1,721,093 research outputs found
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Characterization and surface functionalization of self-assembling lipid- and surfactant- based materials for medical and consumer applications
Get PDFSurfactants and lipids are used in a broad range of applications from basic science to industrial hygiene and food products. These molecules are versatile building blocks for self-assembling materials, with tunable surface properties and nanostructure via the interplay of different forces. Further, surface modification of membranes with specialized lipids can allow for formation of complex, functional materials with hierarchical assembly. This work discusses two different surfactant-based systems and their tunable features.Cationic liposomes (CLs) are a common synthetic carrier of nucleic acids (NA) for gene delivery and silencing. Optimization of NA delivery and expression requires understanding of the interactions between the lipid nanoparticles (NPs) and cellular membranes, affecting NP binding, uptake, endocytic trafficking, and endosomal escape. PEG(polyethylene glycol)-lipid molecules can also be distally modified with peptide binding groups in order to target to different tissue types via specific protein-ligand interactions. In our first study, we modulate the specific and nonspecific binding interactions between peptide-targeted NPs and cells and use flow cytometry to measure the uptake of targeted NPs by several cancer cell lines in vitro. Several optimized formulations were subsequently evaluated in vivo for tumor selectivity, with promising results. After CL-NA NPs bind to targeted cells, surface properties of the NP are still critical for endosomal escape and NA cargo delivery. In two studies, we tuned NP surface properties via addition of cationic lipids or PEG-lipid molecules modified with peptide or hydrophobic binding groups, which can tether the NPs to cellular membranes via polymer bridges, with the goal of promoting endosomal escape. The study on hydrophobically-modified PEG-lipids showed that small variations in chemical structure and membrane composition significantly altered the binding properties of CLs and CL-NA NPs, likely due to the equilibrium balance of different conformations of the PEG-lipid. Formulations were subsequently evaluated in vitro using confocal microscopy and particle localization software to demonstrate the effect of surface properties on cell binding and endosomal trafficking, with GFP-labeled Rab proteins utilized to identify different endocytic pathways.Finally, we explore inter-membrane interactions in a system of single-chain surfactants, similar to those used in the commercial beauty products (specifically, hair conditioner). This system is composed of two fatty alcohols, a monovalent cationic surfactant, and water. Small-angle x-ray scattering and polarized optical microscopy were used to determine structure and phase behavior across composition space. We observed at least four different phases as a function of membrane charge density (σ), including a chain-ordered lamellar phase (LCO) at low σ and a second lamellar phase (Lx) of intermediate order at high σ that coexists with a fiber-like phase of tubular morphology. This study led to the discovery of new structural features and unexpected membrane interactions that better informed our understanding of how composition determines structure and, ultimately, the viscoelastic properties of interest for this application
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Advancing Cationic Micelle Lipid Nanoparticles and Extracellular Vesicles as Vectors for Hydrophobic Anticancer Drug Delivery
Get PDFLipid nanoparticles are promising delivery vehicles for hydrophobic anti-cancer drugs that enable targeted accumulation of drug at specific tissues and reduced systemic side effects. However, their clinical success is limited by challenges in drug loading, targeting in vivo, and endosomal entrapment. Developing next generation lipid nanoparticles that overcome these challenges is critical to realize their full potential to transform cancer chemotherapy.This doctoral work establishes a protocol for isolation of extracellular vesicles (EVs) and expands their ability to load the hydrophobic cancer chemotherapy drug paclitaxel (PTX) for drug delivery applications. EVs are nanoscale, cell-secreted vesicles that facilitate intercellular communication. Exosomes are a subset of EVs with 30 to 150 nm diameters that have high potential as drug delivery vectors because they show low immunogenicity and cell-specific cytosolic delivery of their contents. A serial centrifugation and differential ultracentrifugation protocol was used to isolate vesicles from prostate and melanoma cancer cells that had protein enrichment, diameters, and high membrane rigidity consistent with exosomes. Despite achieving high yields of EVs for both cell types, we observed inefficient loading of PTX in isolated EVs, which restricts their therapeutic application. To overcome this, we adapted a Förster resonance energy transfer (FRET) based lipid mixing assay to study methods for fusion