and Richard M. Epand Department of Biochemistry and Biomedical

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Thermodynamics of methyl-#-cyclodextrin-induced lipid vesicle solubilization: Effect of lipid headgroup and backbone José Carlos Bozelli, yu heng hou, and Richard M. Epand Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03447 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 12, 2017

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Thermodynamics of methyl-β-cyclodextrin-induced lipid vesicle solubilization: Effect of lipid headgroup and backbone

José Carlos Bozelli, Jr; Yu Heng Hou; and Richard M. Epand*

Department of Biochemistry and Biomedical Sciences, McMaster University, Health Sciences Centre, Hamilton, Ontario L8S 4K1 Canada

*Corresponding author: [email protected]

1 ACS Paragon Plus Environment

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Abstract The low aqueous solubility of phospholipids makes necessary the use of lipid carriers in studies ranging from lipid traffic and metabolism to the engineering of model membranes bearing lipid transverse asymmetry. One particular lipid carrier that has proven to be particularly useful is methyl-β-cyclodextrin (MβCD). In order to assess the interaction of MβCD with structurally different phospholipids, the present work reports the results of isothermal titration calorimetry in conjunction with dynamic light scattering measurements. The results showed that the interaction of MβCD with large unilamellar vesicles composed of a single type of lipid led to the solubilization of the lipid vesicle and, consequently, the complexation of MβCD with the lipids. This interaction is dependent on the nature of the lipid headgroup with a preferable interaction with phosphatidylglycerol in comparison to phosphatidylcholine. It was also possible to show a role played by the phospholipid backbone on this interaction. In many cases the differences in the transfer energy between one lipid and another in going from a bilayer to a cyclodextrin-bound state can be qualitatively explained by the energy required to extract the lipid from a bilayer. In all cases the data showed that the solubilization of the vesicles is entropically-driven with a large negative ∆Cp, suggesting a mechanism dependent on the hydrophobic effect.


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Abbreviation list: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG); 1-palmitoyl-2oleoyl-sn-glycero-3-phospho-L-serine (POPS); 1',3'-bis[1,2-dioleoyl-sn-glycero-3phospho]-sn-glycerol (TOCL); Association constant (KB); Bovine heart cardiolipin (BHCL); Cyclodextrins (CD); Dynamic light scattering (DLS); Isothermal titration calorimetry (ITC); Large Unilamellar Vesicles (LUVs); Monolysocardiolipin (MLCL); Multilamellar vesicles (MLV); Phosphatidylcholine (PC); Phosphatidylethanolamine (PE); Phosphatidylglycerol (PG); Phosphatidylinositol (PI); Phosphatidylserine (PS); Methyl-β-cyclodextrin (MβCD); Red blood cells (RBC); Small unilamellar vesicles (SUVs); Sphingomyelin (SM); Tris(hydroxyl-methyl)aminomethane (Tris).


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Keywords: methyl-β-cyclodextrin; isothermal titration calorimetry; dynamic light scattering;

thermodynamics;

monolysocardiolipin;

Cyclodextrin-Phospholipid

complexes


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Introduction Living organisms are prevented from reaching thermodynamic equilibrium by compartmentalizing their contents within cells and organelles. Membranes are the semi-permeable barriers that delimit these compartments. The membrane is arranged as a lipid bilayer that also contains proteins and carbohydrates. Although the membrane is highly dynamic, with lipids presenting many kinds of motions in a broad range of time scales, lipids are usually confined to the membrane due to their low water solubility1. Hence, in order to study how the removal or addition of a particular lipid affects the membrane properties as well as a function of a protein of interest, usually, it is necessary to use a lipid carrier. This is also the case for the study of lipid traffic between different compartments as well as lipid metabolism. For model membranes the lipid composition can be altered simply by assembling the system with different lipids. However, such membranes differ from biological membranes because most procedures result in the formation of membranes with a symmetrical transbilayer distribution of lipids. The formation of model membranes with transbilayer asymmetry requires special methods. Many of these methods utilize lipid carriers to facilitate the formation of these membranes. The most commonly used lipid carrier for these purposes is cyclodextrin Cyclodextrins (CDs) comprise a family of cyclic oligosaccharides with three major representatives, namely α-, β-, and γ-CD2. These are torus-like macro-rings built up from 6, 7, or 8 glucopyranose units, respectively, linked by α(1→4) glycosidic bonds. The torus has a height of 7.9 Å with a cavity volume ranging from 174 to 427 Å3. The CD structure contains a (slightly) hydrophobic cavity, while the external surface is hydrophilic. In an aqueous environment, the 5 ACS Paragon Plus Environment

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energetically disfavored water-apolar interactions in the cavity can be readily prevented by hydrophobic molecules binding to the cavity2. The formation of these so-called inclusion complexes increase the water solubility of these hydrophobic molecules as well as their stability, which is one of the reasons why CDs are extensively used in the cosmetic, food, and pharmaceutical industries. In the 1980´s, a study of CD with red blood cells (RBC), aimed to evaluate their cytotoxicity, showed that these molecules presented hemolytic properties, which later was ascribed to a non-specific extraction of phospholipids from RBC3,4. This finding paved the way for the use of CDs as lipid carriers being employed in a range of applications5-10. There were major efforts to study CD-cholesterol interactions in model and biological membranes. It has been shown that CD could deliver as well as extract cholesterol from membranes and the efficiency was a function of CD concentration, temperature and time of incubation, type of CD and cell type and membrane lipid composition6,11-17. The molecular mechanism of CDmediated cholesterol extraction is not fully understood at the moment. The two widely accepted models to explain CD-cholesterol interaction differ on whether CD interacts with cholesterol at the membrane or in the aqueous phase11,18-20. In comparison to CD-cholesterol interaction, the interaction of CD with phospholipids is less well characterized. It was shown that the efficiency of phospholipid extraction from RBC follows the order α-CD > β-CD >> γ-CD4. This is different from the order observed for the extraction of cholesterol from RBC, which was found to follow the order β-CD >> γ-CD > α-CD4. In this case, the extraction is also highly sensitive to CD concentration12,15. 1H- and

31

P-NMR studies showed

that interaction of α-CD with phospholipids are modulated by the nature of the 6 ACS Paragon Plus Environment

