RESUMEN
Paclitaxel (PTX) is one of the most widely utilized chemotherapeutics globally. However, the extremely poor water solubility of paclitaxel necessitates a mechanism of delivery within blood. Fluid lipid PTX nanocarriers (lipids in the chain-melted state) show promise as PTX delivery vectors, but remain limited by their solubility of PTX within the membrane. To improve pharmacokinetics, membrane surfaces are typically coated with polyethylene glycol (PEG). Recent work has demonstrated the generation of a population of micelles within fluid lipid formulations containing a 2kDa PEG-lipid at a 10 mol% ratio. Driven by the positive curvature of the PEG-lipid (i.e. area of head group > area of tails), micelle-containing formulations were found to exhibit significantly higher uptake in cancer cells, cytotoxicity, and in vivo antitumor efficacy compared to formulations containing solely liposomes. Here, we describe the custom synthesis of a library of high-curvature micelle-inducing PEG-lipids and examine the effects of PEG chain length, chain branching (single- or double-PEG-lipid), and cationic charge on PTX solubility and cytotoxicity. We examined PEG-lipids at standard (10 mol%) and high (100-x mol%, where x=PTX mol%) formulation ratios. Remarkably, all formulations containing the synthesized high-curvature PEG-lipids had improved PTX solubility over unPEGylated formulations and commercially available DOPE-5k. The highest PTX solubility was found within the 100-xPTX mol% PEG-lipid micellar formulations, with particles made from 2k2 (two PEG2k chains) encapsulating 13 mol% PTX for up to 24 h. The pancreatic cancer cell line PC3 exhibited higher sensitivity to formulations containing PEG-lipid at 100-xPTX mol%, the most potent of which being formulations made from 2k2 (IC50 = 14 nM). The work presented here suggests formulations employing high-curvature PEG-lipids, particularly the double-PEG-lipid 2k2, hold great potential as next-generation PTX delivery systems owing to their high PTX solubility, enhanced cell cytotoxicity, and ability for precision targeting by affixation of ligands to the PEG molecules.
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Tau, an intrinsically disordered neuronal protein and polyampholyte with an overall positive charge, is a microtubule (MT) associated protein, which binds to anionic domains of MTs and suppresses their dynamic instability. Aberrant tau-MT interactions are implicated in Alzheimer's and other neurodegenerative diseases. Here, we studied the interactions between full length human protein tau and other negatively charged binding substrates, as revealed by differential-interference-contrast (DIC) and fluorescence microscopy. As a binding substrate, we chose anionic liposomes (ALs) containing either 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS, -1e) or 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG, -1e) mixed with zwitterionic 1,2dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to mimic anionic plasma membranes of axons where tau resides. At low salt concentrations (0 to 10 mM KCl or NaCl) with minimal charge screening, reaction mixtures of tau and ALs resulted in the formation of distinct states of AL-tau complexes coexisting with liquid-liquid phase separated tau self-coacervates arising from the polyampholytic nature of tau containing cationic and anionic domains. AL-tau complexes exhibited distinct types of morphologies. This included, large ≈20-30 micron tau-decorated giant vesicles with additional smaller liposomes with bound tau attached to the giant vesicles, and tau-mediated finite-size assemblies of small liposomes. As the ionic strength of the solution was increased to near and above physiological salt concentrations for 1:1 electrolytes (≈150 mM), AL-tau complexes remained stable while tau self-coacervate droplets were found to dissolve indicative of breaking of (anionic/cationic) electrostatic bonds between tau chains due to increased charge screening. The findings are consistent with the hypothesis that distinct cationic domains of tau may interact with anionic lipid domains of the lumen facing monolayer of the axon plasma membrane suggesting the possibility of transient yet robust interactions at physiologically relevant ionic strengths.
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Lipids, and cationic lipids in particular are of interest as delivery vectors for hydrophobic drugs such as the cancer therapeutic paclitaxel, and the structures of lipid assemblies affect their efficacy. We investigated the effect of incorporating the multivalent cationic lipid MVL5 (+5e) and poly(ethylene glycol)-lipids (PEG-lipids), alone and in combination, on the structure of fluid-phase lipid assemblies of the charge-neutral lipid 1,2-dioleoyl-sn-glycero-phosphocholine (DOPC). This allowed us to elucidate lipid-assembly structure correlations in sonicated formulations with high charge density, which are not accessible with univalent lipids such as the well-studied DOTAP (+1e). Cryogenic transmission electron microscopy (cryo-TEM) allowed us to determine the structure of the lipid assemblies, revealing diverse combinations of vesicles and disc-shaped, worm-like, and spherical micelles. Remarkably, MVL5 forms an essentially pure phase of disc micelles at 50 mol % MVL5. At a higher (75 mol %) content of MVL5, short- and intermediate-length worm-like micellar rods were observed, and in ternary mixtures with PEG-lipid, longer and highly flexible worm-like micelles formed. Independent of their length, the worm-like micelles coexisted with spherical micelles. In stark contrast, DOTAP forms mixtures of vesicles, disc micelles, and spherical micelles at all studied compositions, even when combined with PEG-lipids. The observed similarities and differences in the effects of charge (multivalent versus univalent) and high curvature (multivalent charge versus PEG-lipid) on the assembly structure provide insights into parameters that control the size of fluid lipid nanodiscs, relevant for future applications.
