Effect of Bilayer Partitioning of Curcumin on the Adsorption and

In addition, it is further proposed that at low C/L ratio Curcumin binds to the surface of the ... Richard J. Alsop , Alexander Dhaliwal , and Maikel ...
0 downloads 0 Views 1MB Size
Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Effect of bilayer partitioning of Curcumin on the adsorption and transport of a cationic dye across POPG liposomes probed by Second Harmonic Spectroscopy Gopal K Varshney, Srinivas R Kintali, Pradeep Kumar Gupta, and Kaustuv Das Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02797 • Publication Date (Web): 16 Sep 2016 Downloaded from http://pubs.acs.org on September 20, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Effect of bilayer partitioning of Curcumin on the adsorption and transport of a cationic dye across POPG liposomes probed by Second Harmonic Spectroscopy

G. K. Varshneya,b, S. R. Kintalia,b, P. K. Guptaa,b and K. Dasa,b* a

Optical Spectroscopy & Diagnostic Lab

Laser Bio-Medical Applications Section Raja Ramanna Center for Advanced Technology Indore, M.P., India 452013

b

Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400094, India

*Corresponding author Email: [email protected]; [email protected]

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The effect of Curcumin partitioning into the bilayer during the adsorption and transport of a cationic dye, LDS, across a negatively charged POPG bilayer was investigated by the interface selective Second Harmonic (SH) spectroscopic technique. The intensity of SH electric field (E2ω) arising due to LDS adsorbed on the outer bilayer of the POPG liposome was observed to increase instantaneously (< 1 second) following addition of Curcumin. The fractional increase in the SH electric field (Ef2ω) and the bilayer transport rates (kT) of LDS was studied with respect to the pH of the solution and also with the Curcumin content in the lipid bilayer. Results obtained indicate that compared to the anionic form of the drug, its neutral form is more conducive of increasing the Ef2ω of LDS. With increasing Curcumin content in the lipid bilayer two distinct regimes could be observed in terms of Ef2ω and kT values of LDS. For Curcumin:Lipid (C/L) ratio ≤ 0.02 the Ef2ω of LDS increases rapidly while kT remains unchanged; and for C/L ratio ≥ 0.02; the Ef2ω values remains more or less constant while there is a significant (~40 times) increase followed by a modest increase in the kT values of LDS. The observed results support an earlier two-state binding model of Curcumin with the POPG bilayer. In addition, it is further proposed that at low C/L ratio Curcumin binds to the surface of the bilayer replacing the counter ions (Na+) bound to the lipid head groups which changes the bilayer surface charge density thereby causing more LDS cations to adsorb on the bilayer surface. At high C/L ratio Curcumin intercalates within the hydrophobic domain of the bilayer altering its hydrophobicity inducing enhanced transport of the LDS cation. Results presented in this work provide further insights about how Curcumin alters bilayer properties when it partitions from the aqueous to the bilayer phase.

ACS Paragon Plus Environment

Page 2 of 29

Page 3 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Introduction The lipid bilayer of cell membranes is a natural binding site for several types of amphipathic molecules, like proteins, drugs, detergents etc. It is important to study the binding of a drug to a membrane because drug-membrane binding can cause changes in the bilayer properties and thus affect the functions of embedded proteins1-2. Amphipathic drugs can bind to the membrane-water interface or intercalate into the nonpolar chain region of the membrane. One such example of an amphipathic drug is Curcumin (Scheme 1) which shows a wide spectrum of pharmacological effects3-6. The results of recent studies suggest that Curcumin can regulate various structurallyunrelated membrane proteins by changing the properties of the surrounding lipid bilayer rather than directly binding to proteins7-8. Consequently several studies have been performed in model membrane systems to understand how Curcumin affects the bilayer properties by different biophysical techniques9-14. However majority of these studies9,

11-13

were carried out under an

equilibrium condition, i.e. after Curcumin was bound to the membrane. A few studies10, 14 where direct observation of membrane properties were carried out while Curcumin is added revealed some interesting aspects of the membrane binding properties of this amphipathic drug. It has been observed that surface area of individual giant unilamellar vesicles (GUVs) of DOPG lipid increases upon binding of Curcumin from solution by the micropipette aspiration technique10. The rate of increase is faster up to a Curcumin:Lipid molar ratio (C/L) of 0.02 and becomes slower there after. Combining this with results obtained from earlier studies on X-ray lamellar diffraction measurements of the thickness of DOPG bilayers as a function of Curcumin content9 a two-state model was proposed for Curcumin binding to DOPG bilayer. Curcumin initially binds to the aqueous bilayer interface and then at higher concentrations gradually partition to a state where it is inserted into the hydrocarbon region of the bilayer10. In another