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polar headgroup as well as by the nature of acyl chains21,22. The ability of α-CD to extract lipids from RBC was found to follow the order phosphatidylserine (PS) ≥ phosphatidylethanolamine (PE) >> sphingomyelin (SM) > phosphatidylcholine (PC)22. A theoretical study extended this finding showing that the complexation of α-, β-, and γ-CD with phospholipids bearing different headgroups occurs in the following decreasing order: phosphatidylinositol (PI) > PS > PE23. On the other hand, in brain capillary endothelial cells it has been shown that α−CD and γ-CD lack specificity, while β-CD showed a preferential extraction of SM over PC24. By employing pyrene-labeled phospholipids, Tanhuanpää and collaborators25 showed that the association constant of lipids with γ-CD decreases strongly with increasing acyl chain length. In that study, a change on the complex stoichiometry was also observed as the acyl chain length increased. The β-CD derivative, randomly methylated β-CD (MβCD), has been shown to be a versatile lipid carrier. At low concentrations (< 10 mM) MβCD is cholesterol-specific, while at higher concentrations phospholipids start being extracted from different cell types14-16,26. Like the majority of CD, MβCD binds lipids and can exchange lipids between model membranes or model and biological membranes. Indeed, it has been shown that MβCD mediates efficient transfer of long-chain phospholipids from vesicles to cells without significantly compromising their growth or viability9. However, contrary to other CD, MβCD has also been shown to solubilize lipid vesicles, leading to the formation of soluble CD-lipid complexes27-30. The amount of MβCD needed for lipid vesicle solubilization is dependent on acyl chain length, degree of unsaturation, and lipid headgroup30. MβCD has also been used to measure transbilayer movement of sterols as well as 7 ACS Paragon Plus Environment

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cholesterol affinity to bilayers with different lipid compositions14,16,31. Although MβCD have been used as a lipid carrier for a range of applications, it is still not fully understood how the interaction with lipids occurs at the molecular level, nor has the affinity for different lipids been reported. The relationship between lipid extraction and the efficiency of lipid delivery is intricate and it is very important to properly choose the conditions for the use of MβCD as a lipid carrier, regardless of the application. Ideally, for lipid exchange, one would like to have a moderate binding of the lipids of interest to MβCD, with an intermediate strength of interaction. We report here the interaction of MβCD with large unilamellar vesicles (LUVs) individually prepared with structurally different phospholipids. We have employed isothermal titration calorimetry (ITC) to measure the thermodynamic parameters of the interaction between MβCD and these structurally different phospholipids. In addition, we have employed dynamic light scattering (DLS) to properly understand the structural nature of the species involved.


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Experimental section Materials. Lipids used were: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG),

1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine

(sodium

salt)

(POPS), Sphingomyelin (from Chicken egg) (SM), Bovine heart cardiolipin (sodium salt) (BHCL), 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (sodium salt) (TOCL), and Bovine heart monolysocardiolipin (sodium salt) (MLCL). All lipids were purchased from Avanti Polar Lipid (Alabaster, AL). Methyl-β-cyclodextrin (MβCD)

was

purchased

from

Sigma

(St.

Louis,

MO).

Tris(hydroxyl-

methyl)aminomethane (Tris) and NaCl were from BioShop (Canada). All reagents were used as received. The buffer used in all experiments was 10 mM Tris-HCl pH 7.4, 100 mM NaCl.

Large

unilamellar

vesicles

(LUVs)

preparation.

Lipid

dissolved

in

chloroform/methanol, 2:1 (v/v) was deposited as a film on the wall of a glass test tube by solvent evaporation with nitrogen. Final traces of solvent were removed for 2–3 h in a vacuum chamber attached to a liquid nitrogen trap. The lipid films were then suspended in the appropriate buffer by vortexing at room temperature to form MLVs (multilamellar vesicles). In order to prepare large unilamellar vesicles (LUVs), MLVs were further processed with 11 passages through two stacked polycarbonate membranes (100 nm pore size; Nucleopore Filtration Products, Pleasanton, CA) in a barrel extruder (Lipex Biomembranes, Canada), at a


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temperature above the phase transition of the lipid. LUVs were kept at 4 ºC and used within days of preparation.

Determination of phospholipid concentration. Total phospholipid concentration was determined by measuring the amount of inorganic phosphate released after digestion by the method of Ames32.

Single-lipid LUVs-MβCD reaction equilibrium. The solubilization of LUVs prepared with structurally different lipids by MβCD was studied by titrating LUVs to a final concentration ranging from 0.5 to 2 mM into a 60 mM MβCD solution. In a following experiment, MβCD-lipid complex was prepared based on the solubilization assay and a dilution of these complexes into buffer was made to evaluate the reverse reaction. The size of particles in solution was evaluated by DLS, while the thermodynamic parameters of the reactions were evaluated by ITC.

Dynamic light scattering (DLS). The hydrodynamic size of the particles present in the solution was determined by DLS measurements using a Zeta sizer nano S (Malvern, UK). The measurements were done at the temperature employed in the ITC experiment.

Isothermal titration calorimetry (ITC). LUVs solubilization – ITC experiments have been performed as previously described by Anderson and collaborators28. Briefly, the heat flow resulting from the solubilization of lipid vesicles by MβCD solution was measured using a high-


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sensitivity Nano-ITC low volume instrument (cell effective volume 170 µL; TA Instruments – New Castle, DE). Prior to use, solutions were degassed under vacuum to eliminate air bubbles and dissolved gas. MβCD was placed in the cell at a concentration of 60 mM and LUVs of the desired composition were placed in the syringe at a concentration between 2.5-10 mM. 0.71 μL injections were made every 300 s. Experiments were conducted at several different temperatures. Each injection produced a heat of reaction which was determined by integration of the heat flow tracings. The heat of dilution was determined in control experiments by injecting buffer into the MβCD solution. The data analysis was made according to the thermodynamic model of Anderson and collaborators28. The reader is referred to reference 28 for the full description of the model. Briefly, the titration of lipid vesicles into a concentrated solution of MβCD leads to a constant net heat of reaction per injection up to a point at which further addition of lipid to the MβCD solution produces a marked decrease in the net heat of reaction. The lipid:MβCD molar ratio at this point, the so-called ‘breakpoint’, is RBreakpoint. Right-angle light scattering28 and DLS measurements (present work) showed that at the plateau region lipid vesicles are solubilized by MβCD. Since the critical bilayer concentration (c.b.c.) for diacylphospholipids is known to be very small (< nM)1 it is reasonable to assume that at the plateau region all heat produced in the sample cell is due to the formation of MβCD-lipid complexes. Hence, in the plateau region:

where QObs is the corrected heat of reaction and ∆H0 is the enthalpy of MβCD-lipid complex formation.


