Mechanical injuries to the cell often lead to disruptions of the cell’s phospholipid bilayer membrane and potential detrimental effects including cell death. Understanding the mechanical states required to disrupt the phospholipid bilayer would result in better multiscale constitutive models and further knowledge of cell injury. The objectives of this research were to perform biaxial deformations of the phospholipid bilayer to quantify phospholipid bilayer disruption and to identify potential parameters that can be used in multiscale constitutive equations. We show that the von Mises stress, 26.6-61.1, increases linearly with the von Mises strain rate, 1.7e8-6.7e8, and that the strain at failure is dependent on the stress state with non- and equibiaxial being the most detrimental when failing at <.73 von Mises strain. Understanding the effects of nanoscale mechanical trauma to the cell provides a better understanding of cell injury and may provide insight regarding initiation and progression of cell damage.

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Orientadores: Munir Salomão Skaf, Monica Andrea Pickholz / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-14T17:06:24Z (GMT). No. of bitstreams: 1 Prates_EricaTeixeira_M.pdf: 4627606 bytes, checksum: 4caf846b01f9586ee62d25bee7d6bdc4 (MD5) Previous issue date: 2009 / Resumo: Neste trabalho foi feito o estudo das interações da articaína, um anestésico local de ampla aplicação médico-odontológica, com membranas modelo de POPC (palmitoil-oleil-fosfatidilcolina) em condições próximas às biologicamente relevan- tes empregando-se simulações computacionais de dinâmica molecular. Em uma primeira etapa, empregamos métodos quânticos para modelar a articaína com base no campo de força CHARMM. Das simulações de equilíbrio da articaína em POPC, foi possível obter informações como o seu comportamento conformacional e sua posi- ção transversal na bicamada, assim como suas interações especícas com os lipídios. Os estudos foram realizados para os estados neutro e protonado da articaína, consi- derando também seus isômeros ópticos. Estas análises, em conjunto com resultados experimentais de H-RMN realizados pela Prof. Eneida de Paula (IB-UNICAMP) e pelo Prof. Leonardo F. Fraceto (Dpto. de Eng. Ambiental - UNESP, Sorocaba - SP), demonstram que a articaína, em sua forma neutra, posiciona-se preferencial- mente na interface membrana/água, onde interage frequentemente com os lipídios através de ligações de hidrogênio. Através de ferramentas como perfil de densidade eletrônica do sistema, da parte teórica, e perfil do tempo de relaxação longitudinal para diferentes regiões dos lipídios, da parte experimental, foi discutida a lipossolubilidade da articaína em relação a outros anestésicos. Também foram realizadas simulações de não equilíbrio, utilizando a técnica de Dinâmica Molecular de Caminho Induzido, em que uma molécula de articaína foi removida do interior da membrana para o meio aquoso, através de uma força aplicada em seu centro de massa. Com a aplicação da igualdade de Jarzynski a estas simulações, foi possível estimar a energia livre de partição da ATC neutra (forma mais potente) entre os estados em que encontra-se no seio aquoso e no interior da membrana POPC. / Abstract: We studied the interactions of articaine - a local anesthetic widely used for me- dical and odontological applications - with model membranes of POPC (palmitoyl-oleyl-phosphatidylcholine) at biological relevant conditions. We have employed molecular dynamics technique, which allowed us to investigate the system at molecular level. Firstly, we applied quantum mechanical methods to parametrize articaine molecule based on CHARMM force field. We have done extensive molceular dynamics simulations, taking into account the different ionization states of the drug (neutral and protonated) as well as its optical isomers. From the equilibirum simulations of articaine in POPC membranes, we investigated the conformational behaviour of the drug, its tranversal position and its specific interactions with the lipids and water molecules. Our results show a preferential orientation of the articaine molecule within the membrane. Neutral articaine was mainly found at the lipid head/water interface, in very good agreement with H-RMN experimental results from Prof. Eneida de Paula (IB-UNICAMP) and Prof. Leonardo F. Fraceto (Dpto. de Eng. Ambiental - UNESP, Sorocaba - SP) and from literature (C. Song et al, 2008). By studying properties like electronic density prole and longitudi- nal time relaxation for different regions of the lipid molecules, we discussed the lipossolubility of articaine in comparison to other local anesthetics. We have also performed non-equilibrium simulations, using steered molecular dynamics (SMD) technique. A single articaine molecule was extracted from the membrane to the wa- ter phase, by applying an external force in its mass centre. Coupling the Jarzynski identity to the SMD simulations, we estimated the partition free energy of the neutral drug (the most potent specie) in POPC membranes. / Mestrado / Físico-Química / Mestre em Química