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 29

study using total internal reflection fluorescence microscopic technique, it was observed that Curcumin induces fusion of lipid raft domains at low concentrations by changing the boundary between the ordered and disordered phases14. Second Harominc (SH) spectroscopic technique has the ability to probe the interfacial region exclusively due to symmetry considerations15. Generation of SH light requires that the medium must lack a center of symmetry which is applicable at the lipid bilayer interface. When dye molecules gets adsorbed on the outer surface of a bilayer their SH field adds up coherently to generate an instantaneous increase in the SH signal when the sizes of the liposomes are of the order of the wavelength of the fundamental (~ 800 nm) radiation16. As the dye molecules crosses from the outer to the inner bilayer they becomes oppositely oriented due to the symmetry of the bilayer. The SH field generated from these oppositely oriented dye molecules which are separated by the bilayer thickness (~ 5 nm; which is much less than the coherence length of the process) will cancel out as their second harmonic polarizations will be equal but opposite. Therefore by monitoring the temporal dependence of the SH signal, which is proportional to the population difference of the dye molecules adsorbed between the outer and inner surface the transport process can be monitored. However it is pertinent to note that interfacial adsorption of a molecule/ion and its transport across a bilayer can be monitored by the SH technique only if it possesses a reasonable hyperpolarizability value at the excitation wavelength13,

16-19

. We have

earlier monitored the transport of a cationic hemicyanine dye, LDS, across a negatively charged POPG bilayer by the SH spectroscopic technique and observed that when POPG liposomes were incubated with Curcumin the transport of LDS cation across the bilayer was significantly faster13. Since it has been observed that partitioning of Curcumin from solution to a bilayer is accompanied by membrane area expansion and membrane thinning9-11, 14 it would be interesting

ACS Paragon Plus Environment

Page 5 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

to see whether this will affect the interfacial binding of small organic molecules with lipid bilayer. In this work we report how the adsorption and transport of LDS across a POPG bilayer changes when Curcumin is added to the solution and compare our results with previous studies910

.

Materials & Methods LDS ([2-[4-[4-(dimethylamino)phenyl]-1,3-butadienyl]-1-ethylpyridinium monoperchlorate) was obtained from Exciton and used as received. Curcumin was purified before use by methods described earlier20. 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) i.e. POPG lipid from Avanti Polar Lipids was used as received. Unilamellar liposomes (for preparation details, see SI) were suspended in 20 mM phosphate buffer solution. The size and zeta potential of the liposomes (measured by dynamic light scattering technique) at different pH are provided in the supporting information (Table S1). The absorption, emission and fluorescence lifetime of LDS in the presence and absence of POPG liposomes as well as its SH properties under 800 nm femtosecond laser excitation has already been characterized in our previous work13. The SH experimental set up used for this study was identical to that described in our previous reports13, 19. The average laser power used in the experiments was 600 mW and the generated SH light (at 400 ± 2 nm) was detected at right angles with respect to the fundamental (800 nm) by a PMT using single photon counting technique. The SH signal integration time was kept at one second. The sample in the cuvette was constantly stirred during the measurement using a magnetic stirrer and the sample temperature was controlled by a Neslab circulating water chiller.

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 29

The SH experiments were conducted as follows: The SH intensity of LDS (5 µM) in buffer solution was monitored with time. At t = 50 second, 50 micro-liter concentrated POPG solution was added to make the final concentration of POPG as 50 µM. Unless otherwise stated microliter aliquots from a concentrated stock solution of Curcumin were added to the liposome solution at t = 100 second to get the desired Curcumin:Lipid (C/L) ratio. The electric field of the SH signal (E2ω) were obtained from the observed SH signal (I2ω) as16-18:

E2ω (t ) = I ( 2ω ) dye+liposome (t ) − I ( 2ω )background

(1)

where I(2ω)dye+liposome(t) is the SH signal detected at 2ω at time t and I(2ω)background represents the contributions from the SH field generated by the buffer solution alone. After adding POPG liposome SH electric field (E2ω) of LDS will be proportional to the difference in the populations of the LDS molecules located on the outer surface, N0(t), and the inner surface, Ni(t), of the liposomes at a time t as16-18:

E2ω (t ) ∝ [ N0 (t ) − Ni (t )]Eω Eω

(2)

Results: Figure 1 describes the changes in the SH field (E2ω) of LDS after addition of POPG liposomes (at 50 second) for three different conditions: a) Black curve: Without Curcumin. b) Blue curve: POPG liposomes which were earlier incubated with Curcumin (C/L = 0.1) for few minutes.

ACS Paragon Plus Environment

Page 7 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

c) Red Curve: Curcumin (C/L = 0.1) was added 50 second after the addition of POPG liposomes. The instantaneous increase (< 1 second) in the E2ω signal of LDS after addition of POPG liposomes at 50 second time point for all the three curves in Figure 1 is due to the rapid electrostatic adsorption of the positively charged dye on the negative charged outer bilayer surface as has been demonstrated in numerous studies earlier13,

