Browsing by Subject "Liposomes"
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Item Open Access Bacterial pathogens deliver water- and solute-permeable channels to plant cells.(Nature, 2023-09) Nomura, Kinya; Andreazza, Felipe; Cheng, Jie; Dong, Ke; Zhou, Pei; He, Sheng YangMany animal- and plant-pathogenic bacteria use a type III secretion system to deliver effector proteins into host cells1,2. Elucidation of how these effector proteins function in host cells is critical for understanding infectious diseases in animals and plants3-5. The widely conserved AvrE-family effectors, including DspE in Erwinia amylovora and AvrE in Pseudomonas syringae, have a central role in the pathogenesis of diverse phytopathogenic bacteria6. These conserved effectors are involved in the induction of 'water soaking' and host cell death that are conducive to bacterial multiplication in infected tissues. However, the exact biochemical functions of AvrE-family effectors have been recalcitrant to mechanistic understanding for three decades. Here we show that AvrE-family effectors fold into a β-barrel structure that resembles bacterial porins. Expression of AvrE and DspE in Xenopus oocytes results in inward and outward currents, permeability to water and osmolarity-dependent oocyte swelling and bursting. Liposome reconstitution confirmed that the DspE channel alone is sufficient to allow the passage of small molecules such as fluorescein dye. Targeted screening of chemical blockers based on the predicted pore size (15-20 Å) of the DspE channel identified polyamidoamine dendrimers as inhibitors of the DspE/AvrE channels. Notably, polyamidoamines broadly inhibit AvrE and DspE virulence activities in Xenopus oocytes and during E. amylovora and P. syringae infections. Thus, we have unravelled the biochemical function of a centrally important family of bacterial effectors with broad conceptual and practical implications in the study of bacterial pathogenesis.Item Open Access Chapter 1 - Tubular liposomes with variable permeability for reconstitution of FtsZ rings.(Methods in enzymology, 2009-01) Osawa, Masaki; Erickson, Harold PWe have developed a system for producing tubular multilamellar liposomes that incorporate the protein FtsZ on the inside. We start with a mixture of spherical multilamellar liposomes with FtsZ initially on the outside. Shearing forces generated by applying a coverslip most likely distort some of the spherical liposomes into a tubular shape, and causes some to leak and incorporate FtsZ inside. We describe protocols for liposome preparation, and for preparing membrane-targeted FtsZ that can assemble contractile Z rings inside the tubular liposomes. We also describe the characterization of the multilamellar liposomes in terms of the permeability or leakiness for a small fluorescent dye and larger protein molecules. These liposomes may be useful for reconstitution of other biological systems.Item Open Access Computed tomography imaging of primary lung cancer in mice using a liposomal-iodinated contrast agent.(PLoS One, 2012) Badea, CT; Athreya, KK; Espinosa, G; Clark, D; Ghafoori, AP; Li, Y; Kirsch, DG; Johnson, GA; Annapragada, A; Ghaghada, KBPURPOSE: To investigate the utility of a liposomal-iodinated nanoparticle contrast agent and computed tomography (CT) imaging for characterization of primary nodules in genetically engineered mouse models of non-small cell lung cancer. METHODS: Primary lung cancers with mutations in K-ras alone (Kras(LA1)) or in combination with p53 (LSL-Kras(G12D);p53(FL/FL)) were generated. A liposomal-iodine contrast agent containing 120 mg Iodine/mL was administered systemically at a dose of 16 µl/gm body weight. Longitudinal micro-CT imaging with cardio-respiratory gating was performed pre-contrast and at 0 hr, day 3, and day 7 post-contrast administration. CT-derived nodule sizes were used to assess tumor growth. Signal attenuation was measured in individual nodules to study dynamic enhancement of lung nodules. RESULTS: A good correlation was seen between volume and diameter-based assessment of nodules (R(2)>0.8) for both lung cancer models. The LSL-Kras(G12D);p53(FL/FL) model showed rapid growth as demonstrated by systemically higher volume changes compared to the lung nodules in Kras(LA1) mice (p<0.05). Early phase imaging using the nanoparticle contrast agent enabled visualization of nodule blood supply. Delayed-phase imaging demonstrated significant differential signal enhancement in the lung nodules of