Indiana University-Purdue University Indianapolis (IUPUI) / Tissue cells display mechanosensitivity in their ability to discern and respond to changes in the viscoelastic properties of their surroundings. By anchoring and pulling, cells are capable of translating mechanical stimuli into a biological response through a process known as mechanotransduction, a pathway believed to critically impact cell adhesion, morphology and multiple cellular processes from migration to differentiation. While previous studies on polymeric gels have revealed the influence of substrate elasticity on cellular shape and function, a lack of suitable substrates (i.e. with mobile cell-substrate linkers) has hindered research on the role of substrate viscosity. This work presents the successful design and characterization of lipid-bilayer based cell substrates of tunable viscosity affecting cell-substrate linker mobility through changes in viscous drag. Here, two complementary membrane systems were employed to span a wide range of viscosity. Single polymer-tethered lipid bilayers were used to generate subtle changes in substrate viscosity while multiple, polymer-interconnected lipid bilayer stacks were capable of producing dramatic changes in substrate viscosity. The homogeneity and integrity of these novel multibilayer systems in the presence of adherent cells was confirmed using optical microscopy techniques. Profound changes in cellular growth, phenotype and cytoskeletal organization confirm the ability of cells to sense changes in viscosity. Moreover, increased migration speeds coupled with rapid area fluctuations suggest a transition to a different migration mode in response to the dramatic changes in substrate viscosity.

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Two-dimensional (2D) nanoscale confinement effects on poly(ε-caprolactone) (PCL) crystallization were probed through crystallization studies of PCL-b-poly(tert-butyl acrylate) (PCL-b-PtBA) copolymers, PCL with bulky tri-tert-butyl ester endgroups (PCL triesters), PCL with triacid end groups (PCL triacids), and magnetic nanoparticles stabilized by PCL triacid (PCL MNPs) at the air/water (A/W) interface. Thermodynamic analyses of surface pressure-area per monomer (Π−A)) isotherms for the Langmuir films at the A/W interface showed that PCL-b-PtBA copolymers, PCL triheads and PCL MNPs all formed homogenous monolayers below the dynamic collapse pressure of PCL, Π_C ~11 mN•m⁻¹. For compression past the collapse point, the PCL monolayers underwent a phase transition to three-dimensional (3D) crystals and the nanoscale confinements impacted the PCL crystalline morphologies. Studies of PCL-b-PtBA copolymers revealed that the morphologies of the LB-films became smaller and transitioned to dendrites with defects, stripes and finally nano-scale cylindrical features as the block length of PtBA increased. For the case of PCL triester, irregularly shaped crystals formed at the A/W interface and this was attributed to the accumulation of bulky tert-butyl ester groups around the crystal growth fronts. In contrast, regular, nearly round-shaped lamellar crystals were obtained for PCL triacids. These morphological differences between PCL triacids and PCL triesters were molar mass dependent and attributed to differences in dipole density and the submersion of carboxylic acid groups in the subphase. Nonetheless, enhanced uniformity for PCL triacid crystals was not retained once the polymers were tethered to the spherical surface of a PCL MNP. Instead, the PCL MNPs exhibited small irregularly shaped crystals. This nano-scale confinement effect on the surface morphology at the A/W interface was also molar mass dependent. For the small molar mass PCL MNPs, two layers of collapsed nanoparticles were observed. In a later chapter, studies of polyethylene glycol (PEG) surfactant adsorption onto phospholipid bilayers through quartz crystal microbalance with dissipation monitoring (QCM-D) measurements revealed a strong dependence of the adsorption and desorption kinetics on hydrophobic tail group structure. PEG surfactants with a single linear alkyl tail inserted and saturated the bilayer surface quickly and the surfactants had relatively fast desorption rates. In contrast, PEG lipids, including dioleoyl PEG lipids and cholesterol PEGs, demonstrated slower adsorption and desorption kinetics. The interactions of Pluronics and Nonoxynol surfactants with phospholipid bilayers were also studied. Pluronics showed no apparent affinity for the phospholipid bilayer, while the Nonoxynol surfactants damaged the lipid bilayers as PEG chain length decreased. / Ph. D.