16-19

. After addition of POPG

liposomes E2ω of LDS decreases gradually due to transport of the LDS cation from the outer to inner lipid bilayer (case a; black curve). Consistent with our earlier studies the rate of decrease of the E2ω of LDS gets significantly faster when POPG liposomes contain Curcumin (case b; blue curve). However when Curcumin is added to the solution containing LDS and POPG liposomes, it generates an additional instantaneous increase in the E2ω signal of LDS (case c; red curve). This phenomenon is observed whenever Curcumin is added as shown in Figure S1 in supporting information. In order to see whether this is due to E2ω generated by the Curcumin molecules itself, we have monitored the E2ω signal of POPG liposomes in presence of only Curcumin which is shown as an inset in Figure 1. The time course of E2ω signal coming from a pH 5.0 buffer solution increases marginally after addition of POPG liposomes (added at 50 second) but remains more or less unchanged even after addition of Curcumin (added at 100 second) whose concentration is five times higher than that used in Figure 1b and1c. Therefore the instantaneous increase in the E2ω signal of LDS following addition of Curcumin does not arise due to E2ω of Curcumin. In the presence of POPG liposomes the E2ω signal of LDS at any time is directly proportional to the population difference between the LDS molecules residing at the outer and inner lipid bilayer (Eqn. 2). The additional instantaneous increase in the E2ω signal of LDS indicates that addition of Curcumin affects this population difference. Therefore the

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 29

instantaneous increase of the E2ω signal of LDS, immediately after addition of Curcumin (case c; red curve, Figure 1), can be attributed to an instantaneous increase of the number of LDS molecules adsorbed on the outer bilayer. It would be interesting to see whether this phenomenon is a particular one (for LDS cation only) or a general one (for any organic cations). Therefore we have repeated the same experiment corresponding to curve c of Figure 1, but replaced the LDS cation by either Malachite Green or Auramine-O cation both of which have appreciable SH signals at 800 nm excitation15. Although no instantaneous increase in the E2ω signal of these two cations were observed after addition of Curcumin at room temperature (data not shown), lowering the temperature of the solution (3 0C) indeed resulted in an instantaneous increase in the E2ω signal of these two cations which is shown in Figure 2, along with LDS. These results therefore suggest that addition of Curcumin changes the outer bilayer of the POPG liposomes in such a way so as to induce an increase in the number of cationic molecules adsorbed on the negatively charged outer bilayer. In order to quantify the observed changes in the E2ω signal due to Curcumin, we have chosen LDS as the probe molecule and kept the temperature at 3 0C because as evident from Figure 2, changes in the E2ω signal after addition of Curcumin of LDS molecule is largest among the three cations. Curcumin induced fractional changes in the E2ω signal of LDS can now be quantified as: Ef2ω = ∆E2ω /E02ω

(3)

where ∆E2ω is the maximum observed difference of the SH electric field of LDS before and after addition of Curcumin (Figure 2). Since the E2ω signal of LDS after addition of POPG liposomes remains unchanged (it remains constant for several hundred seconds which is consistent with our earlier report13) before addition of Curcumin it is reasonable to assume (as per Eqn. 1) that the

ACS Paragon Plus Environment

Page 9 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

E2ω signal of LDS (in presence of POPG liposomes at 3 0C) will be directly proportional to the number of LDS molecules adsorbed on the outer bilayer of the liposomes for the initial few hundreds of seconds. Therefore, Curcumin induced fractional increase in the E2ω signal of LDS can be directly correlated to the fractional increase in the number of LDS molecules adsorbed on the outer lipid bilayer. In the following sections we have attempted to investigate Curcumin induced fractional changes of SH electric field (Ef2ω) of LDS under various environmental conditions. Curcumin is an amphiphilic molecule having three acidic hydroxyl groups among which the enolic hydroxyl group is the most reactive having a pKa of ≈ 8.4

21-22

. In order to investigate

whether the ionization of the enolic hydroxyl group should affect the Ef2ω of LDS we did a pH variation study. Figure 3 shows the changes in Ef2ω of LDS with changes in the pH of the medium. As shown in the inset of Figure 3, a change in the pH of the medium (from 5.0 to 8.5) causes Ef2ω of LDS to change significantly. The fractional increase in the SH electric field of LDS at pH 5.0 was observed to be twice (~17%) as much as that observed for pH 8.5 (~8%). Since with increase in pH, the formation of enolic anion of Curcumin is favored it indicates that neutral species of Curcumin is more effective in increasing the Ef2ω of LDS. Therefore in the subsequent studies we have kept the medium pH at 5.0. The changes in the E2ω signal of LDS with changing Curcumin:Lipid (C/L) mole ratio are provided in Figure 4. It is evident from Figure 4 that the transport of the cation remains more or less similar up to a C/L value of 0.02 and starts to become significantly faster thereafter. Transport rate constants of LDS under different C/L mole ratio were obtained by fitting with an exponential decay function as mentioned in our early work13. To extract the decay time constants of the individual curves they were fitted after the Curcumin induced jump in the E2ω signal of