LSL-Kras(G12D);p53(FL/FL) mice compared to nodules in Kras(LA1) mice (p<0.05) indicating higher uptake and accumulation of the nanoparticle contrast agent in rapidly growing nodules. CONCLUSIONS: The nanoparticle iodinated contrast agent enabled visualization of blood supply to the nodules during the early-phase imaging. Delayed-phase imaging enabled characterization of slow growing and rapidly growing nodules based on signal enhancement. The use of this agent could facilitate early detection and diagnosis of pulmonary lesions as well as have implications on treatment response and monitoring.Item Open Access Curved FtsZ protofilaments generate bending forces on liposome membranes.(The EMBO journal, 2009-11) Osawa, Masaki; Anderson, David E; Erickson, Harold PWe have created FtsZ-YFP-mts where an amphipathic helix on the C-terminus tethers FtsZ to the membrane. When incorporated inside multi-lamellar tubular liposomes, FtsZ-YFP-mts can assemble Z rings that generate a constriction force. When added to the outside of liposomes, FtsZ-YFP-mts bound and produced concave depressions, bending the membrane in the same direction as the Z ring inside liposomes. Prominent membrane tubules were then extruded at the intersections of concave depressions. We tested the effect of moving the membrane-targeting sequence (mts) from the C-terminus to the N-terminus, which is approximately 180 degrees from the C-terminal tether. When mts-FtsZ-YFP was applied to the outside of liposomes, it generated convex bulges, bending the membrane in the direction opposite to the concave depressions. We conclude that FtsZ protofilaments have a fixed direction of curvature, and the direction of membrane bending depends on which side of the bent protofilament the mts is attached to. This supports models in which the FtsZ constriction force is generated by protofilament bending.Item Open Access Drug Delivery and Anti-Vascular Effects of Temperature Sensitive Liposomal Doxorubicin(2010) Manzoor, Ashley AnneTraditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumor. Relying on the EPR effect (Enhanced Permeability and Retention), a host of nanoparticles (from micelles and dendrimers to liposomes and lipidic nanoparticles) have been developed and tested for passive accumulation into tumor interstitium. Unfortunately, most nanoparticles achieve only suboptimal drug delivery to tumors, due to heterogeneity of tumor vessel permeability, limited nanoparticle penetration, and relatively slow drug release. However, recent developments in nanoparticle technology have occurred with the design and testing of a fast drug-releasing liposome triggered by local heat. This temperature-sensitive liposome formulation loaded with doxorubicin (Dox-TSL) has already shown substantial anti-tumor efficacy and is currently in clinical trials.
Previous pre-clinical work to understand the mechanism of efficacy has illustrated increases in overall drug concentration in the tumor, and an anti-vascular effect not observed with heat alone. These initial studies have also suggested that these liposomes may be the most efficacious when they are injected into a pre-heated tumor, with the hypothesis that in this treatment scheme the liposomes may be releasing inside the tumor vasculature. However, whether intravascular release is indeed occurring, and the subsequent implications this paradigm change in drug delivery could have are still unanswered questions.
The experiments presented herein aimed to investigate two effects: the existence and influence of intravascular drug release on drug delivery and distribution within the tumor, and the effect of drug delivery on subsequent anti-vascular effects. To investigate drug delivery, two mouse models were used. Dorsal window chambers implanted with FaDu human squamous carcinomas were used with real-time intravital confocal microscopy to evaluate time-resolved delivery of doxorubicin and liposome extravasation over the first 20 minutes of treatment. As a complimentary mouse model, flank FaDu tumors were also treated with Dox-TSL or treatment controls (doxorubicin with and without heat and Doxil with heat), and subsequently sectioned and histologicaly imaged to evaluate drug delivery and penetration depth, as well as impact on hypoxia and perfusion parameters. To investigate vascular effects, a GFP-eNos transgenic mouse model was used, also with window chamber confocal microscopy, to evaluate morphological changes occurring in the tumor vasculature following treatment.
