this suggests COP1 and COP2 promote or favor an increase in the rotational mobility of the acyl chains in the gel phase. Furthermore, in the presence of these copolymers, no effect on the liquid-crystalline phase was observed throughout the range of copolymer concentrations used. The fact that both Tm and the zeta-potential increase in the presence of copolymers suggests a tighter bilayer packing for the DPPC/copolymer dispersions in comparison to DPPC dispersions in the absence of copolymers. Thus a tighter lipid bilayer packing at the level of the acyl chains would increase the mean phase transition temperature of the bilayer due to an increased interaction between the phospholipids and decrease the mean molecular area per lipid at the bilayer surface, thereby increasing the surface charge density and the zeta-potential [62,63]. A previous report of the effect of different polymers in DPPC SUVs determined that the addition of PVPy and PEO did not affect the thermal behavior of the

DPPC SUVs, but poly(MPC) and poly(MPC-co-BMA) addition produced an increase in the Tm [64], which relates well with the results obtained in this work. Hydrophilic polymers produce an increase of the fluorescence anisotropy in gel the phase (below 40 ◦ C), promoting a decrease of membrane fluidity [64]. Contrary to these results, our copolymers produce a fluidization of the lipid bilayer at high copolymer concentrations in the gel phase (Fig. 3b). While it is true that the behavior is different, the systems are not fully comparable because the authors used PVPy and PEO mono-block polymers, unlike our studies where the lipophilic blocks could be inserted into the lipid bilayer with the hydrophilic block interacting with the surface of lipid bilayer. Fluorescence lifetime and anisotropy decay measurements of DPH also can give information about the lipid bilayer-copolymer interaction. Lifetimes ( c ), rotational correlation times (), limiting anisotropy (r∞ ) and the order parameter (S) as a function of copolymer concentration are presented in Table 3 for the gel phase at 20 ◦ C, the liquid-crystalline phase at 50 ◦ C and at 40 ◦ C near the Tm. For homogeneous systems in which the fluorescent probe senses a single environment, a single lifetime is obtained. However, in

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37

Table 3 t1/2 Values and initial rates of water efflux through the bilayer in the presence of different copolymer concentrations. t1/2 values obtained at 1080 s. COP1

COP2

[copolymer] (g/L)

t1/2 a (s)

vi b (A/t) × 10−3

[copolymer] (g/L)

t1/2 a (s)

vi b (A/t) × 10−3

Without 1 × 10−4 0.025 0.05 0.1 0.2 2.0

145 145 148 145 140 145 178

1.74 1.70 1.67 1.65 1.67 1.62 1.49

Without 1 × 10−4 0.025 0.05 0.1 0.2 1.0

145 155 155 153 160 180 240

1.74 1.37 1.32 1.22 0.94 0.59 0.26

a t1/2 : Time that is required to reach half of the maximum absorbance slope obtained from of turbidity at 450 nm for DPPC LUVs in the presence of the co-polymers during the first 120 s after the osmotic shock. b vi : slope obtained from of turbidity at 450 nm for DPPC LUVs in the presence of the co-polymers during the first 120 s after the osmotic shock.