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 29

LDS is completed. Figure 5 shows the changes in Ef2ω and transport rated constant (kT) values of LDS with changing Curcumin:Lipid (C/L) mole ratio. Representative time dependent E2ω signal of LDS at two different C/L ratio (4.0x10-3 to 4.0x10-2) are shown in the inset for a qualitative assessment on the effect of increasing Curcumin concentration. It is interesting to note that with increasing C/L ratio the observed trends of Ef2ω and kT values of LDS, are not similar at lower C/L ratio. While the increase in the Ef2ω of LDS is initially rapid the transport rate of the cation remains quite similar up to a C/L ratio of ≤ 0.02. For C/L ratio being ≥ 0.02, while the Ef2ω values remains more or less constant there is a significant (~40 times) increase followed by a modest increase in the kT values of LDS. It is pertinent to note that the observed trend in the variation of Ef2ω with C/L ratio is quite similar to earlier studies where it was observed that bilayer thinning and surface area expansion of a DOPC bilayer increases rapidly up to a C/L ratio of 0.02 and saturates thereafter9-10. Figure 3 to 5 describes the effect of Curcumin with respect to its concentration and ionic species without the presence of any electrolytes. Since biological membranes are always suspended in presence of different electrolytes such as NaCl, KCl etc. it would be prudent to investigate the effect of Curcumin in presence of these ions. Therefore we have studied the effect of NaCl keeping the Curcumin:Lipid molar ratio at 0.10. Figure 6 shows the effect of increasing NaCl concentration on the Curcumin induced changes in the Ef2ω values of LDS. With increasing NaCl concentration Ef2ω of LDS increases and at the highest NaCl concentration (20 mM) used in this study the magnitude of increase was nearly two fold compared to the situation when no electrolyte (i.e. NaCl) is present.

ACS Paragon Plus Environment

Page 11 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Discussions: In this work we have investigated the adsorption and transport process of the LDS cation across a negatively charged POPG bilayer while Curcumin is added to the bilayer by the interface selective Second-Harmonic spectroscopic technique. Results presented in Figures 1 and 2 suggest that the observed increase in the E2ω signal of LDS upon Curcumin addition is due to creation of additional binding sites for the LDS cation on the outer bilayer of POPG liposomes by Curcumin. Curcumin (Scheme 1) has three acidic protons out of which the enolic proton is most acidic (pKa ~ 8.4)21-22. Results of several photophysical studies carried out on Curcumin suggests that intermolecular hydrogen bonding between the enolic proton of Curcumin and a hydrogen-bondacceptor group in polar protic solvents is possible23-30. Since the molecule also has a strong affinity for membranes8-12 it is quite likely that there will be hydrogen bonding interactions between the drug (Curcumin) and the polar head groups of the lipid when the drug is bound to the membrane. Results obtained from the pH experiment (Figure 3) shows that the observed 50% decrease in the Ef2ω of LDS when the pH is increased from 5.0 to 8.5 correlates with the ≈ 50% decrease in the concentration of the neutral form of the drug at pH 8.5. Therefore our results indicate that hydrogen bonding between the enolic proton of Curcumin and the polar headgroup of the POPG liposomes is crucial for the creation of additional binding sites for LDS. The observed changes in the Ef2ω and kT values of LDS (Figure 5) with changes in C/L ratio indicate the presence of two distinct regimes: 1) C/L ratio ≤ 0.02: At this regime addition of Curcumin generates more binding sites for LDS cation without affecting its transport rate across the bilayer.

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 29

2) C/L ratio ≥ 0.02: In this regime addition of Curcumin does not generates any more binding sites for LDS cation but causes the transport of the cation to become significantly faster across the bilayer. If the proposed two-state binding model of the drug with bilayer (DOPG or POPG) as suggested earlier is correct then results presented in Figure 5 suggests that surface associated state of the drug is responsible for creation of extra binding sites for the LDS cation whereas the bilayer intercalated state of the drug is responsible for the enhanced transport of LDS cation. It is reasonable to expect that when the drug is intercalated in the bilayer the energy barrier (associated within the hydrophobic domain of the bilayer) for the LDS cation to cross the bilayer will be lowered as Curcumin is a polar molecule having a dipole moment of ~10 Debye31. However in order to understand why it should lead to an increment in cation binding sites we need to consider the effect of other ions (i.e. electrolytes) present in the solution. Several Molecular Dynamics (MD) simulation studies have found that strong ion–lipid interactions in POPG lipids stabilize the bilayer by reducing lipid-lipid repulsion arising due to similar charge of the lipid head groups32-35. These interactions occur with positively charged counter ions (e.g. Na+) and carbonyl oxygens present in the POPG head groups via formation of ion-lipid and lipid-ion-lipid complexes. Results obtained from atomistic32 and coarse-grained34 MD simulations show that interactions with anti microbial peptides (AMP) with POPG membranes are associated with the release of these counter ions from the membrane interface. The entropy increase due to the counter ion release from the electric double layer surrounding the membrane is attributed as one of the origins of the attraction between the strongly charged POPG membrane and AMP. In a particular case the mechanism for degradation of a POPG membrane upon binding to a cationic AMP was ascribed to the counter ion release by the AMP thereby

