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

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

A Study of Molecular Adsorption and Transport at Cell Membrane and Biologically Relevant Surfaces by Second Harmonic Generation

Most of the biological processes in living systems involve molecular adsorption and transport at biomembranes. It is highly desired to study the time-resolved transport kinetics through living cell membranes. In this thesis, an experimental means based on a nonlinear optical phenomenon, Second Harmonic Generation (SHG) has been demonstrated to detect the molecular adsorption and transport through living cell membranes in real time and to evaluate the salt ion effects on adsorption processes in biologically relevant colloidal systems. In the case of gram-negative bacteria, E.coli, a hydrophobic cation, Malachite Green (MG) has been observed to adsorb onto the cell surface and then sequentially transport across the double bilayer structures, the bacterial outer membrane and the cytoplasmic membrane. The adsorption characteristics as well as the transport rate constant at each of the membranes have been determined. In contrast to the prokaryotic E.coli cell, the molecular ion can adsorb onto the eukaryotic Murine Erythroleukemia (MEL) cell but cannot penetrate its membrane which has no hydrophobic ion permeable channels and is more tightly packed. MG cation has been used as a SHG indicator to probe the effects of solvent ionic strength and ion specificity on molecular adsorption at model protein systems. Polystyrene sulfate (PSS) microspheres and polystyrene carboxyl (PSC) microspheres have been examined. The electrostatic force dominated molecule-surface interaction between MG cations and the sulfate terminations at PSS surface is largely affected by the ionic strength of the solution but is not sensitive to the ion identity. On the other hand, the hydrophobic force dominated molecule-surface interaction between hydrophobic regions of the MG dye and PSC microsphere shows pronounced specific ion effects but is less affected by ionic strength of the solution

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

Interfacial structure of phospholipids probed by high-resolution, high-repetition-rate broadband vibrational sum-frequency generation spectroscopy

Diese Arbeit konzentriert sich auf die Anwendbarkeit eines hochmodernen 100 kHz BB-VSFG-Spektrometers, das kürzlich im SALSA Photonics Lab entwickelt wurde, für die Analyse der Grenzflächenstruktur von Alkylketten, des sie umgebenden Wassers und der Phosphatkopfgruppen von Phospholipidschichten. Zunächst wurden Phospholipid-Doppelschichten, die mehrere Komponenten enthalten, bei Laserwiederholraten von 5, 10, 50 und 100 kHz mit konstanter Pulsenergie untersucht. Die BB-VSFG-Spektren legen nahe, dass die Phospholipid-Doppelschichten während der Messungen ohne wärmeinduzierte Veränderungen stabil waren. Darüber hinaus bot die Erhöhung der Laserwiederholungsrate eine praktikable Möglichkeit, Spektren in kurzen Datenerfassungszeiten zu erhalten, ohne dass das Signal-Rausch-Verhältnis beeinträchtigt wurde. Die extrem kurze Aufnahmezeit von 500 ms, die hohe spektrale Auflösung und alle verwendeten Pulsparameter sorgen dafür, dass bei Messungen unter Umgebungsbedingungen keine thermisch bedingten Photoschäden auftreten. Es wurde eine systematische Untersuchung von ein- und zweikomponentigen Phospholipid-Monoschichten in Abhängigkeit von der Oberflächenspannung und dem Mischungsverhältnis für verschiedene Kombinationen an Polarisationen durchgeführt und die Abhängigkeit der Schwingungsspektren untersucht. Die Struktur von Alkylketten und umgebendem Wasser wurde anhand derselben Modellsystemen analysiert. Bislang nicht beobachtete Schwingungsbanden und Spektren von Monolagen mit geringer Oberflächenbedeckung wurden mit einem bisher nicht erreichten Signal-Rausch-Verhältnis gemessen und beschrieben. Die Struktur von Phospholipid-Monolagen mit identischen Kopfgruppen und unterschiedlichen Ketten wurde analysiert und verglichen. Die Spektren bestätigten die Anwesenheit von Wassermolekülen in der Nähe der Phosphat- und Cholingruppen der Phospholipid-Monolagen.This thesis focuses on the applicability of a state-of-the-art 100 kHz BB-VSFG spectrometer recently developed at the SALSA Photonics Lab and on the analysis of the interfacial structure of alkyl chains, surrounding water, and the phosphate head groups of phospholipid layers. First, multi-component phospholipid bilayers were studied at laser repetition rates of 5, 10, 50, and 100 kHz at constant pulse energy. The spectra suggest that the phospholipid bilayers were stable during the measurements with no heat-induced distortions. Moreover, an increase in the laser repetition rate provided a feasible route to obtain spectra in short data acquisition times without compromising the signal-to-noise ratio. The extremely short acquisition time of 500 ms, the high spectral resolution, and all applied pulse parameters ensured no thermal induced photodamages occur during the measurements. A systematic study of one- and two-component phospholipid monolayers as a function of surface tension and mixture ratio at different polarization combinations was performed and the dependence of the vibrational spectra was explored. The structure of alkyl chains and surrounding water was analyzed using the same model systems. Vibrational modes that were previously unseen and spectra of monolayers at low surface coverage were reported for the first time with an unprecedented signal-to-noise ratio. The structure of phospholipid monolayers containing identical head groups and different chains was analyzed and compared. The order of the phospholipid molecules as a function of the composition of the monolayers was inferred from the spectral data. The influence of the hydration and/or changes in the orientation of the phosphate group was visible from the spectra as well