Asunto(s)
Micelas , Fosfatidilcolinas , Fosfatidilcolinas/química , Ácidos Grasos Monoinsaturados , Microscopía Electrónica de Transmisión , Liposomas/químicaRESUMEN
Paclitaxel (PTX) is a hydrophobic small-molecule cancer drug that loads into the membrane (tail) region of lipid carriers such as liposomes and micelles. The development of improved lipid-based carriers of PTX is an important objective to generate chemotherapeutics with fewer side effects. The lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and glyceryl monooleate (GMO) show propensity for fusion with other lipid membranes, which has led to their use in lipid vectors of nucleic acids. We hypothesized that DOPE and GMO could enhance PTX delivery to cells through a similar membrane fusion mechanism. As an important measure of drug carrier performance, we evaluated PTX solubility in cationic liposomes containing GMO or DOPE. Solubility was determined by time-dependent kinetic phase diagrams generated from direct observations of PTX crystal formation using differential-interference-contrast optical microscopy. Remarkably, PTX was much less soluble in these liposomes than in control cationic liposomes containing univalent cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), which are not fusogenic. In particular, PTX was not substantially soluble in GMO-based cationic liposomes. The fusogenicity of DOPE and GMO is related to the negative spontaneous curvature of membranes containing these lipids, which drives formation of nonlamellar self-assembled phases (inverted hexagonal or gyroid cubic). To determine whether PTX solubility is governed by lipid membrane structure or by local intermolecular interactions, we used synchrotron small-angle X-ray scattering. To increase the signal/noise ratio, we used DNA to condense the lipid formulations into lipoplex pellets. The results suggest that local intermolecular interactions are of greater importance and that the negative spontaneous curvature-inducing lipids DOPE and GMO are not suitable components of liposomal carriers for PTX delivery.
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Antineoplásicos , Neoplasias , Paclitaxel , Liposomas , Solubilidad , MicelasRESUMEN
Paclitaxel (PTX) is a hydrophobic small-molecule cancer drug that loads into the membrane (tail) region of lipid carriers such as liposomes and micelles. The development of improved lipid-based carriers of PTX is an important objective to generate chemotherapeutics with fewer side effects. The lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and glyceryl monooleate (GMO) show propensity for fusion with other lipid membranes, which has led to their use in lipid vectors of nucleic acids. We hypothesized that DOPE and GMO could enhance PTX delivery to cells through a similar membrane fusion mechanism. As an important measure of drug carrier performance, we evaluated PTX solubility in cationic liposomes containing GMO or DOPE. Solubility was determined by time-dependent kinetic phase diagrams generated from direct observations of PTX crystal formation using differential-interference-contrast optical microscopy. Remarkably, PTX was much less soluble in these liposomes than in control cationic liposomes containing univalent cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), which are not fusogenic. In particular, PTX was not substantially soluble in GMO-based cationic liposomes. The fusogenicity of DOPE and GMO is related to the negative spontaneous curvature of membranes containing these lipids, which drives formation of nonlamellar self-assembled phases (inverted hexagonal or gyroid cubic). We used synchrotron small-angle x-ray scattering to determine whether PTX solubility is governed by lipid membrane structure (condensed with DNA in pellet form) or by local intermolecular interactions. The results suggest that local intermolecular interactions are of greater importance and that the negative spontaneous curvature-inducing lipids DOPE and GMO are not suitable components of lipid carriers for PTX delivery regardless of carrier structure.
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Aggregated and hyperphosphorylated Tau is one of the pathological hallmarks of Alzheimer's disease. Tau is a polyampholytic and intrinsically disordered protein (IDP). In this paper, we present for the first time experimental results on the ionic strength dependence of the radius of gyration (Rg) of human Tau 4RS and 4RL isoforms. Synchrotron X-ray scattering revealed that 4RS Rg is regulated from 65.4 to 58.5 Å and 4RL Rg is regulated from 70.9 to 57.9 Å by varying ionic strength from 0.01 to 0.592 M. The Rg of 4RL Tau is larger than 4RS at lower ionic strength. This result provides an insight into the ion-responsive nature of intrinsically disordered and polyampholytic Tau, and can be implicated to the further study of Tau-Tau and Tau-tubulin intermolecular structure in ionic environments.