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Continuing the titration of lipid vesicles after the ‘breakpoint’ does not lead to further vesicle solubilization as indicated by right-angle light scattering28 and DLS measurements (present work). At the ‘breakpoint’ vesicles start to coexist with free MβCD and MβCD-lipid complexes, while after it the concentration of lipid vesicles start increasing despite the fact that the concentration of free MβCD and MβCD-lipid complexes is hold essentially constant. Thus, the boundary condition for these two cases is represented by the boundary equation (below):

where [MβCD]0, n, [L]0, and KB are, respectively, the total concentration of MβCD, the MβCD:lipid stoichiometry, the total concentration of lipid, and the association constant. Rearrangement of the above equation leads to the equation used to calculate the association constant (KB) for the lipid:MβCD complex formation:

In the present study the values of n is assumed constant; although, it is known to exist a distribution of n values. Anderson and collaborators28 had found an average value for n equal to 4 for the interaction between MβCD and POPC, which led them to propose that there are two MβCD per POPC acyl chain. This was consistent with the length of the acyl chains and the height of MβCD cavity. Here, the average length of the acyl chains used were all similar to POPC (ca. 16.5 Å). Hence, we have assumed a fixed value of n = 4 for diacylphospholipids, while the stoichiometry for the interaction with MLCL, which bears three acyl chains, was fixed at 6. Finally, with the measured ∆H0 and KB we could then calculate the other thermodynamic parameters.


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Dissociation of MβCD-Lipid complexes – The heat flow resulting from the dilution of MβCD-lipid complexes was measured using the Nano-ITC. Prior to use, solutions were degassed under vacuum to eliminate air bubbles and dissolved gas and their hydrodynamic size were measured. Buffer was placed in the cell and MβCD-lipid complex was placed in the syringe. 2.5 μL injections were made every 300 s. Experiments were conducted at 303 K. Control experiments with the titration of MβCD into buffer were also conducted.


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Results Impact of phospholipid headgroup on MβCD-induced lipid vesicle solubilization The interaction of LUVs with a concentrated solution of MβCD was investigated using DLS and ITC measurements. Initially we measured the interaction between MβCD and LUVs composed of phospholipids bearing the same acyl chain composition at the sn-1 (palmitoyl, 16:0) and sn-2 (oleoyl, 18:1) positions, the only difference being their polar headgroup. The headgroups studied were PC, PS, and phosphatidylglycerol (PG). Figure 1 presents the size distribution of a 60 mM MβCD solution in the absence and presence of increasing concentrations of LUVs at 303 K. A control of LUVs alone is also presented. The size distribution of LUVs alone showed a hydrodynamic radius with a peak around 48 nm for PC, PS, and PG LUVs. This was expected since the LUVs were made using 100 nm (diameter) polycarbonate membranes. The MβCD solution in the absence of lipids showed a hydrodynamic radius with a peak around 0.8 nm, in agreement with the presence of MβCD monomers in solution2. For all three lipids, addition of LUVs into the MβCD solution up to a critical lipid:MβCD molar ratio led to a size distribution with the peak centered at around 1 nm, which is the mark of vesicle solubilization. Above this critical lipid:MβCD molar ratio, no more solubilization was observed and the hydrodynamic radius peak shifted to around 50 nm (Figure 1). These results indicate the solubilization of all LUVs studied at low lipid:MβCD molar ratios; in agreement with previous results in


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the literature

28,30

. Furthermore, due to the known low-water solubility of these

lipids (< nM), the results further suggest the formation of MβCD-lipid complexes under those conditions, since lipids alone would not be soluble at those concentrations (the concentration range used in these experiments was 10 to 1,250 µM)1. Although the behavior according to the DLS measurements are similar at low lipid:MβCD ratio, the ratio at which large aggregates start appearing is different for different lipids, suggesting a difference in the degree of interaction between MβCD and each of these lipids. PC and PS showed the presence of large aggregates when the lipid:MβCD ratio was at 0.008; while for PG the ratio was 5fold higher at 0.040. Thus, the result suggests a role for the lipid headgroup in MβCD-lipid interactions. The interaction of MβCD with these lipids was also studied by employing ITC. The heat flow traces measured for the titration of LUVs into a MβCD solution at 303 K are presented in Figure 2. In all cases, heat flows presented both endothermic and exothermic peaks. For PC and PG, titrations started with the presence of endothermic peaks of roughly constant heights up to a lipid:MβCD molar ratio where they started decreasing and eventually become exothermic, as observed by Anderson and collaborators28 (Figure 2A and 2C). For PS the trend was slightly different with a gradual decrease in the endothermic peak height with every injection until it became exothermic (Figure 2B). According to our DLS measurements and in agreement with Anderson and collaborators28, the endothermic peaks are ascribed to LUVs solubilization in the sample cell in the presence of excess MβCD; whereas the exothermic peaks are due to the dilution of MβCD in the sample cell (corroborated by buffer into MβCD titration, not 15 ACS Paragon Plus Environment

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shown). Correction for dilution may thus be achieved by subtracting the exothermic component. In the present work we corrected for dilution by subtracting the average value of the dilution heats (comparison with a injection by injection subtraction yielded similar results). The net heat per injection is presented in Figure 3. The titration curve for PC and PG presented an endothermic plateau region which corresponds to the enthalpy of LUVs solubilization (Figure 3). The enthalpy of PC and PG vesicles solubilization was, respectively, 11.2 and 10.3 kcal.mol-1 at 303 K (Table 1). Once a particular lipid:MβCD molar ratio is achieved there is a rapid drop in the heat released until it reaches zero (after baseline correction). This particular molar ratio is the so-called ‘breakpoint’28. The ´breakpoint´ values for PC and PG at 303 K were 0.008 and 0.030 and they are in reasonable agreement with the molar ratio where large aggregates start to be observed in the DLS measurements (see above). These results corroborate the finding that the phospholipid headgroup plays a role in this interaction as suggested by DLS measurements. The titration curve for PS was different from the other two with a lack in the endothermic plateau and a less evident ´breakpoint´ (Figure 3). However, based on DLS measurements the ‘breakpoint’ should be at a molar ratio similar to that observed for PC. Using the thermodynamic model previous described by Anderson and collaborators28, which is based on the coexistence of membranes and lipid-bound MβCD, the affinity of MβCD for these phospholipids bearing different headgroups was evaluated by ITC at three different temperatures and is summarized in Table 1. The quantification of the affinity between MβCD and PS was not evaluated 16 ACS Paragon Plus Environment

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because of the difficulty in the clear identification of the so-called ‘breakpoint’ and, therefore, it is not presented. We also note that unlike other lipids used, PS forms smaller particles at a POPS/MβCD ratio of 0.003 (see inset in Figure 1C). Thus in at least two respects POPS behaves differently than the other lipids, for reasons we cannot yet explain. The data showed that MβCD has on average ca. 4-fold higher affinity for PG in comparison to PC. In addition, as the temperature is increased the affinity for the lipids is also increased and the enthalpy decreases, in agreement with the literature (Table 1)16,25,28,30. The thermodynamic parameters obtained from those experiments are presented in Table 1 which shows that the MβCD-phospholipid interaction is entropically-driven. Moreover, the heat capacity change (∆CP) for PC and PG was – 300 and – 280 cal.mol-1.K-1, respectively (Table 1).

