Second Harmonic Generation Spectroscopy and Microscopy of Liposomes, Nanoparticles, and Cells

Second harmonic generation (SHG) is used to investigate the factors that impact nanoparticle-based drug-delivery applications. In the first study, molecular adsorption and transport kinetics of a positively-charged dye, malachite green isothiocyanate (MGITC), is characterized at the surface of different colloidal liposomes in water using SHG spectroscopy. The molecular interactions of MGITC is compared to our previous investigations with malachite green (MG). In comparison to MG, MGITC demonstrates stronger adsorption and faster transport through lipid membranes. Correspondingly, the SHG experimental results are in excellent agreement with the molecular dynamics (MD) simulations results. A key finding illustrates the importance of functional groups, such as isothiocyanate, in controlling molecular translocation across the phospholipid-water interface. In a related study, temperature-dependent SHG measurements are performed to investigate the thermodynamics associated with the adsorption and transport kinetics of MG at the surface of 1,2-dioleoyl-sn-glycero-3phospho-(1’rac-glycerol) (DOPG) liposomes. The molecular transport is determined to be approximately 5 times faster at 40 ⁰C in comparison to the molecular transport at 25 ⁰C. Additionally, the changes in adsorption enthalpy and entropy are determined. The change in adsorption entropy is positive and the change in adsorption enthalpy is negative, indicating that the adsorption process is spontaneous at all aqueous temperatures. Similarly, SHG microscopy is used to probe the molecular interactions of MG and MGITC molecules at the surface of living human nonsmall adenosquamous lung cancer cells (H596 cells). The observed molecular translocation in living H596 cells is significantly faster for MGITC in comparison to MG. SHG microscopy is also used to probe fixed, dead H596 cells with MGITC dye molecules. In comparison to fixed cells, living cells have pronounced fluctuations of SHG intensity which is attributed to more complicated interactions, including active transport and cell regulation. Finally, gold, silver, and gold-silver-gold core-shell-shell (CSS) plasmonic nanoparticles having size of 10-100 nm are synthesized and functionalized with miRNA molecules using Diels-Alder chemistry. The retro Diels-Alder thermal release of miRNA from the surface of novel plasmonic nanoparticles is investigated at their corresponding plasmon resonances using surface-specific SHG spectroscopy. In summary, these time-resolved studies highlight the importance of SHG as a sensitive, powerful, and versatile tool to monitor the real-time surface chemistry of colloidal nanoparticle-based drug-delivery systems

Nanoparticle-Based Drug-Delivery Systems Studied by Second Harmonic Generation

Second harmonic generation (SHG) is used to study different types of colloidal nanoparticle drug-delivery systems. The surface charge density, electrostatic surface potentials, and ion adsorptions of 50 nm colloidal gold nanoparticle samples coated with mercaptosuccinic acid are determined using SHG measurements under varying NaCl and MgCl2 concentrations in water. Numerical solutions to the spherical Poisson-Boltzmann equation are fit to the SHG results to account for the nanoparticle surface curvature and ion adsorption to the Stern layer interface, showing excellent agreement with electrophoretic mobility measurements. In another study, nanoparticles of gold, silver and polystyrene are functionalized with microRNA using a nitrobenzyl photocleavable linker that cleaves upon ultraviolet irradiation. The SHG is shown to be a sensitive probe for monitoring the photocleaving dynamics of the oligonucleotides in real time. The photoactivated controlled release is observed to be most efficient on resonance at 365 nm irradiation, with pseudo-first-order rate constants that are linearly proportional to irradiation powers. Silver nanoparticles show an approximate 6-fold plasmon enhancement in photocleaving efficiency over corresponding polystyrene nanoparticles and an approximate 3-fold plasmon enhancement over gold nanoparticles. Additionally, gold-silver-gold core-shell-shell nanoparticles are prepared and are functionalized with miRNA using Diels-Alder chemistry. The plasmonic extinction peak of these nanoparticles, centered at near-infrared (NIR) wavelengths, that can be controlled by varying the thickness of gold and silver shells. Photothermal release of oligonucleotides from the nanoparticle surface under NIR irradiation is studied for drug-delivery applications in the NIR optical window of biological tissue. Lastly, SHG is used to investigate molecular adsorption and transport kinetics of positively charged dyes at the surface of liposomes in water. The adsorption and time-dependent SHG results are analyzed to obtain the free energies of adsorption, the adsorption site densities, and the transport kinetics under varying liposome chemistries and buffer conditions. Parameters such as electrostatic interactions, the chemical structure of the lipid head group, the buffer conductivity, ion-pair formation and adsorbate-adsorbate repulsion are found to influence the adsorption and transport at the liposome surface. In all of these studies, real-time SHG measurements are shown to be highly sensitive for investigating surface dynamics in nanoparticle-based drug delivery systems