heterogeneous systems, several lifetime values are obtained corresponding to different dielectric constant gradients [65]. The center of the Lorentzian distribution,  c , was taken to be the fluorophore lifetime, which it is related to the presence of water molecules, that is, with the polarity of the microenvironment [66]. The value of the fluorescence lifetime (for a discrete analysis) or the mean fluorescence lifetime (for a lifetime distribution) yields information on the average environment of the fluorophore. The presence of water decreases this value due to the higher dielectric constant and yields information on the hydration in the copolymerlipid interface [66]. The effect of water on the fluorescence lifetime of DPH fluorophores was used as a test for the presence of water at the copolymers-lipid interface. The first observation of note in our results is that, in this lamellar system, we obtained a single fluorescence lifetime in both the absence and in the presence of copolymers, i.e., DPH sensed a single average microenvironment rather than several different microenvironments. Furthermore, the lifetime and rotational correlation times of DPH decrease as the temperature increases in DPPC dispersions in the absence of copolymers. In the presence of COP1 and COP2, the fluorescence lifetime also decreases, but the rotational correlation time of DPH increases as the temperature increases at the same copolymer concentration. Analysis of the data at three temperatures indicates that an increase in the amount of COP1 or COP2 in the bilayer promotes an increase in water penetration into the lamellae, in the gel and liquid-crystalline phase. From the anisotropy decay analysis we recovered values of  1 , the rotational correlation time, which is inversely related to the fluorophore rotational rate R1 , and r∞ , the limiting or residual anisotropy at infinite time. These parameters represent dynamic and structural properties, respectively. Because the probe rotational rate reflects the orientational dynamics of the surrounding phospholipid molecules, COP1 and COP2 incorporation produces a decrease in the rotational rate in the gel phase at 20 ◦ C and a subsequent increase in the liquid-crystalline phase at 50 ◦ C at low and high concentration of copolymers. Furthermore, the changes in the residual anisotropy at infinite time, r∞ , related to the lipid packing order, indicate a monotonous disorder increase as the temperature increases in the absence and in the presence of copolymers. However, when the values of r∞ in the presence of COP1 or COP2 are compared to those in DPPC LUVs alone, there is different behavior in the gel and liquid-crystalline phases. COP1 and COP2 elicit a slight increase in the residual anisotropy at infinite time, r∞ , in the gel phase at 20 ◦ C and produce a decrease in this parameter in the liquid-crystalline phase at 50 ◦ C. This suggests that both copolymers interact with the lipid bilayer, where COP1 and COP2 promote an increase in the order of the phospholipid acyl chain packing in the gel phase but a decrease in the liquid-crystalline phase. These results correlate well with the order parameter values, S, which also show that, COP1 and COP2 promote the order of the acyl chains in the gel phase but promote disorder in the liquid-crystalline phase.

3.3. Effect of COP1 and COP2 copolymers upon water permeability in DPPC LUVs dispersion Permeability of water through lipid bilayers in unilamellar vesicles is a favorable process compared to the movement of other molecules, including ions [67–69]. The movement of water molecules through the lipid bilayer in DPPC vesicles, particularly the efflux of water after a hypertonic shock is applied, can be followed by an increase in the suspension turbidity [70,71]. In our case, the hypertonic shock was generated by addition of 1 mM NaCl solution, which produces a fast increase in the absorbance measured at 450 nm. The rate of water influx/outflux following an osmotic shock can be quantified by the parameter t1/2 , defined as the time required for a 50% increase/decrease in the turbidity of the sample relative to the maximum/minimum value reached at long times (≥500s) after the osmotic shock. The effects of COP1 and COP2 on water efflux through the lipid bilayers of DPPC LUVs are shown in Fig. 5. The time-course of turbidity at 450 nm was obtained in the presence of COP1 and COP2 at different concentrations (see Fig. 5). It can been clearly observed that COP1 has a slight effect on water outflow at high concentrations because the turbidity decreases only 0.1 absorbance unit, from 0.5 to 0.4; conversely, COP2 decreases the water outflow in a concentration dependent manner, with a turbidity decrease from 0.55 to 0.20 absorbance units or t1/2 value increase from 145 s to 240 s. Initial rate measurements, obtained from the temporal variation of turbidity during the first 120 s after osmotic shock, are shown in Table 3. As can be seen, COP1 has only a slight effect on water outflow; conversely, COP2 elicits a significant decrease in the rate of water permeability through the lipid bilayer. If we compare the results obtained in the absence and in the presence of COP1 or COP2, the t1/2 values increase by only about 23% for COP1, but in the presence of COP2 the t1/2 values increase 66% (Table 3 and Fig. 5). These results corroborate those obtained by timeresolved fluorescence anisotropy, considering that COP2 promotes a higher packing degree of the lipid bilayer, the resultant increase in the order being reflected in a decrease in the water permeability through the lipid bilayer [72]. The effect of COP1 and COP2 of decreasing water permeability through the lipid bilayer (increasing t1/2 ), does not contradict the effect of these copolymers in increasing the polarity of the bilayer (decrease in the lifetime). Although both copolymers would appear to an increase of the water penetration into the lamellae, both copolymers also enhance the retention of these water molecules in the lipid bilayer and therefore decrease water outflow through the lipid bilayer. This effect was more pronounced for COP2 that for COP1. According to Rabbel and collaborators, the conformational states that PEO-PPO-PEO triblock copolymers can adopt depend of hydrophobicity of the PPO block. Triblock copolymer with high central block hydrophobicity