Impact of phospholipid backbone on MβCD-induced lipid vesicle solubilization In a subsequent step to characterize the interaction between phospholipids and MβCD a study of the interaction between MβCD and sphingomyelin (SM) was carried out. The headgroup structure of SM is identical to that of PC (Figure 4A). but the former has a sphingosine backbone while the later has a glycerol backbone. As a result, one of the differences is that in SM the acyl chain is linked by an amide rather than an ester bond, giving SM more H-bonding capabilities over PC. The SM used in this study was the one found in chicken egg, in which the predominant acyl chain is palmitoyl (16:0). All the experiments were conducted


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above the melting transition temperature of the lipids in order to have all lipids in the liquid crystalline phase (ca. 314 K for egg SM33). The titration of SM LUVs (63 nm hydrodynamic radius obtained from DLS measurements) into a concentrated MβCD solution led to the solubilization of the lipid vesicles as indicated by a size distribution peak centered at 1 nm of hydrodynamic radius (not shown). As observed for the other phospholipids studied this result suggested the presence of SM-MβCD complexes. Increasing the concentration of SM added to the sample led to a shift to large vaues in the size distribution peak after a particular lipid:MβCD molar ratio, ca. 0.015 at 323 K, suggesting the coexistence of lipid-bound MβCD and lipid vesicles in agreement with the previous results and literature16,28.. The solubilization of SM LUVs into a 60 mM MβCD solution was also evaluated by ITC. The heat flow trace was similar to those found for PC and PG with the presence of both endothermic and exothermic peaks representing vesicle solubilization and MβCD dilution, respectively (Figure 4B). As for those lipids the titration curve for SM into MβCD initially showed an endothermic plateau (Figure 4C). The measured ‘breakpoint’ was in good agreement with the DLS measurements (0.020, Table 1). However, contrary to the solubilization of PC and PG LUVs, the solubilization of SM LUVs exhibited a gradual decrease of the heat after the ‘breakpoint’ towards zero, in comparison to the sharp decrease observed for PC and PG. The measured affinity also increases and the enthalpy decreases with increasing the temperature (Table 1). However, when comparing PC and SM (after interpolating the data to correspond to the same temperature) the affinity of MβCD with SM is around 2-fold lower than that with PC and the enthalpy of vesicle 18 ACS Paragon Plus Environment

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solubilization was around 125% higher, evincing the role played by the phospholipid backbone on this interaction and in agreement with the literature16. This result would also be in accord with greater lipid-lipid interaction with SM compared with PC, possibly due to the H-bond capabilities of the amide group. The results showed that MβCD-SM interaction is also entropically-driven and the ∆CP obtained was – 320 cal.mol-1.K-1.

The interaction of MβCD with LUVs composed of CL and MLCL Cardiolipin (CL) is a relevant physiological phospholipid. CL is a dimeric lipid, which consists of two phosphatidyl moieties linked by a glycerol bridge and bears four acyl chains (Figure 5A). We studied the interaction of MβCD with LUVs individually prepared with two molecular species of CL (Figure 5A): (i) a synthetic form (TOCL), which bears four oleoyl acyl chains (18:1); (ii) a natural form extracted from beef heart (BHCL). Since BHCL was extracted from a natural membrane there is a distribution of acyl chains present. However, the predominant acyl chain present in BHCL is linoleoyl (18:2). In addition, we also evaluated the interaction of MβCD with a lysocardiolipin (iii) monolysocardiolipin (MLCL). MLCL is formed by the deacylation of CL and, therefore, presents three acyl chains. The MLCL used was also extracted from a natural membrane and the predominant acyl chain is linoleoyl (Figure 5A).. Figure 5B presents the titration curves (net heat) regarding the titration of LUVs composed of TOCL, BHCL, and MLCL in a 60 mM MβCD solution at 318 K. The integrated heats for TOCL and BHCL showed no heat of interaction taking place in the sample cell even at high temperatures.


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On the other hand, the titration of MLCL LUVs in a concentrated solution of MβCD clearly showed that MβCD is capable of solubilizing LUVs composed of this lipid at 318 K. According to Anderson and collegues28, the MβCD:lipid stoichiometry is on average 4fordiacylphospholipids bearing acyl chains with an average length of 16.5 A; i.e., 2 MβCD per acyl chain. Thus, it was assumed a fixed stoichiometry of 6:1 MβCD:MLCL in order to calculate the affinity of the interaction and other thermodynamic parameters (Table 1). The measured enthalpy was 5.6 kcal.mol-1 and as for the other phospholipids, the interaction is entropically-driven.

Reaction equilibrium The forward reaction when titrating LUVs in a concentrated MβCD solution results in vesicle solubilization up to a point where vesicles start to coexist with lipid-bound MβCD. If these two species are in equilibrium with each other, the reverse reaction should be lipid-bound MβCD yielding lipid aggregates (vesiclelike) and free MβCD. An evaluation of whether the solubilization represents an equilibrium process was assessed by preparing lipid-bound MβCD and titrating this into buffer. As an example PG was used, since the concentration range possible to prepare the complex was higher, making easier the dilution experiment. The particle size was determined by DLS and the thermodynamic parameters were determined by ITC. The MβCD-PG complex was prepared at the molar ratio at the ‘breakpoint’. The size distribution showed that at those conditions the sample contained 20 ACS Paragon Plus Environment

