Impacts of Salt, Buffer, and Lipid Nature on Molecular Adsorption and Transport in Liposomes As Observed by Second Harmonic Generation

Abstract

It is of great importance to interrogate the impact of local environment on the transport of small molecules across lipid bilayers, as they are key to the function and capabilities of eukaryotic cells and liposome-based delivery systems. Herein are described real-time studies of the molecular adsorption and transport kinetics of positively charged small-molecule organic dyes at the surface of liposomes under different buffer and salt conditions, made possible by application of second harmonic generation (SHG). The molecular transport of malachite green (MG) within the liposome bilayer is more rapid in 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPG and DOPS, respectively) liposomes in citrate buffer without added salts, whereas no adsorption or transport of MG is observed in trimethyl quinone-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (QPADOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes. While the transport rate constant increases linearly with concentration of MG for DOPG liposomes, there is much less dependence of the transport rate constant on the concentration of MG in DOPS liposomes. Adsorption site densities and free energies of adsorption are determined using the modified Langmuir adsorption isotherm model by fitting SHG signals resulting from MG addition. The resulting free energy of adsorption for MG to the surface of liposomes is higher in the absence of added salts due to increased electrostatic attractions. However, the corresponding adsorption site densities increase in the presence of salts due to ion-pair formation and decreased repulsions between cationic adsorbates. Comparisons at different citrate buffer and KCl concentrations demonstrate that adsorption and transport of MG in liposomes are influenced by several interrelated factors that include molecular structure of the lipid headgroup, electrostatic interactions between the charged liposome surface and ionic adsorbates and electrolytes, and ion-pair formation. Similar investigations using the structurally similar but more highly charged dicationic dye methyl green point to a lack of its adsorption to and transport within the liposomes, possibly due to a much stronger hydration shell. These findings highlight important considerations for potential liposome-based, drug-delivery applications and the transport of small-molecule drugs across the plasma membrane.

Combinatorial Delivery of miRNA‐Nanoparticle Conjugates in Human Adipose Stem Cells for Amplified Osteogenesis

“…SHG is dipole forbidden in centrosymmetric media, like bulk solution, but can be generated at the surface of nanoparticles where the symmetry is broken. Recently, SHG has been used as an accurate method to determine molecular adsorption and chemical reactions at nanoparticle surfaces, ion‐transport processes in liposomes, and the release of oligonucleotides from the surface of plasmonic nanoparticles …”

Ultrafast and nonlinear spectroscopy of brilliant green-based nanoGUMBOS with enhanced near-infrared emission

“…The SHG signal from the molecular dye dissolved in water is due to incoherent hyper-Rayleigh scattering (HRS), where the SHG signal is proportional to the molecular concentration. [52][53][54][55][56][57][58] Enhanced SHG from the nanoGUMBOS as compared with the molecular dye is attributed primarily to the increased size. To a first approximation, the SHG signal is expected to scale as the radius to the sixth power for small nanoparticles, 51,59 although many other factors are important including the molecular nonlinear susceptibilities and angular scattering distributions.…”

“…While SHG is a coherent process for a single nanoparticle, the measured SHG signal from a colloidal nanoparticle sample is an incoherent sum of the nanoparticles in the laser focus such that the total SHG signal is linearly proportional to the nanoparticle concentration. 50,51,[56][57][58] The nanoGUMBOS also have a higher extinction at 800 nm as compared with the BG molecule and the gold nanospheres, causing some single-photon resonance enhancement in the nonlinear spectroscopy. Local ordering of the BG molecules in the nanoGUMBOS due to aggregation and π-π stacking may also occur causing orientations that are not isotropically distributed, leading to an additional source of increased SHG signals.…”

Molecule‐Membrane Interactions in Biological Cells Studied with Second Harmonic Light Scattering

“…It was expected that the rate of transport across the CM shouldb es imilar to those obtained from prior liposome-based studies. [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] In both cases, the molecular probe has to directly diffusea cross the lipid bilayer.S till, the deduced average moleculart ransport rate for the bacterial CM is found to be an order of magnitude slower compared to unilamellar liposomes (ca. 10 À3 s À1 )w hose lipid composition was homogeneous.…”

“…Following this demonstration,anumber of studies have since examined the effects of lipid composition and av ariety of environmental factorso nt he propensity and characteristics of molecular transport across membranes in model liposome systems. [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] The studies on liposomes suggested the feasibility that SHS could be used for monitoring molecular transport across membranesi nl iving biological cells. Indeed, in 2010, our laboratory reportedt he first demonstration that SHS could be applied to study molecular transport across membranes in living cells.…”