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Proteínas Intrínsecamente Desordenadas , Sincrotrones , Humanos , Rayos XRESUMEN
Novel approaches are required to address the urgent need to develop lipid-based carriers of paclitaxel (PTX) and other hydrophobic drugs for cancer chemotherapy. Carriers based on cationic liposomes (CLs) with fluid (i.e., chain-melted) membranes (e.g., EndoTAG-1) have shown promise in preclinical and late-stage clinical studies. Recent work found that the addition of a cone-shaped poly(ethylene glycol)-lipid (PEG-lipid) to PTX-loaded CLs (CLsPTX) promotes a transition to sterically stabilized, higher-curvature (smaller) nanoparticles consisting of a mixture of PEGylated CLsPTX and PTX-containing fluid lipid nanodiscs (nanodiscsPTX). These CLsPTX and nanodiscsPTX show significantly improved uptake and cytotoxicity in cultured human cancer cells at PEG coverage in the brush regime (10 mol % PEG-lipid). Here, we studied the PTX loading, in vivo circulation half-life, and biodistribution of systemically administered CLsPTX and nanodiscsPTX and assessed their ability to induce apoptosis in triple-negative breast-cancer-bearing immunocompetent mice. We focused on fluid rather than solid lipid nanodiscs because of the significantly higher solubility of PTX in fluid membranes. At 5 and 10 mol % of a PEG-lipid (PEG5K-lipid, molecular weight of PEG 5000 g/mol), the mixture of PEGylated CLsPTX and nanodiscsPTX was able to incorporate up to 2.5 mol % PTX without crystallization for at least 20 h. Remarkably, compared to preparations containing 2 and 5 mol % PEG5K-lipid (with the PEG chains in the mushroom regime), the particles at 10 mol % (with PEG chains in the brush regime) showed significantly higher blood half-life, tumor penetration, and proapoptotic activity. Our study suggests that increasing the PEG coverage of CL-based drug nanoformulations can improve their pharmacokinetics and therapeutic efficacy.
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Antineoplásicos Fitogénicos , Neoplasias de la Mama , Ratones , Humanos , Animales , Femenino , Paclitaxel/química , Liposomas/química , Distribución Tisular , Caspasa 3 , Polietilenglicoles/química , Lípidos , Neoplasias de la Mama/tratamiento farmacológico , Portadores de Fármacos/química , Línea Celular Tumoral , Antineoplásicos Fitogénicos/químicaRESUMEN
Exosomes are cell-secreted vesicles less than ≈150 nm in size that contain gene-encoding and gene-silencing RNA and cytosolic proteins with roles in intercellular communication. Interest in the use of exosomes as targeted drug delivery vehicles has grown since it was shown that they can bind specific cells and deliver intact genetic material to the cytosol of target cells. We isolated extracellular vesicles (EVs), consisting of a mixture of exosomes and microvesicles, from prostate (PC3) and melanoma (M21) cancer cell lines using serial ultracentrifugation. Interrogation via western blot analysis confirmed enrichment of CD63, a widely recognized EV surface protein, in the EV pellet from both cell lines. Nanoparticle tracking analysis (NTA) of EV pellets revealed that the two cell lines produced distinct vesicle size profiles in the ≈30 nm to ≈400 nm range. NTA further showed that the fraction of exosomes to all EVs was constant, suggesting cellular mechanisms that control the fraction of secreted vesicles that are exosomes. Transmission electron microscopy (TEM) images of the unmodified PC3 EVs showed vesicles with cup-like (i.e., nanocapsule) and previously unreported prolate morphologies. The observed non-spherical morphologies for dehydrated exosomal vesicles (size ≈30-100 nm) are most likely related to the dense packing of proteins in exosome membranes. Solubility phase diagram data showed that EVs enhanced the solubility of paclitaxel (PTX) in aqueous solution compared to a water-only control. Combined with their inherent targeting and cytosol delivery properties, these findings highlight the potential advantages of using exosomes as chemotherapeutic drug carriers in vivo.