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particles with a hydrodynamic radius peak centered around 1 nm, corroborating the presence of MβCD-PG complexes (Figure 6A). In a following step the complex was diluted into buffer leading to the appearance of large aggregates. As subsequent dilutions were done, the amount of large particles increased; after 1,000-fold dilution two populations of large aggregates stabilized, with particles having on average 600 and 2,600 nm hydrodynamic radii. Both the size distribution of MβCD-PG complex and the two population of large aggregates did not change during the course of 1 hour (not shown), consistent with the hypothesis that the reaction was in equilibrium. It is interesting to note that in order to produce large lipid aggregates upon MβCD-PG complex dissociation, lipids would need first to dissociate from the complex with MβCD in aqueous solution, after which the free lipid would form large aggregates. We suggest that an equilibrium between free and MβCD-bound lipid is initially present, but once the free lipid aggregates the process becomes irreversible. Thus although the overall process resulting in lipid aggegrates is irreversible, we suggest that there is an initial equilibrium between free and MβCD-bound lipid that is reversible and can be treated thermodynamically. It is common to use an analogous scheme for the thermal denaturation of many proteins.34,35 This process is often irreversible, resulting in a protein aggregate that does not rapid re-equilibrate with a folded protein. However, the irreversible aggregation is suggested to be preceded by a reversible unfolding equilibrium that can be treated thermodynamically. This reversible unfolding equilibrium proposed in the thermal denaturation of proteins is analogous to the reversible binding equilibrium for binding between MβCD and lipid that we propose in this work..


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The dilution of the MβCD-PG complex as measured by ITC is presented in Figure 6B. The heat flow presented exothermic peaks as expected for the reverse reaction (not shown). However, the integrated heats, when the dilution of MβCD is taken into account, show essentially no observed heat of reaction (Figure 6B). As noted previous by Anderson and collaborators28 the dilution of MβCD is highly exothermic and this might contribute to mask the heats of the reverse reaction. In addition, the likely small amount of these large lipid aggregates formed probably does not produce enough heat to be observed in those conditions. For instance, if one assumes: (i) that the large aggregates are unilamellar vesicle bilayers, (ii) the final lipid concentration after 1,000 fold dilution would be 600 nM with around 70% having 600 nm and 30% having 2,600 nm (based on the size distribution) and (iii) an average area of 0.7 nm2 per lipid; the calculated concentration of these large lipid aggregates formed would be 20 and 0.5 nM.


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Discussion In this study, the interaction between structurally different phospholipids and methyl-β-cyclodextrin (MβCD) was assessed. The evaluation was conducted by titrating LUVs into a concentrated MβCD solution. The particle size distribution and thermodynamic parameters resulting from the interaction were characterized by DLS and ITC, respectively. MβCD has often been used as a lipid carrier to study how the removal or addition of a particular lipid affect the membrane properties including modulation of the function of a membrane protein, lipid traffic between different compartments, lipid metabolism, as well as the engineering of model membranes bearing lipid asymmetry5-10. However, the characterization of the interaction between MβCD and structurally different phospholipids was lacking. The use of synthetic phospholipids showed that lipid headgroup and backbone play a role in the interaction with MβCD. The results showed that the phospholipid headgroup and backbone impact on the interaction with MβCD. Huang and London30 showed that the concentration of MβCD needed to solubilize POPG small unilamellar vesicles (SUVs) is around 33% less than that needed to solubilize POPC and POPS SUVs. This result is in agreement with results presented here, where it was shown that MβCD interacts around 4-fold stronger with POPG than POPC (and, probably, POPS). While the entropy of MβCD and PG interaction is similar to that obtained for MβCD and PC interaction, the enthalpy is different (lower endothermic enthalpy for PG in comparison to PC). At the buffer conditions used here PG bears a negative charge, while PC is zwitterionic. Moreover, PG has two –OH groups on the glycerol headgroup, which could act as hydrogen bonding donors and acceptors36. 23 ACS Paragon Plus Environment

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Whether it is the negative charge and/or the hydrogen bonding capability that favors the interaction between PG and MβCD in comparison to PC is not clear at present (see below). However, since the MβCD cavity is slightly apolar and bears an internal volume of 262 Å3 it is highly unlikely that it will bind the lipids by the bulky and polar headgroups (volume is 319 and 304 A3 for PC and PG headgroups, respectively1). When considering PC and PG molecules with the same acyl chain composition, the gel to liquid crystalline phase transition occurs at the same temperature (using physiological pH and buffer conditions), suggesting a similar packing of the acyl chains for those lipids in the bilayer37. The presence of negative charge on PG leads to electrostatic repulsion at the headgroup region, which would act to facilitate the extraction of the lipid by MβCD. Moreover, the MβCD-PG complex would be more stable in solution due to the electrostatic repulsion among them. In addition, the presence of hydrogen bonds between lipid and MβCD would also stabilize the complex favoring extraction. Hence, the role played by the headgroup might act either to destabilize the bilayer facilitating MβCD complexation or to stabilize the MβCD-lipid complex. This interpretation is in accord with our finding that the difference in thermodynamics between POPG and POPC is in the enthalpy term, the entropy being quite similar at the same temperature (Table 1). Experiments with RBC and brain endothelial cells found a preferred interaction with SM over PC for α- and β-CD22,24. Opposed to these findings, it was found in the present work, a less preferred interaction of MβCD with SM in comparison to PC, which is in agreement with the report of Tsamaloukas and collaborators16. The differences found between model and biological systems 24 ACS Paragon Plus Environment

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might be due to the large heterogeneity of the latter, including the presence of proteins and cholesterol, as well as to the different type of CD used in this and other studies22,24. As per the findings reported here, the use of model systems comprised of synthetic lipids showed that lipid headgroup and backbone play a role in its interaction with MβCD. Both SM and PC have an identical choline headgroup with two hydrocarbon chains, roughly of the same size (those used in this study), the difference being mainly their backbone, a sphingosine base for SM and glycerol for PC. The backbone differences mainly affect the membrane-water interfacial region with a stronger capability of inter-molecular hydrogen bonding for SM, since it has an amide instead of an ester group as well as having an additional OH group, which favors SM-SM interactions33. Since a stronger intermolecular hydrogen bonding capability supposedly would also favor the interaction with MβCD, it seems tempting to speculate that the lower affinity observed for SM in comparison to PC is due to a stronger SM-SM H-bonding interaction compared with the SM-MβCD H-bonding interactions. The majority of biological phospholipids bear two acyl chains, but there are a few exceptions, with cardiolipin (CL) being one example. This phospholipid bears four acyl chains and has very important physiological roles38. The results presented here clearly showed that MβCD does not interact with CL. Although, MβCD-CL interaction could not be observed here, there is evidence that MβCD could exchange CL between two populations of model membranes supporting the existence of interaction between these molecules39. It should be pointed out that the presence of four acyl chains on CL could lead to higher activation energy to extract the lipid from the bilayer and the lack of interaction between MβCD and CL 25 ACS Paragon Plus Environment