The results presented herein demonstrate that contrary to the traditional liposome paradigm of extravasation and subsequent drug release, thermally sensitive liposomes release drug inside the tumor vasculature, and that the released free drug diffuses into the tumor interstitium. Real-time confocal imaging of doxorubicin delivery to murine tumor window chambers illustrates that intravascular drug release provides a mechanism to increase both the time that tumor cells are exposed to maximum drug levels and the penetration distance achievable by free drug diffusion. Histological analysis further confirms this finding, illustrating that drug delivered with Dox-TSL intravascular release can result in drug penetration levels up to 80 µm from vessels, in comparison with 40 µm achievable with free drug with heat. Further, Dox-TSL delivers drug to a higher percentage of a tumor's hypoxic area than possible with free drug with or without heat. Endothelial cells display marked morphological changes apparent immediately following treatment, with significant vascular destruction at 6 hours. However, heat had a similar influence on vascular morphology, underscoring the complexity of the anti-vascular effect, particularly in the more sensitive vasculature of a mouse model compared with reported human vascular heat tolerances. This work establishes intravascular release as a new paradigm in drug delivery to solid tumors, resulting in improved drug bioavailability, penetration depth, and enhanced delivery of drug to hypoxic regions of tumors.
Item Open Access Dual-energy micro-CT functional imaging of primary lung cancer in mice using gold and iodine nanoparticle contrast agents: a validation study.(PLoS One, 2014) Ashton, Jeffrey R; Clark, Darin P; Moding, Everett J; Ghaghada, Ketan; Kirsch, David G; West, Jennifer L; Badea, Cristian TPURPOSE: To provide additional functional information for tumor characterization, we investigated the use of dual-energy computed tomography for imaging murine lung tumors. Tumor blood volume and vascular permeability were quantified using gold and iodine nanoparticles. This approach was compared with a single contrast agent/single-energy CT method. Ex vivo validation studies were performed to demonstrate the accuracy of in vivo contrast agent quantification by CT. METHODS: Primary lung tumors were generated in LSL-Kras(G12D); p53(FL/FL) mice. Gold nanoparticles were injected, followed by iodine nanoparticles two days later. The gold accumulated in tumors, while the iodine provided intravascular contrast. Three dual-energy CT scans were performed-two for the single contrast agent method and one for the dual contrast agent method. Gold and iodine concentrations in each scan were calculated using a dual-energy decomposition. For each method, the tumor fractional blood volume was calculated based on iodine concentration, and tumor vascular permeability was estimated based on accumulated gold concentration. For validation, the CT-derived measurements were compared with histology and inductively-coupled plasma optical emission spectroscopy measurements of gold concentrations in tissues. RESULTS: Dual-energy CT enabled in vivo separation of gold and iodine contrast agents and showed uptake of gold nanoparticles in the spleen, liver, and tumors. The tumor fractional blood volume measurements determined from the two imaging methods were in agreement, and a high correlation (R(2) = 0.81) was found between measured fractional blood volume and histology-derived microvascular density. Vascular permeability measurements obtained from the two imaging methods agreed well with ex vivo measurements. CONCLUSIONS: Dual-energy CT using two types of nanoparticles is equivalent to the single nanoparticle method, but allows for measurement of fractional blood volume and permeability with a single scan. As confirmed by ex vivo methods, CT-derived nanoparticle concentrations are accurate. This method could play an important role in lung tumor characterization by CT.Item Open Access Inside-out Z rings--constriction with and without GTP hydrolysis.