ACS Paragon Plus Environment

Page 13 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

destroying the cationic bridges between charged lipid head groups and de-stabilizing the bilayer35. The adsorption and subsequently the transport of LDS cation is initiated by electrostatic force of attraction between the dye and the negatively charged bilayer. For reasons discussed above, the negative charge of the bilayer will be somewhat screened due to the presence of Na+–lipid interactions as the liposomes are suspended in sodium phosphate buffer solution. Therefore any factor that affects this interaction is expected to alter the bilayer surface charge density which in turn will affect the electrostatic adsorption of LDS cation with the bilayer. Since it has been reported that Curcumin forms hydrogen bond with the carbonyl oxygens in a DPPC bilayer11-12 we propose that addition of Curcumin to a POPG liposome solution suspended in sodium phosphate buffer medium disturbs the Na+–lipid interactions by formation of intermolecular hydrogen bond between the enolic hydrogen of Curcumin and carbonyl oxygen of the DOPG lipid. This hypothesis is consistent with our experimental observations (Figure 3) that the enolate anion of Curcumin is less conducive for increasing the Ef2ω of LDS. Replacement of the Na+ ions with electrically neutral Curcumin molecules is expected to enhance the electrostatic force of attraction between LDS cation and the negatively charged POPG bilayer by increasing the bilayer surface charge density which would explain our experimental observations. It is interesting to note that although the bilayer surface charge screening increases with increasing Na+ ion concentration (by adding NaCl to the solution), Curcumin is still able to disturb the Na+– lipid interaction (Figure 6) which demonstrates the lipophilic character of the molecule. It is pertinent to note that this alteration of bilayer surface charge density by Curcumin should depend upon relative orientation of the molecule with the bilayer. The chemical structure of Curcumin (Scheme I) indicates that interaction between the enolic hydrogen of Curcumin and the carbonyl

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 29

oxygens of the POPG bilayer is likely to be maximum when the drug will reside at the surface of the bilayer rather than intercalate within hydrocarbon region of the bilayer. It is pertinent to note here that water near the bilayer also plays a crucial role for several biochemical processes such as adsorption, ionic transport and membrane protein orientation. Several recent studies using the heterodyne detected electronic sum frequency generation technique have shed light upon how lipid head groups affect the interfacial pH and orientation of interfacial water molecules36-38. In addition MD studies also showed that formation of the Na+-lipid complexes becomes energetically more stable when a water molecule is shared between them32-35. Finally, the results obtained by us are summarized in a cartoon (Figure 7) which tries to explain the experimental observations. Addition of LDS cation to a POPG liposome solution results in electrostatic adsorption of the dye to the negatively charged bilayer surface. The amount of adsorption of the cation depends upon the surface charge density of the bilayer governed by the Na+–lipid interactions. Addition of Curcumin (C/L ratio ≤ 0.02) results in replacement of Na+–lipid complexes by formation of Curcumin-lipid complexes which helps further adsorption of the LDS cation at the outer bilayer due to an increase in the negative charge density of the bilayer surface. Further addition of Curcumin results in partitioning of the drug inside the bilayer where the long axis of the molecule is oriented perpendicular to aqueous-bilayer interfacial plane. In this geometry the polar Curcumin molecule reduces the hydrophobic character of the hydrophobic domain of the bilayer and as a result the transport of the LDS cation across the bilayer gets faster.

Summary and conclusions:

ACS Paragon Plus Environment

Page 15 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

The function of membrane bound proteins is sensitive to their hydrophobic as well as hydrophilic interactions with the bilayer39. The hydrophobic interaction happens inside the bilayer, with the hydrophobic part of the bilayer whereas the hydrophilic interaction occurs at the aqueous-bilayer interface with the polar head groups of the lipids and the surrounding ions. A disturbance in these interactions is expected to affect their functionality and for Curcumin our results combined with previous results9-10 are expected to provide further insights about the ability of this drug to regulate various structurally-unrelated membrane proteins by changing the properties of the lipid bilayer. Finally, we note that a closer inspection of Figures 1 and the inset of Figure 5 reveal that the increase in the E2ω signal of LDS, immediately following addition of Curcumin, is not instantaneous at low temperature. This indicates that disruption of Na+–lipid interactions and formation of Curcumin-lipid hydrogen bonding is a kinetically controlled process. Attempts will be made to investigate this aspect in a future study. Supporting Information Available: Detail procedures for preparation and characterization (with respect to their size and zeta potential) of POPG liposomes. SH electric field intensity profiles of LDS under various conditions. This material is available free of charge via the Internet at http://pubs.acs.org.