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observed here could be due to kinetic reasons. The increase in the number of acyl chains is analogous to the increase in the length of the acyl chain. For the later it is known that increasing the length of the acyl chain leads to an increase in the activation energy of lipid dissociation from the bilayer1. In addition, a decrease in the interaction between γ-CD and phospholipids as the acyl chain length is increased has been reported25. Likewise, the stoichiometry of interaction between MβCD and these lipids would be different. In contrast to CL, we observed solubilization of LUVs prepared with MLCL at high temperatures. MLCL is a lysolipid that forms bilayers and can readily be incorporated into bilayers of PC due to the fact it bears three acyl chains41. in vivo only individuals with Barth Syndrome, a disease caused by mutation of tafazzin42, accumulate significant quantities of MLCL43,44. MLCL plays an important role in destabilizing oxidative phosphorylation super-complexes45. In addition, MLCL binds specifically to the BCL-2 protein Bid; however, in order to do so it must translocate from the inner to the outer mitochondrial membrane46. The transfer of lipids between these membranes are facilitaded by membrane contact sites and/or proteins47,48. Hence, the ease of extraction of MLCL by MβCD, shown in the present work, suggests a mechanism whereby MLCL can be exchanged between membranes relatively more easily than fully acylated cardiolipin. In all cases the interaction of MβCD with phospholipids was entropicallydriven and presented large negative ∆CP values. According to Sturtevant49, large negative ∆CP values are characteristic of the so called “hydrophobic shielding effect”; i.e., the transfer of a hydrophobic surface from water to an apolar environment. When applying these ∆CP values to the equation given by Spolar et 26 ACS Paragon Plus Environment

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al.50, a surface area of ca. 10 nm2 is obtained. The apolar surface area (ASA) of MβCD cavity is ca. 1.55 nm2, assuming it as a cylinder with diameter of 6.3 Å and a height of 7.9 Å. Based on the MβCD:acyl chain stoichiometry of 2:1 for acyl chains on average 16.5 Å long, there would be in total four MβCD molecules per diacyl phospholipid, which gives a total ASA of 6.2 nm2. Thus, the release of the MβCD cavity-bound water molecules due to the insertion of the acyl chains leading to complex formation would explain only in part (ca. 60%) the observed ∆CP values. That is, MβCD-induced LUVs solubilization and, consequently, MβCD lipid interaction, is driven in part by a hydrophobic effect. The identification of the other factors that could be contributing for the positive entropy change is speculative. However, one would not expect a large change in the hydration of the lipid headgroup in the bilayer when compared to the lipid in CD complex; although it is not likely that the change in headgroup hydration in the two environments is zero, it might not be large. Another factor that could contribute to the entropy change is the decreased motional order of the phospholipid molecule when it is in the CD complex compared with a more restrained bilayer. Finally, there is acyl chain flexibility. The motional freedom of the acyl chains is likely to be different in the CD complex in comparison to the bilayer. This would also affect the entropy, but the magnitude or even sign of the change is difficult to accurately predict. Also, the extraction of lipids from the bilayer might increase the acyl chain flexibility of the remaining lipids in the bilayer. Interaction of phospholipids with MβCD has some particular properties. Of the many types of CD, this is the only one that leads to vesicle solubilization at high concentrations; although all the other types of CD have been shown to bind 27 ACS Paragon Plus Environment

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and exchange lipids among model membranes27-30. Furthermore, although partition to the bilayer has not been shown for this CD, surface tension measurements indicate that this CD is surface active to some extent; contrary to the other CD, which do not show such activity51-54. Hence, it seems reasonable that MβCD could bind phospholipids both at the bulk water and at the bilayer. In addition, the fact that the MβCD will interact only with the outer monolayer of the membrane could also lead to bilayer instability as a result of a mass imbalance between the lipids of the opposing monolayers. This could lead to fast depletion of outer leaflet lipids, possibly leading to the exposure of the inner leaflet lipid acyl chains to bulk water. This would raise significantly the energy of the system and, consequently, favor the dissolution of these lipid vesicles in favor of lipid-bound MβCD in order to decrease the free energy of the system. This phenomenon would also be entropically-driven and in agreement with the results presented here. In conclusion, the solubilization of LUVs has been studied in order to understand the role of structurally different parts of phospholipids to the interaction with MβCD. The results showed that the lipid headgroup and backbone play a role in the interaction with MβCD. The complexation of lipids by MβCD is likely favored by a destabilization of the lipid in the bilayer and/or a stabilization of the MβCDlipid complex. The thermodynamic analysis showed that vesicle solubilization is entropically-driven, suggesting a release of water, probably from the CD core, when a lipid is transferred from a bilayer to CD. In addition, there would be increased flexibility of the phospholipid acyl chains in the MβCD complex than in the bilayer. 28 ACS Paragon Plus Environment

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Acknowledgments We would like to acknowledge Dr. Alba Guarne (McMaster University) for the use of the zetasizer instrument and Dr. Philippe Dumas (University of Strasbourg) for critically reading the manuscript. R.M.E. acknowledges NSERC (grant 9848).


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Author Contributions J.C.B.Jr. designed experiments, conducted experiments, analyzed data, and wrote the paper; Y.H. conducted experiments and analyzed data; R.M.E. provided laboratory facilities, designed experiments, analyzed data, and wrote the paper.


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Figure legends

Fig. 1 – Effect of lipid headgroup on vesicle solubilization by concentrated MβCD. (A) Chemical structure of POPC with its headgroup outlined in red. For PS and PG only headgroup chemical structures are presented. Size distribution of 60 mM MβCD in absence and presence of increasing concentrations of LUVs composed of (B) POPC, (C) POPS, and (D) POPG. The size distribution of the LUVs alone is also presented. Volume % is the relative proportion of each component of the size distribution based on their volume. This was obtained by applying the Mie theory to the scatter intensity. Inserts are an expansion of the xaxis to better visualize the overlapped curves between 0 and 2 nm in main panels. Experiments performed at 303 K for all phospholipids.

Fig. 2 – Calorimetric measurements of the role of lipid headgroup on MβCDinduced lipid vesicles solubilization. Heat flow trace of the titration of LUVs composed of (A) POPC, (B) POPS, and (C) POPG into 60 mM MβCD. [Phospholipid] = 7.5 mM. Each peak corresponds to the injection of 0.71 µL of vesicle suspension into MβCD in the sample cell. Titrations were performed at 303 K.