of EVs with synthetic, PTX-loaded liposomes to create hybrid PTX delivery vesicles. We discovered that acidic conditions enhanced the fusion of EVs with bare synthetic liposomes while avoiding contaminating depletants and preserving EV membrane proteins. Remarkably, acidic conditions also induced clustering of EVs with themselves. These findings reveal a previously unexplored protein-lipid or lipid-lipid component to EV content release and enables minimally perturbative modification of EV contents towards a hybrid drug delivery vesicle.In parallel, we investigated the capacity of cationic liposomes (CLs) and cationic lipid nanoparticles (CLNPs) with novel membrane lipids and micellar structures to load and deliver PTX to cancer cells in vitro and in vivo. We found that lipid tails containing two cis double bonds enhanced the PTX loading of CLs by nearly 3-fold over conventional single cis double bond tails used in clinical stage CLs currently (e.g. EndoTAG-1TM), while maintaining or enhancing their cytotoxic efficacy against cancer cells. With respect to lipid headgroups, prior work revealed that incorporation of polyethylene glycol (PEG)-conjugated lipids (PEGylation) above a threshold membrane content drives the formation of disc-shaped micelles (nanodiscs), a novel structure for CLNPs with fluid-phase membranes, and improves cellular uptake and cytotoxic efficacy of CLs. Building on this, we demonstrated that CLs with PEG-lipid contents above this threshold show 10 to 35-fold greater tumor accumulation than those with sub-threshold PEG-lipid contents in vivo. This led us to explore using the novel multivalent lipid MVL5 in PTX-loaded CLNPs following the observation that CLNPs containing 50 mol% MVL5 form particle populations consisting of entirely nanodiscs, with rod and spherical micelles forming at 75 mol% or greater MVL5. Unexpectedly, a nearly 2-fold improvement in PTX solubility was observed for micellar MVL5 CLNPs over CLs based on EndoTAG-1TM containing the univalent cationic lipid DOTAP. This enhanced solubility translated to improved cytotoxic efficacy at high PTX content in vitro, with PEGylation driving further improvement. Finally, we found that steric stabilization of sub-200 nm diameter particles by PEGylation significantly improves MVL5 CLNP cell uptake and penetration depth. This supports a model where the rate limiting steps of PTX delivery by CLNPs are diffusion of endocytic vesicles containing CLNPs across the actin mesh near the cell surface, combined with hopping of PTX from endosomal vesicle membranes to nearby microtubules. By identifying and enhancing physicochemical properties of CLNPs critical to their performance as PTX delivery vehicles, these findings provide actionable steps which would improve the cost, safety, and efficacy of CLNPs as hydrophobic drug delivery vehicles in clinical applications
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Probing forces generated and architectures mediated by Tau on microtubules
Get PDFTau, a developmentally regulated protein localized to the axon of mature neurons, stabilizes axonal microtubules but has been implicated in many neurodegenerative diseases (“tauopathies”) including Alzheimer’s, Pick’s, and, more recently, chronic traumatic encephalopathy. Despite its importance in both development and disease, difficulty in understanding Tau is due, in part, to its intrinsically disordered nature; Tau does not assume a secondary structure in solution. However, lack of structure does not imply lack of function, as Tau binds to microtubules, thereby regulating microtubule assembly/stability and affecting inter-microtubule interactions, although the latter remains controversial. Herein, through microscopy and synchrotron small-angle X-ray scattering of cell free Tau/microtubule reconstitutions under various conditions, we report on the nature of the Tau structure and Tau-mediated interactions between microtubules. By examining the force-response of Tau-coated paclitaxel-stabilized microtubules by osmotic depletants, we observed that longer isoforms of Tau at high (and physiologically relevant) coverage on microtubules more effectively sterically stabilize microtubules against microtubule bundling. This steric stabilization occurs by the amino-terminal tail of Tau assuming the conformational (and repulsive) properties of a polyelectrolyte brush. Furthermore, the coverage at which this transition into a polyelectrolyte brush occurs gives the first direct measurement of the size of the longer isoforms of Tau (~20-23 nm) on microtubule surfaces. To understand the molecular mechanism of Tau-mediated inter-microtubule interactions in dissipative, out-of-equilibrium conditions, we co-polymerized Tau with microtubules in the absence of stabilizing agents (i.e. paclitaxel) and found that Tau mediates microtubule bundles with resultant architectures mimicking fascicles of microtubules found in the axonal initial segment. These bundles confirmed an attractive component to the Tau-mediated microtubule interaction through an aggregate of sub-kBT interactions along the microtubule length heretofore unreported in intrinsically disordered systems. The interaction dependence on microtubule length reconciles previous (unsuccessful) attempts at reconstituting Tau-mediated bundles, as stabilizing agents often promoted more, but shorter, microtubules. These novel biophysical characterizations of Tau on the microtubule surface give insight to the physiological function of Tau inside the neuronal axon and represent possible properties to investigate the role of mutations and post-translational modifications of Tau that lead to neurodegenerative disease