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 29

References: 1) Schreier, S.; Malheiros, S. V. P.; Paula, E.; Surface active drugs: self-association and interaction with membranes and surfactants. Physicochemical and biological aspects. Biochimica et Biophysica Acta 2000, 1508, 210-234. 2) Peetla, C.; Stine, A.; Labhasetwar, V. Biophysical Interactions with Model Lipid Membranes: Applications in Drug Discovery and Drug Delivery. Molecular Pharmaceutics 2009, 6, 1264–1276. 3) Gupta, S. C.; Patchva, S.; Aggarwal, B. B. Therapeutic Roles of Curcumin: Lessons Learned from Clinical Trials. The AAPS Journal 2013, 15, 195-218. 4) Ravindran, J.; Prasad, S.; Aggarwal, B. B. Curcumin and Cancer Cells: How Many Ways Can Curry Kill Tumor Cells Selectively? The AAPS Journal 2009, 11, 495-510. 5) Hatcher, H.; Planalp, R.; Cho, J.; Torti, F. M.; Torti, S. V. Curcumin: From ancient medicine to current clinical trials. Cellular and Molecular Life Sciences 2008, 65, 16311652. 6) Maheshwari, R. K.; Singh, A. K.; Gaddipati, J.; Srimal, R. C. Multiple biological activities of curcumin: A short review. Life Sciences 2006, 78, 2081–2087. 7) Bilmen, J. G.; Khan, S. Z.; Javed, M. U. H.; Michelangeli, F. Inhibition of the SERCA Ca2+ pumps by Curcumin. Eur. J. Biochem 2001, 268, 6318–6327. 8) Ingolfsson, H. I.; Koeppe, R. E.; Andersen, O. S. Curcumin is a modulator of bilayer material properties. Biochemistry 2007, 46, 10384–10391. 9) Hung, W. C.; Chen, F. Y.; Lee, C. C.; Sun, Y.; Lee, M. T.; Huang, H. W. MembraneThinning Effect of Curcumin. Biophys. J. 2008, 94, 4331-4338.

ACS Paragon Plus Environment

Page 17 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

10) Sun, Y.; Lee, C-C.; Hung, W-C.; Chen, F-Y.; Lee, M-T.;Huang, H. W. The Bound States of Amphipathic Drugs in Lipid Bilayers: Study of Curcumin. Biophys. J. 2008, 95, 2318– 2324. 11) Barry, J.; Fritz, M.; Brender, J. R.; Smith, P. E.; Lee, D. K.; Ramamoorthy, A. Determining the Effects of Lipophilic Drugs on Membrane Structure by Solid-State NMR Spectroscopy: The Case of the Antioxidant Curcumin. J. Am. Chem. Soc. 2009, 131, 4490-4498. 12) Perez-Lara, A.; Ausili, A.; Aranda, F. J.; Godos, A.; Torrecillas, A.; Corbalan-Garcia, S.; Gomez-Fernandez,

J.

C.

Curcumin

Disorders

1,2-Dipalmitoyl-sn-glycero-3-

phosphocholine Membranes and Favors the Formation of Nonlamellar Structures by 1,2Dielaidoyl-sn-glycero-3-phosphoethanolamine. J. Phys. Chem. B 2010, 114, 9778–9786. 13) Varshney, G. K.; Saini, R. K.; Gupta, P. K.; Das, K. Effect of Curcumin on the Diffusion Kinetics of a Hemicyanine Dye, LDS-698, across a Lipid Bilayer Probed by Second Harmonic Spectroscopy. Langmuir 2013, 29, 2912−2918. 14) Tsukamotoa, M.; Kuroda, K.; Ramamoorthy, A.; Yasuhara, K. Modulation of raft domains in a lipid bilayer by boundary-active Curcumin. Chem. Comm. 2014, 50, 3427– 3430. 15) Eisenthal, K. B. Second Harmonic Spectroscopy of Aqueous Nano- and Microparticle Interfaces. Chem. Rev. 2006, 106, 1462-1477. 16) Srivastava, A.; Eisenthal, K. B. Kinetics of molecular transport across a liposome bilayer. Chem. Phys. Lett. 1998, 292, 345−351.

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 29

17) Liu, Y.; Yan, E. C. Y.; Eisenthal, K. B. Effects of Bilayer Surface Charge Density on Molecular Adsorption and Transport across Liposome Bilayers. Biophys. J. 2001, 80, 1004−1012. 18) Liu, J.; Subir, M.; Nguyen, K.; Eisenthal, K. B. Second Harmonic Studies of Ions Crossing Liposome Membranes in Real Time. J. Phys. Chem. B 2008, 112, 15263−15266. 19) Saini, R. K.; Dube, A.; Gupta, P. K.; Das, K. Diffusion of Chlorin-p6 Across Phosphatidyl Choline Liposome Bilayer Probed by Second Harmonic Generation. J. Phys. Chem. B 2012, 116, 4199-4205. 20) Peret-Almeida, L.; Cherubino, A. P. F.; Alves, R. J.; Dufosse, L.; Gloria, M. B. A. Separation and determination of the physico-chemical characteristics of curcumin, demethoxycurcumin and bisdemethoxycurcumin. Food Res. Int. 2005, 38, 1039–1044. 21) Bernabe-Pineda, M.; Ramirez-Silva, M. T.; Romero-Romob, M.; Gonzalez-Vergara, E.; Rojas-Hernandez, A. Determination of acidity constants of curcumin in aqueous solution and apparent rate constant of its decomposition. Spectrochim. Acta Part A 2004, 60, 1091–1097. 22) Wang, Z.; Leung, M. H. M.; Kee, T. W.; English, D. S. The Role of Charge in the Surfactant-Assisted Stabilization of the Natural Product Curcumin. Langmuir 2010, 26, 5520–5526. 23) Adhikary, R.; Mukherjee, P.; Kee, T. W.; Petrich, J. W. Excited-State Intramolecular Hydrogen Atom Transfer and Solvation Dynamics of the Medicinal Pigment Curcumin. J. Phys. Chem. B 2009, 113, 5255-5261.