Fig. 3 – Calorimetric measurements of the role of lipid headgroup on MβCDinduced lipid vesicles solubilization. Corrected heat of reaction (QObs) as a function of the phospholipid:MβCD molar ratio. LUVs composed of POPC, POPS, or POPG were injected into a 60 mM MβCD solution and the heat of reaction was corrected by the dilution of MβCD. Titrations were performed at 303 K.

Fig. 4 – Calorimetric measurements of the role of lipid backbone on MβCDinduced lipid vesicles solubilization. (A) Chemical structure of POPC and SM with their backbones outlined in olive. (B) Heat flow trace and (C) corrected heat of reaction (QObs) of the titration of LUVs composed of egg SM into 60 mM MβCD. [Phospholipid] = 10 mM. Each peak corresponds to the injection of 0.71 µL of vesicle suspension into MβCD in the sample cell. Titrations were performed at 323 K. 31 ACS Paragon Plus Environment

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Fig. 5 – Calorimetric measurements of the role of lipid number of acyl chains on MβCD-induced lipid vesicles solubilization. (A) Chemical structure of TOCL, BHCL, and MLCL. (B) Corrected heat of reaction (QObs) per mole of TOCL, BHCL, and MLCL injected as a function of the phospholipid:MβCD molar ratio.

Fig. 6 – Dilution of MβCD-POPG complexes. (A) Size distribution of 60 mM MβCD + 600 µM POPG (MβCD-POPG complexes) at the ‘breakpoint’ and after serial dilution of the complex into buffer. Volume % is the relative proportion of each component of the size distribution based on their volume. This was obtained by applying the Mie theory to the scatter intensity. (B) Corrected heat (QOBS) as a function of the fold dilution of MβCD-POPG complexes into buffer (corrected by MβCD dilution). Titrations were performed at 303 K.

TOC – Cartoon depicting a suggested reaction scheme. LUVs exist in equilibrium with free lipids (L) in aqueous solution with the equilibrium favoring the former. In the presence of excess MβCD, the CD would interact with both, LUV and free lipid. Upon MβCD-L complex dissociation, large heterogeneous lipid aggregates, probably as bilayers, would be formed, but lipids in these aggregates would be morphologically different, being larger and more heterogeneous, from monodisperse LUVs.


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Table 1 – Thermodynamic parameters of the interaction between different phospholipids and MβCD. Breakpoiont (molar ratio) 0.008

K (M-3) 42

∆G0 (kcal.mol-1)

POPC

Temperature (K) 303

T∆S0 (kcal.mol-1)

-2.2

∆H0 (kcal.mol-1) 11.2

POPC

310

0.016

97

-2.8

8.0

POPC

318

0.026

10.8

188

-3.3

6.7

10.0

POPG

303

0.030

232

-3.3

10.3

13.6

POPG POPG

310

0.034

282

-3.5

6.5

10.0

318

0.046

480

-3.9

6.1

10.0

eSM

323

0.020

129

-3.1

7.0

10.1

eSM

328

0.016

97

-3.0

5.2

8.2

eSM

333

0.022

147

-3.3

3.8

7.1

MLCL

318

0.012

24,162*

-6.4

5.6

12.0

Lipid

13.4

∆CP (cal.mol-1.K-1) -300

-280

-320

-5

* Due to reaction stoichiometry the unit is M .


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References [1] Marsh, D. Handbook of Lipid bilayers. CRC Press 2013, Second edition, FL. [2] Szejtli, J. Introduction and general overview of cyclodextrin chemistry. Chem. Rev. 1998, 98, 1743-1753. [3] Irie, T.; Otagiri, M.; Sunada, M.; Uekama, K.; Ohtani, Y.; Yamada, Y.; Sugiyama Y. Cyclodextrin-induced hemolysis and shape changes of human erythrocytes in vitro. J. Pharmacobiodyn. 1982, 5, 741-744. [4] Ohtani, Y.; Irie, T.; Uekama, K.; Fukunaga, K.; Pitha, J. Differential effects of a-, b- and g-cyclodextrins on human erythrocytes. Eur. J. Biochem. 1989, 186, 17-22. [5] Yancey, P.G.; Rodrigueza, W.V.; Kilsdonk, E.P.C.; Stoudt, G.W.; Johnson, W.J.; Phillips, M.C.; Rothblat, G.H. Cellular cholesterol efflux mediated by cyclodextrins. J. Biol. Chem. 1996, 271, 16026-16034. [6] Atger, V.M.; Moya, M.L.; Stoudt, G.W.; Rodrigueza, W.V.; Phillips, M.C.; Rothblat, G.H. Cyclodextrins as catalysts for the removal of cholesterol from macrophage foam cells. J. Clin. Invest. 1997, 99, 773-780. [7] Tanhuanpää, K.; Somerharju, P. γ-cyclodextrins greatly enhance translocation of hydrophobic fluorescent phospholipids from vesicles to cells in culture. J. Biol. Chem. 1999, 274, 35359-35366. [8] Cheng, H.T.; Megha; London, E. Preparation and properties of asymmetric vesicles that mimic cell membranes: effect upon lipid raft formation and transmembrane helix orientation. J. Biol. Chem. 2009, 284, 6079-6092. [9] Kainu, V.; Hermansson, M.; Somerharju, P. Introduction of phospholipids to cultured cells with cyclodextrin. J. Lipid Res. 2010, 51, 3533-3541. [10] Li, G.; Kim, J.H.; Huang, Z.; Clair, J.R.St.; Brown, D.A.; London, E. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogeneous lipids. Proc. Natl. Acad. Sci. U.S.A. 2016, 113, 14025-14030. [11] Kilsdonk, E.P.C.; Yancey, P.G.; Stoudt, G.W.; Bangerter, F.W.; Johnson, W.J.; Phillips, M.C.; Rothblat, G.H. Cellular cholesterol efflux mediated by cyclodextrins. J. Biol. Chem. 1995, 270, 17250-17256. [12] Ohvo, H.; Slotte, J.P. Cyclodextrin-mediated removal of sterols from monolayers: effects of sterol structure and phospholipids on desorption rate. Biochemistry 1996, 35, 8018-8024. [13] Radhakrishna, A.; McConnell, H.M. Chemical activity of cholesterol in membranes. Biochemistry 2000, 39, 8119-8124.