On the Development of Analytical Methodologies to Interrogate the Lipid Dynamics and Phase Transition Resulting from the Reduction of Stimuli-responsive Vesicles

The potential is great for liposome drug delivery systems that provide specific contents release at diseased tissue sites upon activation by upregulated enzymes; however, this potential will only come to fruition with mechanistic knowledge of the contents release process. NAD(P)H:quinone oxidoreductase type 1 (NQO1) is a target for reductively-responsive liposomes, as it is an enzyme upregulated in numerous cancer tissues and is capable of reducing quinone propionic acid (QPA) trigger groups to hydroquinones that self-cleave from dioleolylphosphatidylethanolamine (DOPE) liposome surfaces, thereby initiating contents release. This research targets the development of analytical methodologies to observe and characterize the dynamics and resulting phase change of the QPA-DOPE liposomal system. It is known that after reduction, QPA-DOPE vesicles aggregate and that the aggregation is correlated with release of their encapsulated contents. While postulated, the final phase identity of this system has not been identified as the conventional methods used to make this measurement are not capable of studying such a unique and dynamic system. Presented herein are the analytical methods, both developed and adapted, which have been used to investigate a liposomal system capable of redox stimulated contents release. The purpose of this work was to utilize these tools to (1) study the terminal phase identity of QPA-DOPE vesicles after reduction, (2) manipulate the QPA-DOPE liposomal system for triggerable inter-vesical fusion, and (3) investigate the liposome bilayer behavior post-reduction and pre-release. The findings of this work are presented and their significance discussed

A two-domain elevator mechanism for sodium/proton antiport

Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis1. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets2. The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli1, 3, for which both electron microscopy and crystal structures are available4, 5, 6. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein1, 4. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur7. The only reported NhaA crystal structure so far is of the low pH inactivated form4. Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding1, 8, 9 directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20° against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second3, Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general

In Situ Label-Free Study of Protein Adsorption on Nanoparticles

[Image: see text] Improving the design of nanoparticles for use as drug carriers or biosensors requires a better understanding of the protein–nanoparticle interaction. Here, we present a new tool to investigate this interaction in situ and without additional labeling of the proteins and/or nanoparticles. By combining nonresonant second-harmonic light scattering with a modified Langmuir model, we show that it is possible to gain insight into the adsorption behavior of blood proteins, namely fibrinogen, human serum albumin, and transferrin, onto negatively charged polystyrene nanoparticles. The modified Langmuir model gives us access to the maximum amount of adsorbed protein, the apparent binding constant, and Gibbs free energy. Furthermore, we employ the method to investigate the influence of the nanoparticle size on the adsorption of human serum albumin and find that the amount of adsorbed protein increases more than the surface area per nanoparticle for larger diameters

Lipid membrane characterization with second harmonic scattering:surface potentials, ionization, membrane asymmetry and hydration