<p>When drug molecules are passively absorbed through the cell membrane in the small intestine, the first key step is partitioning of the drug into the membrane. Partition data can therefore be used to predict drug absorption. The partitioning of a solute can be analyzed by drug partition chromatography on immobilized model membranes, where the chromatographic retention of the solute reflects the partitioning. The aims of this thesis were to develop the model membranes used in drug partition chromatography and to study the effects of different membrane components and membrane structures on drug partitioning, in order to characterize drug–membrane interactions.</p><p>Electrostatic effects were observed on the partitioning of charged drugs into liposomes containing charged detergent, lipid or phospholipid; bilayer disks; proteoliposomes and porcine intestinal brush border membrane vesicles (BBMVs), and on the retention of an oligonucleotide on positive liposomes. Biological membranes are naturally charged, which will affect drug partitioning in the human body.</p><p>Proteoliposomes containing transmembrane proteins and cholesterol, BBMVs and bilayer disks were used as novel model membranes in drug partition chromatography. Partition data obtained on proteoliposomes and BBMVs demonstrated how cholesterol and transmembrane proteins interact with drug molecules. Such interactions will occur between drugs and natural cell membranes. In the use of immobilized BBMVs for drug partition chromatography, yet unsolved problems with the stability of the membrane were encountered. A comparison of partition data obtained on bilayer disks with data on multi- and unilamellar liposomes indicated that the structure of the membrane affect the partitioning. The most accurate partition values might be obtained on bilayer disks.</p><p>Drug partition data obtained on immobilized model membranes include both hydrophobic and electrostatic interactions. Such partition data should preferably be used when deriving algorithms or computer programs for prediction of drug absorption.</p>

When drug molecules are passively absorbed through the cell membrane in the small intestine, the first key step is partitioning of the drug into the membrane. Partition data can therefore be used to predict drug absorption. The partitioning of a solute can be analyzed by drug partition chromatography on immobilized model membranes, where the chromatographic retention of the solute reflects the partitioning. The aims of this thesis were to develop the model membranes used in drug partition chromatography and to study the effects of different membrane components and membrane structures on drug partitioning, in order to characterize drug–membrane interactions. Electrostatic effects were observed on the partitioning of charged drugs into liposomes containing charged detergent, lipid or phospholipid; bilayer disks; proteoliposomes and porcine intestinal brush border membrane vesicles (BBMVs), and on the retention of an oligonucleotide on positive liposomes. Biological membranes are naturally charged, which will affect drug partitioning in the human body. Proteoliposomes containing transmembrane proteins and cholesterol, BBMVs and bilayer disks were used as novel model membranes in drug partition chromatography. Partition data obtained on proteoliposomes and BBMVs demonstrated how cholesterol and transmembrane proteins interact with drug molecules. Such interactions will occur between drugs and natural cell membranes. In the use of immobilized BBMVs for drug partition chromatography, yet unsolved problems with the stability of the membrane were encountered. A comparison of partition data obtained on bilayer disks with data on multi- and unilamellar liposomes indicated that the structure of the membrane affect the partitioning. The most accurate partition values might be obtained on bilayer disks. Drug partition data obtained on immobilized model membranes include both hydrophobic and electrostatic interactions. Such partition data should preferably be used when deriving algorithms or computer programs for prediction of drug absorption.