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Vesículas Extracelulares/química , Paclitaxel/química , Línea Celular Tumoral , Portadores de Fármacos/química , Vesículas Extracelulares/metabolismo , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patología , Microscopía Electrónica de Transmisión , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , SolubilidadRESUMEN
Cationic liposomes (CLs) are effective carriers of a variety of therapeutics. Their applications as vectors of nucleic acids (NAs), from long DNA and mRNA to short interfering RNA (siRNA), have been pursued for decades to realize the promise of gene therapy, with approvals of the siRNA therapeutic patisiran and two mRNA vaccines against COVID-19 as recent milestones. The long-term goal of developing optimized CL-based NA carriers for a broad range of medical applications requires a comprehensive understanding of the structure of these vectors and their interactions with cell membranes and components that lead to the release and activity of the NAs within the cell. Structure-activity relationships of lipids for CL-based NA and drug delivery must take into account that these lipids act not individually but as components of an assembly of many molecules. This review summarizes our current understanding of how the choice of the constituting lipids governs the structure of their CL-NA self-assemblies, which constitute distinct liquid crystalline phases, and the relation of these structures to their efficacy for delivery. In addition, we review progress toward CL-NA nanoparticles for targeted NA delivery in vivo and close with an outlook on CL-based carriers of hydrophobic drugs, which may eventually lead to combination therapies with NAs and drugs for cancer and other diseases.
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Lipid carriers of hydrophobic paclitaxel (PTX) are used in clinical trials for cancer chemotherapy. Improving their loading capacity requires enhanced PTX solubilization. We compared the time-dependence of PTX membrane solubility as a function of PTX content in cationic liposomes (CLs) with lipid tails containing one (oleoyl; DOPC/DOTAP) or two (linoleoyl; DLinPC/newly synthesized DLinTAP) cis double bonds by using microscopy to generate kinetic phase diagrams. The DLin lipids displayed significantly increased PTX membrane solubility over DO lipids. Remarkably, 8 mol% PTX in DLinTAP/DLinPC CLs remained soluble for approximately as long as 3 mol% PTX (the solubility limit, which has been the focus of most previous studies and clinical trials) in DOTAP/DOPC CLs. The increase in solubility is likely caused by enhanced molecular affinity between lipid tails and PTX, rather than by the transition in membrane structure from bilayers to inverse cylindrical micelles observed with small-angle X-ray scattering. Importantly, the efficacy of PTX-loaded CLs against prostate cancer cells (their IC50 of PTX cytotoxicity) was unaffected by changing the lipid tails, and toxicity of the CL carrier was negligible. Moreover, efficacy was approximately doubled against melanoma cells for PTX-loaded DLinTAP/DLinPC over DOTAP/DOPC CLs. Our findings demonstrate the potential of chemical modifications of the lipid tails to increase the PTX membrane loading while maintaining (and in some cases even increasing) the efficacy of CLs. The increased PTX solubility will aid the development of liposomal PTX carriers that require significantly less lipid to deliver a given amount of PTX, reducing side effects and costs.
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Antineoplásicos/administración & dosificación , Ácidos Linoleicos/química , Liposomas/química , Ácido Oléico/química , Paclitaxel/administración & dosificación , Antineoplásicos/toxicidad , Ácidos Grasos Monoinsaturados/química , Humanos , Células PC-3 , Paclitaxel/toxicidad , Fosfatidilcolinas/química , Compuestos de Amonio Cuaternario/químicaRESUMEN
Hierarchical assembly of building blocks via competing, orthogonal interactions is a hallmark of many of nature's composite materials that do not require highly specific ligand-receptor interactions. To mimic this assembly mechanism requires the development of building blocks capable of tunable interactions. In the present work, we explored the interplay between repulsive (steric and electrostatic) and attractive hydrophobic forces. The designed building blocks allow hydrophobic forces to effectively act at controlled, large distances, to create and tune the assembly of membrane-based building blocks under dilute conditions, and to affect their interactions with cellular membranes via physical cross-bridges. Specifically, we employed double-end-anchored poly(ethylene glycol)s (DEA-PEGs)-hydrophilic PEG tethers with hydrophobic tails on both ends. Using differential-interference-contrast optical microscopy, synchrotron small-angle X-ray scattering (SAXS), and cryogenic electron microscopy, we investigated the ability of DEA-PEGs to mediate assembly in the dilute regime on multiple length scales and on practical time scales. The PEG length, anchor hydrophobicity, and molar fraction of DEA-PEG molecules within a membrane strongly affect the assembly properties. Additional tuning of the intermembrane interactions can be achieved by adding repulsive interactions via PEG-lipids (steric) or cationic lipids to the DEA-PEG-mediated attractions. While the optical and electron microscopy imaging methods provided qualitative evidence of the ability of DEA-PEGs to assemble liposomes, the SAXS measurements and quantitative line-shape analysis in dilute preparations demonstrated that the ensemble average of loosely organized liposomal assemblies maintains DEA-PEG concentration-dependent tethering on defined nanometer length scales. For cationic liposome-DNA nanoparticles (CL-DNA NPs), aggregation induced by DEA-PEGs decreased internalization of NPs by cells, but tuning the DEA-PEG-induced attractions by adding repulsive steric interactions via PEG-lipids limited aggregation and increased NP uptake. Furthermore, confocal microscopy imaging together with colocalization studies with Rab11 and LysoTracker as markers of intracellular pathways showed that modifying CL-DNA NPs with DEA-PEGs alters their interactions with the plasma and endosomal membranes.