(Molecular microbiology, 2011-07) Osawa, Masaki; Erickson, Harold PThe bacterial tubulin homologue FtsZ forms a ring-like structure called the Z ring that drives cytokinesis. We showed previously that FtsZ-YFP-mts, which has a short amphipathic helix (mts) on its C terminus that inserts into the membrane, can assemble contractile Z rings in tubular liposomes without any other protein. Here we study mts-FtsZ-YFP, where the membrane tether is switched to the opposite side of the protofilament. This assembled 'inside-out' Z rings that wrapped around the outside surface of tubular liposomes. The inside-out Z rings were highly dynamic, and generated a constriction force that squeezed the tubular liposomes from outside. This is consistent with models where the constriction force is generated by curved protofilaments bending the membrane. We used this system to test how GTP hydrolysis by FtsZ is involved in Z-ring constriction. Without GTP hydrolysis, Z rings could still assemble and generate an initial constriction. However, the constriction quickly stopped, suggesting that Z rings became rigidly stabilized in the absence of GTP hydrolysis. We propose that remodelling of the Z ring, mediated by GTP hydrolysis and exchange of subunits, is necessary for the continuous constriction.Item Open Access Liposomal Drug Delivery Mediated by MR-guided High Intensity Focused Ultrasound: Drug Dose Painting and Influence of Local Tissue Transport Parameters(2014) Yarmolenko, Pavel SergeyevichUse of chemotherapeutics in treatment of solid tumors suffers from insufficient and heterogeneous drug delivery, systemic toxicity and lack of knowledge of delivered drug concentration. The overall objectives of this work were: 1) to address these shortcomings through development and characterization of a treatment system capable of real-time spatiotemporal control of drug distribution and 2) to investigate the role of MR-image-able tissue transport parameters in predicting drug distribution following hyperthermia-triggered drug release from nanoparticles. Towards these objectives, a combination of potentially synergetic technologies was used: 1) image-able low temperature-sensitive liposomes (iLTSLs) for drug delivery, 2) quantitative drug delivery and transport parameter imaging with magnetic resonance imaging (MRI), and 3) control over drug release with magnetic resonance-guided high intensity focused ultrasound (MR-HIFU). The overall hypothesis of this work is that the drug distribution in the targeted zone spatially correlates with the image-able transport-related parameters as well as contrast enhancement due to release of contrast agent during treatment.
We began by developing and characterizing iLTSLs, which were designed using a lipid formulation similar to one that is in clinical trials in the US (ThermoDox®) and a gadolinium-based MR contrast agent that is in widespread clinical use (Prohance®) and least likelihood of toxicity due to nephrogenic systemic fibrosis (NSF). The resulting liposome was found to stably encapsulate both an anthracycline chemotherapeutic, doxorubicin, and the MR contrast agent. Release rates were similar for these two species in physiologic buffer as well as in human plasma. The next step towards control and imaging of release with this drug delivery system (DDS) was development of algorithms that allowed for large-volume mild hyperthermia with MR-HIFU that would be required to move this combination of technologies into the clinic.
Optimal drug delivery with iLTSL requires a sustained period of heating of the entire target to the range of temperatures that are optimal for liposomal release and maintenance of perfusion (40 - 45 C). The MR-HIFU technology was developed and used mainly for rapid thermal ablation or mechanical disruption of tissue in small ellipsoid volumes. Variability and size of common clinical lesions called for modifications that would enable stable conformal heating of large tumor volumes to the sub-ablative temperature range of mild hyperthermia (40 - 45 C). Therefore, we set out to develop an algorithm that would allow rapid attainment and maintenance of mild hyperthermia in larger volumes of variable shape that were typically encountered in the clinic. We approached this goal through a series of successive steps that addressed different aspects of mild hyperthermia treatment: 1) controlled heating to mild hyperthermia, 2) conformity of heating and 3) ability to heat large volumes.
To achieve controlled heating to mild hyperthermia we implemented a simple binary mild hyperthermia feedback mechanism that adequately maintained mild hyperthermia for extended periods of time in small ellipsoidal volumes. We then developed a conformal small-volume mild hyperthermia algorithm that could provide spatial control over heating in an environment with spatially heterogeneous perfusion. This algorithm used electronic steering of the HIFU focus to heat each MR image voxel with different power, depending on temperature measured within that voxel. Finally, to heat large volumes conformally, we developed an algorithm that combined mechanical displacement of the MR-HIFU transducer (to cover large areas) with electronic deflection of the HIFU beam (to heat sub-volumes conformally). This advancement allowed us to quickly attain mild hyperthermia (<8.1 min to steady state) in larger volumes (cross-sectional area = 8.4 cm, ~12 times larger than previous methods).