Nonequilibrium work methods for determining potentials of mean force (PMF) w(z) have recently gained popularity as an alternative to standard equilibrium based methods. Introduced by Kosztin et al., the forward-reverse (FR) method is a bidirectional work method in that it requires the work to be sampled in both forward and reverse directions along the reaction coordinate z. This bidirectional sampling leads to much faster convergence than other nonequilibrium methods such as the Jarzynski equality, and the calculation itself is extremely simple, making the FR method an attractive way of determining the PMF. Presented here is an extension to the FR method that deals with sampling problems along essentially irreversible reaction coordinates. By oscillating a particle as it is steered along a reaction coordinate, both forward and reverse work samples are obtained as the particle progresses. Dubbed the oscillating forward-reverse (OFR) method, this new method overcomes the issue of irreversibility that is present in numerous soft-matter and biological systems, particularly in the stretching or unfolding of proteins. The data analysis of the OFR method is non-trivial however, and to this end a software package named the ‘OFR Analysis Tool’ has been created. This software performs all of the complicated analysis necessary, as well as a complete error analysis that considers correlations in the data, thus streamlining the use of the OFR method for potential end users. Another attractive feature of the FR method is that the dissipative work is collected at the same time as the free energy changes, making it possible to also calculate local diffusion coefficients, D(z), from the same simulation as the PMF through the Stokes-Nernst-Einstein relation Fdrag = −γv, with γ = kB T /D. While working with the OFR method, however, the D(z) results never matched known values or those obtained through other methods, including the mean square displacement (or Einstein) method. After a reformulation of the procedure to obtain D(z), i.e. by including the correct path length and particle speeds, results were obtained that were much closer to the correct values. The results however showed very little variation over the length of the reaction coordinate, even when D(z) was known to vary drastically. It seemed that the highly variable and noncontinuous velocity function of the particle being steered through the “stiff-spring” method was incompatible with the macroscopic definition of the drag coefficient, γ. The drag coefficient requires at most a slowly varying velocity so that the assumption of a linearly related dissipative work remains valid at all times. To address this, a new dynamic constraint steering protocol (DCP) was developed to replace the previously used “stiff-spring” method, now referred to as a dynamic restraint protocol (DRP). We present here the results for diffusion in bulk water, and both the PMF and diffusion results from the permeation of a water molecule through a DPPC membrane. We also consider the issue of ergodicity and sampling, and propose that to obtain an accurate w(z) (and D(z)) from even a moderately complex system, the final result should be a weighted average obtained from numerous pulls. An additional utility of the FR and OFR methods is that the permeability across lipid bilayers can be calculated from w(z) and D(z) using the inhomogeneous solubility-diffusion (ISD) model. As tests, the permeability was first calculated for H2O and O2 through DPPC. From the simulations, the permeability coefficients for H2O were found to be 0.129 ± 0.075 cm/s and 0.141 ± 0.043 cm/s, at 323 K and 350 K respectively, while the permeability coefficients for O2 were 114 ± 40 cm/s and 101 ± 27 cm/s, again at 323 K and 350 K respectively. As a final, more challenging system, the permeability of tyramine – a positively charged trace amine at physiological pH – was calculated. The final value of P = 0.89 ± 0.24 Ang/ns is over two orders of magnitude lower than that obtained from experiment (22 ± 4 Ang/ns), although it is clear that the permeability as calculated through the ISD is extremely sensitive to the PMF, as scaling the PMF by ∼ 20% allowed the simulation and experimental values to agree within uncertainty. With accurate predictions for free energies and permeabilities, the OFR method could potentially be used for many valuable endeavors such as rational drug design.