ACS Paragon Plus Environment

Page 19 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

24) Adhikary, R.; Carlson, P. J.; Kee, T. W.; Petrich, J. W. Excited-State Intramolecular Hydrogen Atom Transfer of Curcumin in Surfactant Micelles, J. Phys. Chem. B 2010, 114, 2997-3004. 25) Ghosh, R.; Mondal, J. A.; Palit, D. K. Ultrafast Dynamics of the Excited States of Curcumin in Solution. J. Phys. Chem. B 2010, 114, 12129-12143. 26) Ke, D.; Wang, X.; Yang, Q.; Niu, Y.; Chai, S.; Chen, Z.; An, X.; Shen, W. Spectrometric Study on the Interaction of Dodecyltrimethylammonium Bromide with Curcumin. Langmuir 2011, 27, 14112-14117. 27) Erez, Y.; Presiado, I.; Gepshtein, R.; Huppert, D. Temperature Dependence of the Fluorescence Properties of Curcumin. J. Phys. Chem. A 2011, 115, 10962-10971. 28) Erez, Y.; Presiado, I.; Gepshtein, R.; Huppert, D. The Effect of a Mild Base on Curcumin in Methanol and Ethanol. J. Phys. Chem. A 2012, 116, 2039-2048. 29) Saini, R. K.; Das, K. Picosecond Spectral Relaxation of Curcumin Excited State in a Binary Solvent Mixture of Toluene and Methanol. J. Phys. Chem. B, 2012, 116, 1035710363. 30) Saini, R. K.; Das, K. Photophysics of Curcumin excited state in toluene-polar solvent mixtures: Role of H-bonding properties of the polar solvent. J. Lumin., 2014, 145, 832837. 31) Shen, L.; Ji, H.-F. Theoretical study on physicochemical properties of Curcumin. Spectrochim. Acta, Part A 2007, 67, 619-623. 32) Zhao, W.; Rog, T.; Gurtovenko, A. A.; Vattulainen, I.; Karttunen, M. Atomic-Scale Structure and Electrostatics of Anionic Palmitoyloleoylphosphatidylglycerol Lipid Bilayers with Na+ Counterions. Biophys. J. 2007, 92, 1114–1124.

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 29

33) Zhao, W.; Rog, T.; Gurtovenko, A. A.; Vattulainen, I.; Karttunen, M. Role of phosphatidylglycerols in the stability of bacterial membranes. Biochimie 2008, 90, 930– 938. 34) Tolokh, I. S.; Vivcharuk, V.; Tomberli, B.;

Gray, C. G. Binding free energy and

counterion release for adsorption of the antimicrobial peptide lactoferricin B on a POPG membrane. Phys. Rev. E 2009, 80, 031911. 35) Von Deuster, C. I.; Knecht, V. Competing interactions for antimicrobial selectivity based on charge complementarity. Biochim. Biophys. Acta Biomembr. 2011, 1808, 2867–2876. 36) Mondal, J. A.; Nihonyanagi, S.; Yamaguchi, S.; Tahara, T. Structure and Orientation of Water at Charged Lipid Monolayer/Water Interfaces Probed by Heterodyne-Detected Vibrational Sum Frequency Generation Spectroscopy J. Am. Chem. Soc. 2010, 132, 10656–10657. 37) Mondal, J. A.; Nihonyanagi, S.; Yamaguchi, S.; Tahara, T. Three Distinct Water Structures at a Zwitterionic Lipid/Water Interface Revealed by Heterodyne-Detected Vibrational Sum Frequency Generation J. Am. Chem. Soc. 2012, 134, 7842-7850. 38) Kundu, A.; Yamaguchi, S.; Tahara, T. Evaluation of pH at Charged Lipid/Water Interfaces by Heterodyne-Detected Electronic Sum Frequency Generation J. Phys. Chem. Lett. 2014, 5, 762-766. 39) Sachs, J. N.; Engelman, D. M. Introduction to the Membrane Protein Reviews: The Interplay of Structure, Dynamics, and Environment in Membrane Protein Function. Ann. Rev. Biochem. 2006, 75, 707–12.