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[14] Leventis, R.; Silvius, J.R. Use of cyclodextrins to monitor transbilayer movemet and differential lipid affinities of cholesterol. Biophy. J. 2001, 81, 22572267. [15] Niu, S.-L.; Litman, B.J. Determination of membrane cholesterol partition coefficient using a lipid vesicle-cyclodextrin binary system: Effect of phospholipid acyl chain unsaturation and headgroup composition. Biophys. J. 2002, 83, 34083415. [16] Tsamaloukas, A.; Szadkowska, H.; Heerklotz, H. Thermodynamic comparison of the interaction of cholesterol with unsaturated phospholipids and sphingomyelins. Biophys. J. 2006, 90, 4479-4487. [17] Besenicar, M.P.; Bavdek, A.; Kladnik, A.; Macek, P.; Anderluh, G. Kinetics of cholesterol extraction from lipid membranes by methyl-b-cyclodextrin – A surface plasmon resonance approach. Biochim. Biophys. Acta 2008, 1778, 175184. [18] Steck, T.L.; Ye, J.; Lange, Y. Probing red cell membrane cholesterol movement with cyclodextrin. Biophys. J. 2002, 83, 2118-2125. [19] López, C.A.; de Vries, A.H.; Marrink, S.J. Molecular mechanism of cyclodextrin mediated cholesterol extraction. PLoS Comput. Biol. 2011, 7, e1002020. [20] López, C.A.; de Vries, A.H.; Marrink, S.J. Computational microscopy of cyclodextrin mediated cholesterol extraction from lipid model membranes. Sci. Rep. 2013, 3, 2071. [21] Fauvelle, F.; Debouzy, J.C.; Nardin, R.; Gadelle, A. Nuclear magnetic resonance study of a polar headgroup determined α-cyclodextrin-phospholipid association. Bioelectroch. Bioener. 1994, 33, 95-99. [22] Debouzy, J.C.; Fauvelle, F.; Crouozy, S.; Girault, L.; Chapron, Y.; Göschl, M.; Gadelle, A. Mechanisms of α-cyclodextrin induced hemolysis. 2. A study of the factors controlling the association with Serine-, Ethanolamine-, and Cholinephospholipids. J. Pharm. Sci. 1998, 87, 59-66. [23] Yu, Y.-M.; Cai, W.; Shao, X. A simulation on the complexation of cyclodextrins with phospholipids headgroups. J. Incl. Phenom. Macrocycl. Chem. 2006, 56, 225-235. [24] Monnaert, V.; Tilloy, S.; Bricout, H.; Fenart, L.; Cecchelli, R.; Monflier, E. Behavior of alpha-, beta-, and gamma-cyclodextrins and their derivatives on an in vitro model of blood-brain barrier. J. Pharmacol. Exp. Ther. 2004, 310, 745-751. [25] Tanhuanpää, K.; Cheng, K.H.; Anttonen, K.; Virtanen, J.A.; Somerharju, P. Characteristics of pyrene phospholipid/γ-cyclodextrin complex. Biophys. J. 2001, 81, 1501-1510.


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Effect of lipid headgroup on vesicle solubilization by concentrated MβCD. (A) Chemical structure of POPC with its headgroup outlined in red. For PS and PG only headgroup chemical structures are presented. Size distribution of 60 mM MβCD in absence and presence of increasing concentrations of LUVs composed of (B) POPC, (C) POPS, and (D) POPG. The size distribution of the LUVs alone is also presented. Insert is an expansion of the x-axis between 0 and 2 nm. Experiments performed at 303 K for all phospholipids. 254x190mm (96 x 96 DPI)

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Langmuir

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Calorimetric measurements of the role of lipid headgroup on MβCD-induced lipid vesicles solubilization. Heat flow trace of the titration of LUVs composed of (A) POPC, (B) POPS, and (C) POPG into 60 mM MβCD. [Phospholipid] = 7.5 mM. Each peak corresponds to the injection of 0.71 µL of vesicle suspension into MβCD in the sample cell. Titrations were performed at 303 K. 254x190mm (96 x 96 DPI)


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Langmuir

Calorimetric measurements of the role of lipid headgroup on MβCD-induced lipid vesicles solubilization. Corrected heat of reaction (QObs) as a function of the phospholipid:MβCD molar ratio. LUVs composed of POPC, POPS, or POPG were injected into a 60 mM MβCD solution and the heat of reaction was corrected by the dilution of MβCD. Titrations were performed at 303 K. 254x190mm (96 x 96 DPI)


Langmuir

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Calorimetric measurements of the role of lipid backbone on MβCD-induced lipid vesicles solubilization. (A) Chemical structure of POPC and SM with their backbones outlined in olive. (B) Heat flow trace and (C) corrected heat of reaction (QObs) of the titration of LUVs composed of egg SM into 60 mM MβCD. [Phospholipid] = 10 mM. Each peak corresponds to the injection of 0.71 µL of vesicle suspension into MβCD in the sample cell. Titrations were performed at 323 K. 254x190mm (96 x 96 DPI)


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Langmuir

Calorimetric measurements of the role of lipid number of acyl chains on MβCD-induced lipid vesicles solubilization. (A) Chemical structure of TOCL, BHCL, and MLCL. (B) Corrected heat of reaction (QObs) per mole of TOCL, BHCL, and MLCL injected as a function of the phospholipid:MβCD molar ratio. 254x190mm (96 x 96 DPI)


Langmuir

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Dilution of MβCD-POPG complexes. (A) Size distribution of 60 mM MβCD + 600 µM POPG (MβCD-POPG complexes) at the ‘breakpoint’ and after serial dilution of the complex into buffer. Insert is an expansion of the x-axis between 0 and 2 nm. (B) Corrected heat (QOBS) as a function of the fold dilution of MβCD-POPG complexes into buffer (corrected by MβCD dilution). Titrations were performed at 303 K. 254x190mm (96 x 96 DPI)


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Cartoon depicting a suggested reaction scheme. LUVs exist in equilibrium with free lipids (L) in aqueous solution with the equilibrium favoring the former. In the presence of excess MβCD, the CD would interact with both, LUV and free lipid. Upon MβCD-L complex dissociation, large heterogeneous lipid aggregates, probably as bilayers, would be formed, but lipids in these aggregates would be morphologically different, being larger and more heterogeneous, from monodisperse LUVs. 254x190mm (96 x 96 DPI)


and Richard M. Epand Department of Biochemistry and Biomedical

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