La membrane cellulaire est principalement une bicouche phospholipidique constituant une barrière qui régule les échanges entre la cellule et son environnement. Son intérieur hydrophobe empêche le passage d’espèces hydrophiles, chargées, de grande masse moléculaire et polaires, qui sont généralement transportées par des protéines à travers la bicouche. Dans certains cas de systèmes défectueux (e.g. les canalopathies), l’équilibre des concentrations en ions à l’intérieur et à l’extérieur des cellules est perturbé et les cellules sont compromises. C’est pourquoi le développement de transporteurs transmembranaires synthétiques est nécessaire. De nombreux travaux ont été faits dans le développement de transporteurs synthétiques d’anions (particulièrement du chlorure). Dans cette thèse, nous présentons nos travaux sur un nouveau transporteur d’anion appelé axe parapluie, capable de changer de conformation dépendamment de la polarité de son environnement. Dans un premier temps, nous avons conçu le design, puis synthétisé ces axes parapluie qui montrent une importante activité en tant que transporteur de chlorures. Ces composés réunissent deux concepts : - Le parapluie, constitué d’acides biliaires amphiphiles (une face hydrophile, une face hydrophobe). La flexibilité des articulations combinée à la grande surface des acides choliques permettent d’empêcher les interactions défavorables entre les parties hydrophiles et hydrophobes, ce qui facilite l’insertion dans la bicouche. - Un site ammonium secondaire en tant que site de reconnaissance, capable de former des ponts hydrogène avec des ions chlorure. De plus, l’axe peut complexer une roue de type éther couronne pour former un pseudo-rotaxane ou rotaxane parapluie ce qui résulte en l’inhibition partielle de leurs propriétés de transport. Ceci nous mène au second objectif de cette thèse, le développement d’un nouveau moyen de transport pour les médicaments cycliques. Certains macrocycles polaires et biologiquement actifs tels que les nactines ont besoin d’atteindre leur objectif à l’intérieur de la cellule pour jouer leur rôle. La membrane cellulaire est alors un obstacle. Nous avons imaginé tirer profit de notre axe parapluie pour transporter un médicament cyclique (en tant que roue d’un rotaxane parapluie). Les assemblages des rotaxanes parapluie furent accomplis par la méthode de clipage. Le comportement de l’axe et du rotaxane parapluie fut étudié par RMN et fluorimétrie. Le mouvement du parapluie passant d’une conformation fermée à exposée dépendamment du milieu fut observé pour le rotaxane parapluie. Il en fut de même pour les interactions entre le rotaxane parapluie et des vésicules constituées de phospholipides. Finalement, la capacité du rotaxane parapluie à franchir la bicouche lipidique pour transporter la roue à l’intérieur de la vésicule fut démontrée à l’aide de liposomes contenant de la α-chymotrypsine. Cette dernière pu cliver certains liens amide de l’axe parapluie afin de relarguer la roue. / The cell membrane is a phospholipid bilayer barrier that controls the exchanges between the cell and its environment. Its hydrophobic core prevents the entrance of hydrophilic, charged or large polar species that are transported through the bilayer by proteins. In some dysfunctional systems e.g. channelopathies), the balance of ion concentrations between the interior and exterior of the cell is no longer insured and the cell’s health is compromised. That is why the synthesis of synthetic transmembrane transporters is needed. There have been many synthetic anion carriers (especially chloride) developed in this area using different strategies. In this thesis we present our work on a new anion transporter, an umbrella thread. First, we designed and synthesized umbrella threads that showed significant chloride transport activity. These compounds combine two concepts: - the umbrella moiety, made from facial amphiphilic bile acids. The flexibility and large surface of the cholic acids can shield disfavored interactions between hydrophilic and hydrophobic elements that should ease their insertion into the bilayer. - a secondary ammonium recognition site on the thread that can form hydrogen bonds with chloride ions. Furthermore, the thread moiety is able to complex a crown-ether like wheel to form an umbrella pseudo-rotaxane or rotaxane that showed partially inhibited properties for chloride transport. This leads us to the second goal of this thesis, i.e. the development of a new vehicle for drug delivery. Some biologically active polar macrocycles (e.g. nactins) need to reach their target inside the cell to be efficient. The cell membrane also represents an obstacle here. In our work, we imagined using an umbrella thread as the vehicle for the cyclic drug as the wheel of the umbrella rotaxane). The umbrella rotaxanes were successfully assembled by the clipping method. The behavior of both the umbrella thread and umbrella rotaxane was extensively studied by NMR and fluorimetry. The umbrella motion from a shield conformation to an exposed one depending on the environment was observed for the rotaxane. Interactions between the umbrella rotaxane and phospholipid vesicles were also noticed. Finally, its ability to cross the lipid bilayer to deliver the wheel inside the vesicle was shown with α-chymotrypsin-filled liposome assays. This enzyme was able to cleave amide bonds on the umbrella thread to release the wheel.