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Fig. 5. Time-course of turbidity at 450 nm of DPPC LUVs in the presence of (a) COP1 and (b) COP2. Different concentrations of copolymer (g/L) are represented by (a) without copolymer; (b) 1 × 10−4 ; (c) 0.025; (d) 0.05; (e) 0.1; (f) 0.2 and (g) 2.0 for COP1 and (g) 1.0 for COP2. (h) Dispersion of COP1 2.0 g/L and COP2 1.0 g/L alone. The arrow indicates the time when 1 mM NaCl solution was added.

60

(a)

10

9

Calcein Release (%)

-9

Permeability coefficient, P s*1x10 (cm/s)

50

(b)

40

30

20

10

0

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Apparent Rate Constant, k (s)*10-3

6

COP1 2,36 2,21 2,23 2,07 2,24 2,13 2,15 2,31

[Copolymer] (g/L) Without

5

1*10-4 0,025

4

0,05 0,1 0,2 1 2

COP2 2,61 2,42 2,43 2,39 2,51 2,09 1,48 ND

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Fig. 6. Copolymer concentration dependence of the (a) Calcein Release Factor Percentage (RF%) and (b) Permeability Coefficient value (Ps) for calcein release from DPPC liposomes. COP1 effect is represented by (-䊐-) and COP2 by (--). Data were obtained at 40 min after 1 mM 1-octanol addition. Standard deviations were obtained from at least three independent experiments at 25 ◦ C. Table inset, Apparent Rate Constant for calcein efflux through the bilayer in the presence of different copolymer concentrations. Apparent Rate Constant (k) was obtained using Eq. (9) and the Permeability Coefficient (Ps) was obtained using Eq. (8) and the data of Table 1.

produce an increase of the permeability barrier, as do, copolymers with a central block length that not match the thickness of the lipid bilayer [61]. On the other hand, copolymers in the hairpin state have a minimal effect on the membrane permeability. The insertion of such copolymers into the lipid bilayer results in an increased permeability, while adsorption of copolymers has no effect [59,60]. Because our triblock copolymers have the reverse architecture, i.e., a central hydrophilic block and two hydrophobic blocks at the ends, it is much more difficult, if not impossible, for our copolymers to adopt a transmembrane conformation. Although copolymers with a central hydrophilic block may adopt a hairpin conformation and be adsorbed on the surface of the lipid bilayer, the most reasonable assumption is that the hydrophobic blocks insert into the bilayer, exposing the hydrophilic block to the aqueous medium. The behavior of COP1 and COP2 with respect to the difference of water permeability would then be related with the difference in the PEO/PCL ratios for each copolymer. In this context, as mentioned above, COP2 is a more hydrophilic copolymer than COP1 since the PCL blocks have similar numbers of monomers and the PEO/PCL ratio is 3.25 for COP2 compared to 1.875 for COP1.