Following their characterization, we examined iLTSL pharmacokinetics and combined MR-HIFU large volume mild hyperthermia with iLTSL to deliver doxorubicin to large Vx2 carcinomas in the hindlimb muscle of rabbits. To determine MR image-able correlates to the intratumoral drug distribution, we assessed the spatial pattern of drug distribution with fluorescence microscopy and examined spatial correlations of this pattern to several parameters measured with MRI, including the spatial distributions of temperature, contrast enhancement following injection of iLTSL, dynamic contrast-enhanced MRI (DCE-MRI) parameters, and maps of apparent diffusion coefficient (ADC). Dynamic contrast-enhanced MRI parameters have been used extensively in literature to approximate a mixture of parameters critical to drug delivery, such as perfusion (F), permeability-vascular surface area product (PS) and vascular volume and ADC has been previously correlated with cellular density in tumors. Possible utility of such spatial correlations was examined for future use in treatment planning, intraprocedural feedback control and post-treatment evaluation.
Highly perfused peripheral regions of Vx2 tumors in rabbit hindlimb displayed high Ktrans and ve, indicative of high perfusion. Maps of ADC obtained with low b-values also showed high ADC in the periphery of these tumors, indicating high perfusion there. ADC maps that were weighted more towards diffusion (using higher b-values) showed that diffusion was largest in the tumor core, indicating destruction of the cellular membranes and greater mobility of water. Microscopic examination of excised tumors was spatially registered to the MRI datasets and showed that most of the tumor core is necrotic, though some highly vascularized and viable tissue was present in strands or segments that traversed the necrotic regions. Those segments also showed bright doxorubicin fluorescence following treatment with MR-HIFU and iLTSL. The two control groups - free drug and iLTSL without mild hyperthermia - showed minimal to no doxorubicin fluorescence in the tumor.
Susceptibility effects due to use of contrast agent caused large errors (up to 15 °C) in MR thermometry measurements. To address this phenomenon, experiments were designed to arrive at steady state heating (target temperature = 41 °C), and employ an algorithm to learn the spatiotemporal distribution of power that was needed to maintain steady state heating. This heating pattern was then played back several times to verify maintenance of steady state, and if satisfactory, image-able liposomes were injected. Since temperature feedback was replaced by the learned steady-state heating, injection of image-able liposomes likely did not alter the heating performance. Following injection, changes in T1 and magnetic susceptibility were most pronounced in regions that previously showed greatest enhancement during DCE-MRI and displayed larger values of ADC with perfusion-weighted, low b-value scans. Maps of T1 were obtained in real time using a variable flip angle sequence during heating, and were corrected for inhomogeneity of the B1 field and calibrated against a more accurate, T1 mapping technique.
After treatment with MR-HIFU and iLTSL, the drug was preferentially distributed in the viable tissue, in and around the tumor. Doxorubicin fluorescence was greatest in zones that were heated, though the drug distribution did not display a clear boundary between heated and unheated tissue. While iLTSL provided intraprocedural feedback via enhancement of T1-weighted image intensity, susceptibility-related effects of iLTSL on MR thermometry complicate their prospects of clinical use, where precise temperature feedback is required for control of therapy and MR thermometry techniques that are in widespread use would be affected. Spatial correlations between drug delivery with iLTSL and MR-imageable parameters may serve as a predictive tool to identify areas that will not receive adequate drug. Such a-priori knowledge of correlates to the approximate tumor drug distribution has the potential to inform treatment planning by revealing the extent to which drug dose could be painted with a combination of LTSL and MR-HIFU. These studies point to an adjustment of course in further development of drug dose painting this combination of technologies, towards informing treatment planning, and not only painting the dose, but predicting it. These results also point to the need to develop rational combinations other treatments, such as ablation and radiation, to treat regions that will not receive sufficient drug.
