The diffusion kinetics of a hemicyanine dye, LDS-698, across model membrane bilayers was studied in real time by the surface specific second harmonic technique. Using liposomes made from different headgroups, it has been established that the diffusion is initiated by electrostatic adsorption of the positively charged dye to the outer surface of negatively charged liposomes and its time constant is affected by the rigidity of the bilayer. In the presence of the liphophilic drug curcumin (curcumin/lipid mole ratio ~ 0.2), the diffusion of LDS-698 was observed to be faster by ~56 times (from 780 to 14 s) at 25 °C. Under similar curcumin concentration, when cholesterol containing liposomes are used at 2 °C, the observed diffusion time constant increases from 14 to 65 s, showing that the effect of curcumin is superior to the effect of increasing bilayer rigidity on the diffusion process. Control experiments with other lipophilic molecules such as DPH and Nile Red showed that the effect of liposomal curcumin is superior. Consistent with previous reports of curcumin affecting the bilayer organization, this study additionally demonstrates increased permeability of liposomal curcumin, in particular against organic cations. It is speculated that origin of this enhanced membrane permeability by lipophilic molecules may depend upon the interaction of the molecule with the polar headgroup region of the lipid which, in turn, is expected to depend on the chemical structure of the molecule.
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 (E) arising due to LDS adsorbed on the outer bilayer of the POPG liposome was observed to increase instantaneously (<1 s) following the addition of Curcumin. The fractional increase in the SH electric field (E) and the bilayer transport rates (k) of LDS were studied with respect to the pH of the solution and also with the Curcumin content in the lipid bilayer. Results obtained indicate that compared with the anionic form of the drug, its neutral form is more conducive of increasing the E of LDS. With increasing Curcumin content in the lipid bilayer, two distinct regimes could be observed in terms of E and k values of LDS. For Curcumin:Lipid (C/L) ratio ≤0.02, the E of LDS increases rapidly, while k remains unchanged; and for C/L ratio ≥0.02, the E values remains more or less constant, while there is a significant (∼40 times) increase followed by a modest increase in the k 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 counterions (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 and inducing enhanced transport of the LDS cation. Results presented in this work provide further insights into how Curcumin alters bilayer properties when it partitions from the aqueous to the bilayer phase.
The effect of addition of curcumin on the adsorption and transport characteristics of a cationic dye, LDS, across negatively charged bilayers composed of POPG and DPPG lipids were investigated by the interface selective second harmonic (SH) spectroscopic technique. Curcumin induced changes in the SH electric field signal of the LDS ions (E (LDS)) were observed to depend critically on the bilayer acyl chain saturation/unsaturation ratio (S/U). Following earlier works, the increase in the E (LDS) signal is attributed to the release of the Na counterions present in the head group region of the bilayer by curcumin and the decay of the E (LDS) signal is attributed to the bilayer intercalated state of curcumin. While the changes observed in the E (LDS) signal in the presence of POPG liposomes were consistent with our earlier study ( Varshney, G. K. et al. Langmuir , 2016 , 32 , 10415 - 10421 ), they were significantly different for DPPG liposomes, following curcumin addition. While the increase in the E (LDS) signal in the presence of POPG liposomes, is marginal (∼10-20%) and instantaneous (<1 s) followed by a rapid decay (completed within ∼100 s), in the presence of DPPG liposomes it was observed to increase slowly and at saturation shows a substantial increase (100-200%), following curcumin addition. When liposomes consisting of a mixture of POPG and DPPG lipids are used, curcumin induced kinetic characteristics of the E (LDS) signal showed a mixture of the individual kinetic characteristics observed for the unsaturated (POPG) and saturated (DPPG) liposomes. The observed kinetic trends of the E (LDS) signal following curcumin addition are explained on the basis of the relative strength of the Na-POPG and Na-DPPG interaction. Higher ordering of the lipid acyl chain region in DPPG liposome makes the Na-DPPG interaction much stronger than the Na-POPG interaction. Further, it is proposed that, in POPG-DPPG liposomes, individual domains of POPG and DPPG lipids exist at low temperature as suggested by the observed temperature dependent kinetic characteristics of the E (LDS) signal following curcumin addition. These domains are dependent on the S/U ratio and phase state of the bilayer. The gel phase was observed to be more conducive for individual domain formation. Results presented in this work not only support the notion that biological activity of curcumin is associated with its bilayer altering properties, but more interestingly it provides a qualitative insight about how bilayer phase separation can be achieved by modulating the hydrophobic interactions between the lipid acyl chains.
Cells respond to external stress by altering their membrane lipid
composition to maintain fluidity, integrity and net charge. However,
in interactions with charged nanoparticles (NPs), altering membrane
charge could adversely affect its ability to transport ions across
the cell membrane. Hence, it is important to understand possible pathways
by which cells could alter zwitterionic lipid composition to respond
to NPs without compromising membrane integrity and charge. Here, we
report in situ synchrotron X-ray reflectivity (XR) measurements to
monitor the interaction of cationic NPs in the form of quantum dots,
with phase-separated supported lipid bilayers of different compositions
containing an anionic lipid and zwitterionic lipids having variable
degrees of stiffness. We observe that the extent of NP penetration
into the respective membranes, as estimated from XR data analysis,
is inversely related to membrane compression moduli, which was tuned
by altering the stiffness of
the zwitterionic lipid component. For a particular membrane composition
with a discernible height difference between ordered and disordered
phases, we were able to observe subtle correlations between the extent
of charge on the NPs and the specificity to bind to the charged and
ordered phase, contrary to that observed earlier for phase-separated
model biomembranes containing no charged lipids. Our results provide
microscopic insight into the role of membrane rigidity and electrostatics
in determining membrane permeation. This can lead to great potential
benefits in rational designing of NPs for bioimaging and drug delivery
applications as well as in assessing and alleviating cytotoxicity
of NPs.
The membrane permeability induced by the polyene antibiotic Amphotericin B (AmB) against negatively charged L‐α‐phosphatidyl DL‐glycerol (POPG) membranes containing either Ergosterol (Ergo) or Cholesterol (Chol) has been investigated by monitoring the bilayer transport of the LDS‐698 (LDS+) ion using the interfacial selective second harmonic (SH) spectroscopy. The transport of LDS+ becomes faster at very low AmB/Liposome (A/L) ratio whereas Ergo present in the bilayer. As increasing A/L ratio, the average transport time constants (Tav) of the LDS+ ion fall into two kinetic regimes which observed for the different liposomes. In POPG‐Ergo liposomes, the effect of neutralizing either of the two charged functional groups of AmB reveals that interaction of AmB with Ergo is primarily dependent upon the charged amino group present in the mycosamine moiety of AmB. The spectral (absorption and fluorescence) properties of AmB were also studied to supplement the results obtained from SH studies. In summary, at low A/L ratio (< 1), the membrane permeability of the POPG membrane were observed to dependent upon the membrane composition or the pH of the medium, whereas at higher A/L ratio (> 1) the permeability becomes similar for all the cases. The spectroscopic properties of AmB with increasing A/L ratio suggest that the latter phenomena may be due to the aggregated states of AmB in the bilayer which enhance the bilayer permeability irrespective of the bilayer composition and solution pH.
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