3.4. Effect of copolymers on calcein release behavior Calcein is a fluorescent dye that has been widely employed to study solute permeation through lipid bilayers after entrapping it in the inner aqueous compartment of liposomes [44]. Typical behavior for the time-course of the percentage Release Factor (RF%) for calcein in DPPC LUVs in the absence and in the presence of COP1 and COP2 can be seen in Fig. S5 in the ESI. The kinetics of calcein release at room temperature is very slow, involving many hours. For this reason, calcein release experiments were performed in the presence of 1 mM 1-octanol as a membrane fluidizing agent [42] to facilitate calcein release through lipid bilayers. Considering that all DPPC/copolymer dispersions were prepared in the same manner and maintained in same conditions, the differences observed represent the effect of the different copolymers co-solubilized with DPPC lipids. The Release Factor percentage value obtained at 1080 s after 1-octanol addition is plotted as a function of copolymer concentration in Fig. 6a. The first observation from our results is that neither copolymer has a monotonic effect on the concentration dependence. The RF% value decreases to a minimum at low copoly-

M.A. Palominos et al. / Colloids and Surfaces B: Biointerfaces 148 (2016) 30–40

mer concentrations, but at high concentration of copolymers RF% increases without reaching the values obtained in the absence of copolymers. Therefore, at high concentrations both copolymers produce retention or decrease release of calcein across of the lipid bilayers. Similar results were reported by Alves and co-workers using other polymers and unilamellar vesicles [73]. Fig. 6b shows permeability coefficient data and the apparent rate constants are tabulated in the table in the inset (Fig. 6b). Data were obtained according to the description in Material and Methods. The behavior of the permeability coefficients obtained as a function of copolymer concentration shows that both copolymers produce a slight decrease in the calcein permeability at low copolymer concentrations, followed by a clear increase at high concentrations. This increase is reached at 2 gr/L for COP1 and at 0.2 gr/L for COP2 and thereafter it remains somewhat constant up to 1 gr/L, without reaching the values obtained in the absence of copolymers. Despite the slight increase in the permeability coefficient of calcein observed at high both copolymer concentrations, the apparent rate constant obtained for both copolymers shows only slight variations, remaining almost constant, suggesting that, the calcein permeability coefficient depends on the size of the large unilamellar vesicles. 4. Conclusions The results obtained in the present study show that the hydrophobic-hydrophilic-hydrophobic triblock copolymers COP1 and COP2 interact with lipid bilayer of DPPC LUVs. Based on several different fluorescence approaches, both copolymers are found to affect the different physicochemical properties of the bilayer. While, COP2 affects the interface of the bilayer, sensed by Laurdan, and the inner part of the bilayer, sensed by DPH, COP1 showed an effect only on the inner part of the bilayer. Furthermore, both copolymers increase the main phase transition, pointing to an increase in the thermodynamic stability of the lipid bilayer. This behavior may be related to the promoter effect that COP1 and COP2 induce, increasing the order of the phospholipid acyl chain packing (increase of the S parameter) in the gel phase. On the other hand, both copolymers produce a decrease in order in the liquid-crystalline phase. Membrane permeability studies of water outflow and calcein release show that COP2 has a somewhat greater effect than COP1, although both copolymers prevent water outflux and release of calcein from the inner part of liposome, we cannot entirely rule out the possibility that the copolymers are adsorbed to some extent on the surface of the liposomes. The effects that the copolymers induce on the membrane and the fact that the copolymers are co-solubilized together with lipids during vesicles preparation, strongly suggest that the copolymers are predominantly inserted into the bilayer, without causing the collapse of the bilayer in the range of copolymers concentration studied. Because the difference in structure of COP1 and COP2 is basically restricted to the length of the central hydrophilic PEO block, the observed differences between the effects induced by COP1 and COP2 are ascribable to the different hydrophilicities of the PEO block. Acknowledgements This work was supported by the FONDECYT Research Program of CONICYT, project 1141012 (MS-A). Also acknowledged are VRI and Dirección de Investigación y Posgrado (DIPOG), Facultad de Química, Pontificia Universidad Católica de Chile (MS-A). MS-A would like to thank Professor Luis Felipe Aguilar for the use of his laboratory equipment to perform time-resolved fluorescence measurement and Professor Denis Fuentealba for review and Professor Frank Quina for critical review suggestions comments and discussion.

39

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.08. 038.

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