Membranes, composed of a variety of lipids and other biomolecules, mediate signaling processes between cells and their aqueous environment. To fulfill this function, membranes can vary their composition leaflet-specific and thus alter their surface properties. To fully understand the impact of these processes on the molecular level, it is necessary to develop tools that can access the molecular properties of free-floating model membranes label-free. These tools are ideally surface-specific. In this thesis, we apply the nonlinear optical techniques second harmonic scattering (SHS) and vibrational sum-frequency scattering (SFS) together with electrokinetic measurements to label-free characterize the interfacial properties, hydration structure and surface potentials of liposomes in aqueous solutions. First, we generalize the nonlinear optical theory to describe the second-order surface response from interfaces with aqueous solutions independent of the ionic strength for reflection, transmission and scattering geometries. We demonstrate that interference effects from oriented water molecules in the bulk aqueous solution alter the probing depth and the expected second-order response at low ionic strengths. Then, we apply this theory to demonstrate that SHS patterns of liposomes and oil droplets contain all necessary information to extract the absolute surface potential of the respective particles without assuming a model for the interfacial structure. By analyzing scattering patterns that capture the orientational distribution of water around the particles, we find surface potentials of -38 mV for bare oil-droplets and -11 mV for zwitterionic liposomes in water. For anionic liposomes the surface potential varies between -150 mV and -23 mV in solutions containing different amounts of NaCl ranging from $\sim$0 mM to 10 mM. These values are remarkably different for solutions to the Gouy-Chapman model considering a fixed surface charge density. Next, we characterize the hydration and lipid asymmetries in binary mixed membranes using SHS and SFS. The liposomes exhibit hydration asymmetry between the inner and outer leaflet. The lipid number density between the inner and outer leaflet is the same, although geometrical packing arguments would suggest a different density. However, an asymmetric lipid distribution between the leaflets can be induced by fine tuning specific intermolecular interactions between the lipids. This is shown with dipalmitoylphosphoserine and dioleoylphosphocholine mixtures creating a membrane structure that allows intermolecular H-bonding between the phosphate and amine groups of the lipids. Finally, we quantify the surface properties of membranes composed of lipids containing phosphoserine and phosphocholine headgroups. Surprisingly, we find a very high degree of counterion condensation on anionic membranes in pure water: only 1 \% of all lipids are ionized. This indicates a tightly packed layer of ions around the membrane that needs to be considered when modelling the interfacial structure around membranes

Surface characterization of lipid biomimetic systems

Zeta potential and dipole potential measures are direct operational methodologies to determine the adsorption, insertion and penetration of ions, amphipathic and neutral compounds into the membranes of cells and model systems. From these results, the contribution of charged and dipole groups can be deduced. However, although each method may give apparent affinity or binding constants, care should be taken to interpret them in terms of physical meaning because they are not independent properties. On the base of a recent model in which the lipid bilayer is considered as composed by two interphase regions at each side of the hydrocarbon core, this review describes how dipole potential and zeta potential are correlated due to water reorganization. From this analysis, considering that in a cell the interphase region the membrane extends to the cell interior or overlaps with the interphase region of another supramolecular structure, the correlation of dipole and electrostatic forces can be taken as responsible of the propagation of perturbations between membrane and cytoplasm and vice versa. Thus, this picture gives the membrane a responsive character in addition to that of a selective permeability barrier when integrated to a complex system.Fil: Disalvo, Edgardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Centro de Investigación en Biofísica Aplicada y Alimentos. - Universidad Nacional de Santiago del Estero. Centro de Investigación en Biofísica Aplicada y Alimentos; ArgentinaFil: Frías, María de los Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Centro de Investigación en Biofísica Aplicada y Alimentos. - Universidad Nacional de Santiago del Estero. Centro de Investigación en Biofísica Aplicada y Alimentos; Argentin

Liposomes characterization for market approval as pharmaceutical products: Analytical methods, guidelines and standardized protocols

Liposomes are nano-sized lipid-based vesicles widely studied for their drug delivery capabilities. Compared to standard carries they exhibit better properties such as improved site-targeting and drug release, protection of drugs from degradation and clearance, and lower toxic side effects. At present, scientific literature is rich of studies regarding liposomes-based systems, while 14 types of liposomal products have been authorized to the market by EMA and FDA and many others have been approved by national agencies. Although the interest in nanodevices and nanomedicine has steadily increased in the last two decades the development of documentation regulating and standardizing all the phases of their development and quality control still suffers from major inadequacy due to the intrinsic complexity of nano-systems characterization. Many generic documents (Type 1) discussing guidelines for the study of nano-systems (lipidic and not) have been proposed while there is a lack of robust and standardized methods (Type 2 documents). As a result, a widespread of different techniques, approaches and methodologies are being used, generating results of variable quality and hard to compare with each other. Additionally, such documents are often subject to updates and rewriting further complicating the topic. Within this context the aim of this work is focused on bridging the gap in liposome characterization: the most recent standardized methodologies suitable for liposomes characterization are here reported (with the corresponding Type 2 documents) and revised in a short and pragmatical way focused on providing the reader with a practical background of the state of the art. In particular, this paper will put the accent on the methodologies developed to evaluate the main critical quality attributes (CQAs) necessary for liposomes market approval