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A novel role for free tubulin oligomers in cross-linking tau-mediated microtubule bundles: A structural study on protein assembly states
Get PDFMicrotubules are self-assembling cytoskeletal protein structures composed of tubulin dimer subunits and are ubiquitous in mammalian cells, where they serve a multitude of functions such as maintaining cellular shape, providing pathways for intracellular transport, and driving neuronal outgrowth in early development. Their ability to elongate with the addition of tubulin, shorten by peeling away individual or oligomeric subunits, and switch between these two states is called dynamic instability and provides a focal point for cellular regulation of microtubule-driven processes. Indeed, many microtubule-associated proteins (MAPs) are known to bind microtubules and alter dynamic instability by promoting or suppressing either the polymerization or depolymerization of individual microtubules. One heavily studied MAP is tau, an intrinsically disordered protein primarily localized to the axonal compartment of mature neurons. Scientific focus on the properties and function of tau stems from the discovery many decades ago of neurofibrillary tangles as a hallmark of Alzheimer’s disease and, later of tau’s involvement in those tangles and in several other neurodegenerative diseases. Pathological and physiological roles for tau have been studied extensively. For example, we know that different tau isoforms are differentially expressed in development and maturity, that tau binds to and modifies microtubule dynamic instability, and that chemical alterations to tau leading to dysfunction are sufficient to induce disease-like pathology in cell cultures, animal studies, and humans. One poorly understood tau function is its ability to mediate the phase separation and regular spacing of microtubules in string-like arrays or bundles. Such microtubule structures are found at the axon initial segment of mature neurons, where they are referred to as “fascicles,” and have been re-produced with the addition of tau in non-neuronal cell cultures and in cell-free protein experiments involving only tubulin and recombinant human tau. Interestingly, the sorting of tau to the axon has been shown to rely on the structural integrity of the AIS, including microtubules, and is dependent on binding of tau to microtubules, implying a possible link between tau-mediated microtubule bundling and the subcellular localization of tau itself. Thus, imperative to understanding the physiological relevance of tau-mediated microtubule bundling is determining the mechanism by which tau mediates inter-microtubule interactions within bundles.
Several models have been proposed, creating a dogma of tau-tau interactions driving bundling, but are incompatible with polyelectrolyte theory and the structural features of microtubule bundles, namely wall-to-wall distances between neighboring microtubules. To better understand the underlying mechanism of tau-mediated bundling, we sought to monitor the structures of microtubule bundles as a function of time in the presence of several factors or conditions we thought would alter the interactions underlying bundle formation. First, experiments were designed to probe the electrostatic-component of tau-mediated bundling. Several tau-tau models for microtubule bundling rely on the dipole-like distribution of charge along tau’s N-terminal tail, where a relative abundance of acidic residues are followed by an abundance of basic residues, providing a possible mechanism for two opposing tau molecules to overlap and favorably interact to hold adjacent microtubules together. By increasing the ionic strength of the buffers, we sought to weaken such charge-charge interactions, which would predict a decrease in bundling strength or an outright inability of tau to mediate bundle formation in high-salt buffers. Instead, our data show that wall-to-wall distances between microtubules is largely unaffected by increases in monovalent cation species. Interestingly, with the addition of excess divalent cations, Mg2+ or Ca2+, an unexpected transformation of the hexagonal bundle lattice was observed. Specifically, we found that above threshold concentrations of either divalent cation, a phase transition is induced as a function of time or increased cation content. The phase transition is marked by the sudden and simultaneous drop in average wall-to-wall spacing, increase in lattice parameter (bundle size, or number of microtubules per bundle), and proliferation of inverted tubulin rings.