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Polímeros/química , ADN/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas/química , Microscopía Confocal , Nanopartículas/química , Células PC-3 , Tamaño de la Partícula , Propiedades de Superficie , Células Tumorales CultivadasRESUMEN
Poly(ethylene glycol) (PEG) is a polymer used widely in drug delivery to create "stealth" nanoparticles (NPs); PEG coatings suppress NP detection and clearance by the immune system and beneficially increase NP circulation time in vivo. However, NP PEGylation typically obstructs cell attachment and uptake in vitro compared to the uncoated equivalent. Here, we report on a cationic liposome (CL) NP system loaded with the hydrophobic cancer drug paclitaxel (PTX) in which PEGylation (i.e., PEG-CLPTX NPs) unexpectedly enhances, rather than diminishes, delivery efficacy and cytotoxicity to human cancer cells. This highly unexpected enhancement occurs even when the PEG-chains coating the NP are in the transition regime between the mushroom and brush conformations. Cryogenic transmission electron microscopy (TEM) of PEG-CLPTX NPs shows that PEG causes the proliferation of a mixture of sterically stabilized nanometer-scale vesicles and anisotropic micelles (e.g., bicelles). Remarkably, the onset of bicelles at sub-monolayer concentrations of the PEG coat has to our knowledge not been previously reported; it was previously thought that PEG-lipid in this composition regime was incorporated into vesicles but did not alter their shape. Confocal microscopy and flow cytometry reveal significantly greater PTX cell uptake from stabilized PEG-CLPTX NPs (vesicles and bicelles) in contrast to bare CLPTX NPs, which can aggregate in cell medium. This underscores the ability of steric stabilization to facilitate NP entry into cells via distinct size-dependent endocytic pathways, some of which cannot transport large NP aggregates into cells. This study highlights the value of understanding how PEGylation alters NP shape and structure, and thus NP efficacy, to design next-generation stealth drug carriers that integrate active cell-targeting strategies into NPs for in vivo delivery.
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Neoplasias , Polietilenglicoles/química , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Preparaciones de Acción Retardada/farmacología , Humanos , Liposomas , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Células PC-3 , Paclitaxel/química , Paclitaxel/farmacocinética , Paclitaxel/farmacologíaRESUMEN
Active targeting and precise control of drug release based on nanoparticle therapies are urgently required to precisely treat cancer. We have custom-synthesized a functional lipid (termed Fa-ONB) by introducing a folic acid-targeting group into an o-nitro-benzyl ester lipid. As designed, the liposomes formed by Fa-ONB combine active targeting and dual trigger release capabilities, which help to improve drug efficacy and reduce the toxicity of traditional liposomes. We first verified that both pH-induced hydrolysis and light treatment were able to cleave the Fa-ONB lipid. We then prepared a series of liposomes (termed FOBD liposomes) by compounding the Fa-ONB lipid with DOPC at different ratios. After encapsulation of doxorubicin (DOX), we found that the particle size of DOX-loaded FOBD liposomes (DOX/FOBD) first increased (290 to 700 nm) and then decreased again (to 400 nm) under continuous UV irradiation (120 min). The photocatalytic release efficiency under different pH conditions was investigated by dialysis experiments, and it was found that the release efficiency in an acidic environment was significantly increased relative to neutral pH. This pH-triggered release response helps distinguish pathological tissues such as lysosomal compartments and tumors. The light-induced formation of a DOX precipitate increases in efficiency with increasing UV exposure time as well as with increasing environmental acidity or alkalinity. In addition, confocal imaging and flow cytometry showed that the ability of FOBD lipids to actively target HeLa cells increased with increasing Fa-ONB lipid content. Real-time in vivo fluorescence small animal experiments proved targeting to tumors and pH- and photo-induced release properties. Furthermore, therapeutic experiments using a mouse model found a significant tumor inhibitory effect for DOX/FOBD55 liposomes with UV irradiation, clearly demonstrating the benefit of light treatment: the tumor size of the control (PBS) group was 7.59 times that of the light treatment group. Therefore, this research demonstrates the benefits of combining triggerable release functions and effective active tumor targeting in one small lipid molecule for precise cancer treatment.