ACS Paragon Plus Environment

Page 21 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figures with captions:

Scheme I

+ N Et

NMe2

LDS-698 cation

H O

O OMe

MeO HO

OH

Curcumin

Chemical structures of LDS-698 cation and Curcumin

ACS Paragon Plus Environment

Langmuir

120

14 12

E2ω

10

100

8 6 4 2 0

80

50

100

150

200

time (second)

250

300

E2ω

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 29

60

40

20 0

100

200

300

400

500

time (second)

Figure 1: SH electric field (E2ω) of LDS (5 µM) under different conditions at pH 5.0 and room temperature. Black: In presence of POPG liposomes (50 µM) which were added at 50 second time point; Blue: In presence of POPG liposomes (50 µM) incubated with Curcumin (C/L = 0.10), which were added at 50 second time point; Red: In presence of POPG liposomes (50 µM) added at 50 second time point and Curcumin (C/L = 0.10) added at 100 second time point. Inset: SH electric field (E2ω) arising from pH 5.0 buffer to which POPG liposomes (50 µM) was added at 50 second time point and Curcumin (C/L = 0.50) was added at 100 second time point.

ACS Paragon Plus Environment

Page 23 of 29

100 after Curcumin

E2ω

90

∆E2ω

80

before Curcumin

E2ω

70

LDS

60

E2ω

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

50 40

Malachite Green

30 20 10

Auramine O

0 100

200

300

time (second)

Figure 2: Changes in E2ω of LDS (circles), Malachite Green (squares) and Auramine-O (triangles) in the presence of POPG liposomes (50 µM) before and after addition of Curcumin (C/L = 0.1; added at 100 second time point) in pH 5.0 buffer at 3 0C. Estimation of Curcumin associated fractional changes in the E2ω of LDS is also shown.

ACS Paragon Plus Environment

Langmuir

1.25 1.00

0.20 E2ω

0.75 0.50 0.25

0.15

100

E2 ω

200

300

time (second)

f

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 29

0.10

0.05 4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

pH

Figure 3: The variation in the Ef2ω of LDS in presence of POPG liposomes (C/L = 0.1) with pH of the medium at 3 0C. Inset: Time dependent changes of the E2ω signal of LDS in presence of POPG liposomes before and after addition of Curcumin at pH 5.0 (black circles) and at pH 8.5 (hollow circles). The E2ω signals of LDS were normalized before addition of Curcumin to unity for clarity.

ACS Paragon Plus Environment

Page 25 of 29

120

C/L = 0.001 to 0.0160

100 80

60

E2ω

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

40

C/L = 0.02 to 0.20

20 100

200

300

400

500

time (second)

Figure 4: Changes in the E2ω signal of LDS due to addition of Curcumin (C/L varied from 1x10-3 to 0.2) to a solution containing LDS and POPG liposomes. Curcumin was added at t = 100 second time point.

ACS Paragon Plus Environment

Langmuir

0.30

110 100 90

E2ω

80

f

0.25

4

10

70 60 50 40

3

30

10 200

250

300

3

150

f

time (second)

0.15

2

10 0.10

-1

100

kT (sec ) x 10

0.20

E2ω

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 29

1

10 0.05

0

10 0.00 0.00

0.04

0.08

0.12

0.16

0.20

Curcumin:Lipid mole ratio

Figure 5: Changes in the Ef2ω (filled, solid lines) and transport rate constant (kT) values (hollow, dashed lines) of LDS in presence of POPG liposomes at pH 5.0 with variation in the C/L mole ratio at 3 0C. Inset: Representative time dependent E2ω signal of LDS for C/L mole ratio of 4.0x10-2 (solid circles) and 4.0x10-3 (hollow transparent circles).

ACS Paragon Plus Environment

Page 27 of 29

0.45

1.25

0.40

E



1.00 0.75 0.50

0.35 0.25 100

f

E2ω

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

0.30

150 200 time (second)

250

300

0.25

0.20

0.15 0

5

10

15

20

NaCl (mM)

Figure 6: Changes in the Ef2ω of LDS in presence of POPG liposomes and Curcumin (C/L = 0.1) with increasing amounts of an electrolyte, NaCl at pH 5.0 and 3 0C. Inset: Representative time dependent changes of the E2ω signal of LDS in presence of POPG liposomes before and after addition of Curcumin at pH 5.0 and 3 0C. Solid and hollow circles represent NaCl concentrations of 0 and 20 mM respectively. The E2ω signals were normalized before addition of Curcumin to unity for clarity.

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 29

Figure 7: Cartoon showing how Curcumin binds and disturbs the POPG bilayer and affects the adsorption and transport of the LDS cation. The LDS cations adsorb initially on the outer bilayer (top) of the POPG liposomes and then transports to the inner bilayer (bottom).

ACS Paragon Plus Environment

Page 29 of 29

TOC graphic:

Addition of Curcumin at t = 100 s

SH electric field of LDS cation (arb. unit)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

+

Release of counter-ions (Na ): Adsorption increases

No Curcumin

Faster transport

Curcumin

Curcumin

C/L ratio ≤ 0.02

POPG lipid

50

C/L ratio ≥ 0.02

Na+ ion

LDS cation

100

150

PO4 -2 anion

200

250

time (second)

Bilayer partitioning of Curcumin affects the adsorption and transport of a cationic dye

ACS Paragon Plus Environment