Based on these results, we propose and test a model where free tubulin oligomers participate in and are necessary for the bundling of microtubules by tau protein. In our model, tau binds to multiple species of tubulin to join together free (non-lattice-bound) tubulin oligomers and lattice-bound tubulin within microtubules. The multivalent nature of tau’s interactions with various tubulin oligomers and microtubules creates a network of tubulin and tau that cross-links microtubules within bundles. Within this model, an absence or depletion of free tubulin should prevent tau-mediated bundling, while increases to the free tubulin content should enhance bundle properties. Consistent with this model, experiments designed to induce rapid microtubule depolymerization by dropping the sample temperature reproduced the phase transitions observed in the depolymerization events induced by divalent cations. Similarly, samples prepared with increasing GTP content showed resistance to microtubule depolymerization over time, corresponding to the delay and in some cases elimination of the phase transition. Taken together, our results and the model we propose for tau-mediated microtubule bundling represent a novel role for tubulin in the bundling process. We are excited by the prospect of these new findings and their physiological relevance both to the understanding of developmental and mature tau function in human neurons but also to tau dysfunction in neurodegenerative diseases
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Designing lipid nanoparticles toward targeted drug delivery: Fundamental studies identify key compositional properties to improve formulations for the hydrophobic cancer drug paclitaxel
Get PDFIn order to increase the efficacy of the cancer drug paclitaxel (PTX), scientists must develop more effective drug carriers that well solubilize PTX and achieve targeted delivery. Loading cytotoxic drugs like PTX into nanoparticles that are engineered to accumulate in cancer tissue by physical or chemical means is expected to increase local drug concentrations and minimize systemic side effects. Due to its hydrophobic nature, PTX loads into the actual membrane of lipid nanoparticles (LNPs)—closely associating with lipid tails—and has limited solubility in lipid membranes. In vitro cell viability experiments demonstrate a direct correlation between delivery efficacy and the duration of PTX solubility in LNPs. This study importantly reveals enhanced delivery when PTX is loaded below, rather than at, its drug-loading saturation level. Based on PTX loading into membranes, we expect and indeed observe that PTX solubility depends strongly on the types of lipids used in LNP formulations. Here we report the PTX-loading and delivery efficacy of a selection of lipids that enhance delivery of other therapeutics (hydrophilic drugs, nucleic acids) to see if they similarly improve PTX delivery: inverted cone-shaped lipids (DOPE, GMO), poly-unsaturated (18:2, 18:3) lipids, cationic lipids (DOTAP, MVL5), and polymer-conjugated lipids (2k and 5k PEG-lipids). We developed a complementary approach using physical and cell biology characterization methods to elucidate structure-function relationships of LNP composition. DIC microscopy observations are used to generate kinetic phase diagrams to compare PTX solubility in different LNP formulations. Small-angle x-ray scattering (SAXS) shows that PTX-loading thins lipid membranes and determines the self-assembly structures of LNPs. The in vitro delivery efficacy of LNP formulations is determined by incubating PTX-loaded LNPs with PC3 (prostate) and M21 (melanoma) immortalized human cancer cell lines and measuring the resultant cell death. Fluorescent microscopy and flow cytometry provide qualitative and quantitative information about how LNPs interact with cells based on their composition. Finally, cryoTEM images offer important clues about how LNP structure and stability directly impact cell interactions and PTX delivery. In particular, we highlight how LNP vesicles transition to various disk, worm, and sphere micelles as the fraction of cationic or PEG-lipid increases. Using these methods, we have found several promising strategies to improve LNPs for PTX delivery. Studying fundamental LNP properties to determine composition-function relationships is a smarter strategy than empirical discovery because it identifies specific ways to thoughtfully design better drug delivery systems
Ion specific effects in bundling and depolymerization of taxol-stabilized microtubules
No full textMicrotubules (MTs) are nanometer scale hollow cylindrical biological polyelectrolytes. They are assembled from alpha/beta-tubulin dimers, which stack to form protofilaments (PFs) with lateral interactions between PFs resulting in the curved MT. In cells, MTs and their assemblies are critical components in a range of functions from providing tracks for the transport of cargo to forming the spindle structure during mitosis. Previous studies have shown that while cations with valence equal to or larger than 3+ tend to assemble tight 3D bundles of taxol-stabilized MTs, certain divalent cations induce relatively loose 2D bundles of different symmetry (D. J. Needleman et al., Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 16099). Similarly, divalent cations form 2D bundles of DNA adsorbed on cationic membranes (I. Koltover et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 14046). The bundling behavior for these biological polyelectrolyte systems is qualitatively in agreement with current theory. Here, we present results which show that, unlike the case for DNA adsorbed on cationic membranes, bundling of taxol-stabilized MTs occurs only for certain divalent cations above a critical ion concentration (e. g. Ca2+, Sr2+, Ba2+). Instead, many divalent cations pre-empt the bundling transition and depolymerize taxol-stabilized MTs at a lower counterion concentration. Although previous cryogenic TEM has shown that, in the absence of taxol, Ca2+ depolymerizes MTs assembling in buffers containing GTP (guanosine triphosphate), our finding is surprising given the known stabilizing effects of taxol on GDP (guanosine diphosphate)-MTs. The ion concentration required for MT depolymerization decreases with increasing atomic number for the divalents Mg2+, Mn2+, Co2+, and Zn2+. GdCl3 (3+) is found to be extremely efficient at MT depolymerization requiring ion concentrations of about 1 mM, while oligolysine (2+), is observed not to depolymerize MTs at concentrations as high as 144 mM. The surprising MT depolymerization results are discussed in the context of divalents either disrupting lateral interactions between PFs (which are strengthened for taxol containing beta-tubulin), or interfering with taxol's ability to induce flexibility at the interface between two tubulin dimers in the same PF (which has been recently suggested as a mechanism by which taxol stabilizes MTs post-hydrolysis with the induced flexibility counteracting the kink between GDP-tubulin dimers in a PF)
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