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Doxorrubicina/análogos & derivados , Nanopartículas , Neoplasias Experimentales/tratamiento farmacológico , Animales , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Preparaciones de Acción Retardada/farmacología , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Liposomas , Células MCF-7 , Ratones , Nanopartículas/química , Nanopartículas/uso terapéutico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Polietilenglicoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this minireview, which is part of a special issue in honor of Jacob N. Israelachvili's remarkable research career on intermolecular forces and interfacial science, we present studies of structures, phase behavior, and forces in reaction mixtures of microtubules (MTs) and tubulin oligomers with either intrinsically disordered protein (IDP) Tau, cationic vesicles, or the polyamine spermine (4+). Bare MTs consist of 13 protofilaments (PFs), on average, where each PF is made of a linear stack of αß-tubulin dimers (i.e., tubulin oligomers). We begin with a series of experiments which demonstrate the flexibility of PFs toward shape changes in response to local environmental cues. First, studies show that MT-associated protein (MAP) Tau controls the diameter of microtubules upon binding to the outer surface, implying a shape change in the cross-sectional area of PFs forming the MT perimeter. The diameter of a MT may also be controlled by the charge density of a lipid bilayer membrane that coats the outer surface. We further describe an experimental study where it is unexpectedly found that the biologically relevant polyamine spermine (+4e) is able to depolymerize taxol-stabilized microtubules with efficiency that increases with decreasing temperature. This MT destabilization drives a dynamical structural transition where inside-out curving of PFs, during the depolymerization peeling process, is followed by reassembly of ring-like curved PF building blocks into an array of helical inverted tubulin tubules. We finally turn to a very recent study on pressure-distance measurements in bundles of MTs employing the small-angle X-ray scattering (SAXS)-osmotic pressure technique, which complements the surface-forces-apparatus technique developed by Jacob N. Israelachvili. These latter studies are among the very few which are beginning to shed light on the precise nature of the interactions between MTs mediated by MAP Tau in 37 °C reaction mixtures containing GTP and lacking taxol.
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Biopolímeros/química , Proteínas Intrínsecamente Desordenadas/química , Microtúbulos/química , Tubulina (Proteína)/química , Proteínas tau/química , Cationes , Paclitaxel/química , Conformación ProteicaRESUMEN
We report on the discovery of a new organized lipid-nucleic acid phase upon intercalation of blunt duplexes of short DNA (sDNA) within cationic multilayer fluid membranes. End-to-end interactions between sDNA leads to columnar stacks. At high membrane charge density, with the inter-sDNA column spacing (dsDNA) comparable but larger than the diameter of sDNA, a 2D columnar phase (i.e., a 2D smectic) is found similar to the phase in cationic liposome-DNA complexes with long lambda-phage DNA. Remarkably, with increasing dsDNA as the membrane charge density is lowered, a transition is observed to a 3D columnar phase of stacked sDNA. This occurs even though direct DNA-DNA electrostatic interactions across layers are screened by diffusing cationic lipids near the phosphate groups of sDNA. Softening of the membrane bending rigidity (κ), which further promotes membrane undulations, significantly enhances the 3D columnar phase. These observations are consistent with a model by Schiessel and Aranda-Espinoza where local membrane undulations, due to electrostatically induced membrane wrapping around sDNA columns, phase lock from layer-to-layer, thereby precipitating coherent "crystal-like" undulations coupled to sDNA columns with long-range position and orientation order. The finding that this new phase is stable at large dsDNA and enhanced with decreasing κ is further supportive of the model where the elastic cost of membrane deformation per unit area around sDNA columns (â κh2/dsDNA4, h2 = sum of square of amplitudes of the inner and outer monolayer undulations) is strongly reduced relative to the favorable electrostatic attractions of partially wrapped membrane around sDNA columns. The findings have broad implications in the design of membrane-mediated assembly of functional nanoparticles in 3D.