Item Open Access Micro-CT imaging of breast tumors in rodents using a liposomal, nanoparticle contrast agent.(Int J Nanomedicine, 2009) Samei, Ehsan; Saunders, Robert S; Badea, Cristian T; Ghaghada, Ketan B; Hedlund, Laurence W; Qi, Yi; Yuan, Hong; Bentley, Rex C; Mukundan, SrinivasanA long circulating liposomal, nanoscale blood pool agent encapsulating traditional iodinated contrast agent (65 mg I/mL) was used for micro-computed tomography (CT) imaging of rats implanted with R3230AC mammary carcinoma. Three-dimensional vascular architecture of tumors was imaged at 100-micron isotropic resolution. The image data showed good qualitative correlation with pathologic findings. The approach holds promise for studying tumor angiogenesis and for evaluating anti-angiogenesis therapies.Item Open Access Negative-stain electron microscopy of inside-out FtsZ rings reconstituted on artificial membrane tubules show ribbons of protofilaments.(Biophysical journal, 2012-07) Milam, Sara L; Osawa, Masaki; Erickson, Harold PFtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by negative-stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for negative-stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments.Item Open Access Recording Information into DNA(2009) Tanner, Maria ElisaThe objective of this research is to develop the concept of "genetic memory", the storage of information into genetic material, through the demonstration of the feasibility and benefits of recording the time-history of one or more environmental state variables into genetic material. First, the amount of information that could be stored into non-coding DNA using a lossy mechanism such as regulated diffusion is determined by developing a mathematical model. Next, a mechanism through which this concept of sensing, recording, and storing information on the nanoscale could be accomplished is proposed.
In conjunction with DNA, which is the actual means of storing information, the proposed approach for the realization of genetic memory also uses thermosensitive liposomes as a means of sensing state variables and acting as a valve to transport and release the DNA in response to the appropriate stimuli. Each variant of thermally sensitive liposomes contains a unique DNA sequence that serves as an identifier. Upon release, the DNA encounters ligase, ATP, and other cofactors and binds - preserving a record of the stimulus experienced by the liposomes. Liposomes, through careful design, can be developed to have unique transition temperatures at which the permeability rate of the contents encapsulated by the liposome increases. It is only at or above this transition temperature that a liposome will become porous.
Through modeling and experimentation, the feasibility of the liposome/DNA system as a mechanism for information storage is successfully demonstrated.
Item Open Access Unique Contributions of iDQC MR Contrast to Stimuli-Sensitive Liposomal Chemotherapy and Imaging(2012) Howell, Darya Elizabeth RezaLiposomes are excellent chemotherapy drug delivery agents, on the cutting edge of cancer treatment technology. Since liposomes are already used to deploy cancer drugs in patients, imaging capacity would make them dual-purpose "theranostic" vesicles. Intermolecular double quantum coherence (iDQC) MRI is uniquely suited to this application, as its contrast does not require any additional chemicals. Adding contrast agents to liposomes can be time-consuming, add to toxicity, interfere with membrane function, or adversely affect drug loading. Furthermore, iDQC contrast measures diffusion and thus directly depends on membrane permeability and related properties. In this set of experiments, it has been shown that iDQC signal from intra-liposomal water can be distinguished from that of bulk water, and that the T2 dynamics of intra-liposomal water are predictable and dependent on the percent of water encapsulated. These techniques to distinguish between water molecules based on their current physical circumstances lead to many novel possibilities in MRI, as nearly all the signal in conventional MRI is from water protons. Based on the signal to noise ratio in the aforementioned iDQC experiments, we predict that iDQC contrast from liposomes will be visible in vivo, and propose to prove this in a murine model. By examining intra-liposomal water, iDQC can be used to improve chemotherapy delivery via real time monitoring of liposome location and drug release.