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ADN/química , Ácidos Grasos Monoinsaturados/química , Fosfatidilcolinas/química , Compuestos de Amonio Cuaternario/química , Liposomas/química , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Monitoring of nanoparticle-based therapy in vivo and controlled drug release are urgently needed for the precise treatment of disease. We have synthesized a multifunctional Gd-DTPA-ONB (GDO) lipid by introducing the Gd-DTPA contrast agent moiety into an o-nitro-benzyl ester lipid. By design, liposomes formed from the GDO lipid combine MRI tracking ability and dual-trigger release capabilities with maximum sensitivity (because all lipids bear the cleavable moiety) without reducing the drug encapsulation rate. We first confirmed that both photo-treatment and pH-triggered hydrolysis are able to cleave the GDO lipid and lyse GDO liposomes. We then investigated the efficiency of drug release via the combined release processes for GDO liposomes loaded with doxorubicin (DOX). Relative to neutral pH, the release efficiency in acidic environment increased by 10.4% (at pHâ¯=â¯6.5) and 13.3% (at pHâ¯=â¯4.2). This pH-dependent release response is conducive to distinguishing pathological tissue such as tumors and endolysosomal compartments. The photo-induced release efficiency increases with illumination time as well as with distance of the pH from neutral. Photolysis increased the release efficiency by 13.8% at pHâ¯=â¯4.2, which is remarkable considering the already increased amount of drug release in the acidic environment. In addition, the relaxation time of GDO liposomes was 4.1 times that of clinical Gd-DTPA, with brighter T1-weighted imaging in vitro and in vivo. Real-time MRI imaging and in vivo fluorescence experiments demonstrated tumor targeting and MRI guided release. Furthermore, significant tumor growth inhibition in a treatment experiment using DOX-loaded GDO liposomes clearly demonstrated the benefit of photo-treatment for efficacy: the tumor size in the photo-treatment group was 3.7 times smaller than in the control group. The present study thus highlights the benefit of the design idea of combining efficient imaging/guiding, targeting, and triggerable release functions in one lipid molecule for drug delivery applications.
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Medios de Contraste/química , Liposomas/química , Imagen por Resonancia Magnética/métodos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada/farmacología , Sistemas de Liberación de Medicamentos/métodos , Citometría de Flujo , Células HEK293 , Humanos , Indoles/farmacología , Células Asesinas Naturales/efectos de los fármacos , Polímeros/farmacologíaRESUMEN
Complex materials often achieve their remarkable functional properties by hierarchical assembly of building blocks via competing and/or synergistic interactions. Here, we describe the properties of new double-end-anchored poly(ethylene glycol)s (DEA-PEGs)-macromolecules designed to impart hydrophobically mediated tethering attractions between charged lipid membranes. We synthesized DEA-PEGs (MW 2000 (2K) and 4.6K) with two double-tail (symmetric) or a double-tail and a single-tail (asymmetric) hydrophobic end anchors and characterized their equilibrium and kinetic properties using small-angle X-ray scattering. Control multilayer membranes without and with PEG lipid (i.e., single-end-anchored PEG) swelled continuously, with the interlayer spacing increasing between 30 and 90 wt % water content due to electrostatic as well as, in the case of PEG lipid, steric repulsion. In contrast, interlayer spacings in lamellar membrane hydrogels containing DEA-PEGs expanded over a limited water dilution range and reached a "locked" state, which displayed a near constant membrane wall-to-wall spacing (δw) with further increases in water content. Remarkably, the locked state displays a simple relation to the PEG radius of gyration δw ≈ 1.6 RG for both 2K and 4.6K PEG. Nevertheless, δw being considerably less than the physical size of PEG (2(5/3)1/2 RG) is highly unexpected and implies that, compared to free PEG, anchoring of the PEG tether at both ends leads to a considerable distortion of the PEG conformation confined between layers. Significantly, the lamellar hydrogel may be designed to reversibly transition from a locked to an unlocked (membrane unbinding) state by variations in the DEA-PEG concentration, controlling the strength of the interlayer attractions due to bridging conformations. The findings with DEA-PEGs have broad implications for hydrophobic-mediated assembly of lipid- or surfactant-coated building blocks with distinct shape and size, at predictable spacing, in aqueous environments.
RESUMEN
Cationic liposome-nucleic acid (CL-NA) complexes, which form spontaneously, are a highly modular gene delivery system. These complexes can be sterically stabilized via PEGylation [PEG: poly (ethylene glycol)] into nanoparticles (NPs) and targeted to specific tissues and cell types via the conjugation of an affinity ligand. However, there are currently no guidelines on how to effectively navigate the large space of compositional parameters that modulate the specific and nonspecific binding interactions of peptide-targeted NPs with cells. Such guidelines are desirable to accelerate the optimization of formulations with novel peptides. Using PEG-lipids functionalized with a library of prototypical tumor-homing peptides, we varied the peptide density and other parameters (binding motif, peptide charge, CL/DNA charge ratio) to study their effect on the binding and uptake of the corresponding NPs. We used flow cytometry to quantitatively assess binding as well as internalization of NPs by cultured cancer cells. Surprisingly, full peptide coverage resulted in less binding and internalization than intermediate coverage, with the optimum coverage varying between cell lines. In, addition, our data revealed that great care must be taken to prevent nonspecific electrostatic interactions from interfering with the desired specific binding and internalization. Importantly, such considerations must take into account the charge of the peptide ligand as well as the membrane charge density and the CL/DNA charge ratio. To test our guidelines, we evaluated the in vivo tumor selectivity of selected NP formulations in a mouse model of peritoneally disseminated human gastric cancer. Intraperitoneally administered peptide-tagged CL-DNA NPs showed tumor binding, minimal accumulation in healthy control tissues, and preferential penetration of smaller tumor nodules, a highly clinically relevant target known to drive recurrence of the peritoneal cancer.
Asunto(s)
ADN , Técnicas de Transferencia de Gen , Liposomas , Nanopartículas , Péptidos , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Cationes , ADN/química , Terapia Genética/métodos , Humanos , Lípidos/química , Liposomas/química , Nanopartículas/química , Péptidos/químicaRESUMEN
Lipid-based particles are used worldwide in clinical trials as carriers of hydrophobic paclitaxel (PTXL) for cancer chemotherapy, albeit with little improvement over the standard-of-care. Improving efficacy requires an understanding of intramembrane interactions between PTXL and lipids to enhance PTXL solubilization and suppress PTXL phase separation into crystals. We studied the solubility of PTXL in cationic liposomes (CLs) composed of positively charged 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) and neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) as a function of PTXL membrane content and its relation to efficacy. Time-dependent kinetic phase diagrams were generated from observations of PTXL crystal formation by differential-interference-contrast microscopy. Furthermore, a new synchrotron small-angle x-ray scattering in situ methodology applied to DOTAP/DOPC/PTXL membranes condensed with DNA enabled us to detect the incorporation and time-dependent depletion of PTXL from membranes by measurements of variations in the membrane interlayer and DNA interaxial spacings. Our results revealed three regimes with distinct time scales for PTXL membrane solubility: hours for >3 mol% PTXL (low), days for ≈ 3 mol% PTXL (moderate), and ≥20 days for < 3 mol% PTXL (long-term). Cell viability experiments on human cancer cell lines using CLPTXL nanoparticles (NPs) in the distinct CLPTXL solubility regimes reveal an unexpected dependence of efficacy on PTXL content in NPs. Remarkably, formulations with lower PTXL content and thus higher stability show higher efficacy than those formulated at the membrane solubility limit of ≈3 mol% PTXL (which has been the focus of most previous physicochemical studies and clinical trials of PTXL-loaded CLs). Furthermore, an additional high-efficacy regime is seen on occasion for liposome compositions with PTXL ≥9 mol% applied to cells at short time scales (hours) after formation. At longer time scales (days), CLPTXL NPs with ≥3 mol% PTXL lose efficacy while formulations with 1-2 mol% PTXL maintain high efficacy. Our findings underscore the importance of understanding the relationship of the kinetic phase behavior and physicochemical properties of CLPTXL NPs to efficacy.
Asunto(s)
Liposomas/toxicidad , Neoplasias/patología , Paclitaxel/farmacología , Cationes , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalización , ADN/metabolismo , Humanos , Cinética , Liposomas/química , Paclitaxel/química , Dispersión del Ángulo Pequeño , Solubilidad , Sincrotrones , Difracción de Rayos XRESUMEN
Current activity in developing synthetic carriers of nucleic acids (NA) and small molecule drugs for therapeutic applications is unprecedented. One promising class of synthetic vectors for the delivery of therapeutic NA is PEGylated cationic liposome (CL)-NA nanoparticles (NPs). Chemically modified PEG-lipids can be used to surface-functionalize lipid-NA nanoparticles, allowing researchers to design active nanoparticles that can overcome the various intracellular and extracellular barriers to efficient delivery. Optimization of these functionalized vectors requires a comprehensive understanding of their intracellular pathways. In this chapter we present two distinct methods for investigating the intracellular activity of PEGylated CL-NA NPs using quantitative analysis with fluorescence microscopy.The first method, spatial localization, describes how to prepare fluorescently labeled CL-NA NPs, perform fluorescence microscopy and properly analyze the data to measure the intracellular distribution of nanoparticles and fluorescent signal. We provide software which allows data from multiple cells to be averaged together and yield statistically significant results. The second method, fluorescence colocalization, describes how to label endocytic organelles via Rab-GFPs and generate micrographs for software-assisted NP-endocytic marker colocalization measurements. These tools will allow researchers to study the endosomal trafficking of CL-NA NPs which can guide their design and improve their efficiency.