Michael Wright

Lyn deficiency reduces GATA-1, EKLF and STAT5, and induces extramedullary stress erythropoiesis

In vitro studies have implicated the Lyn tyrosine kinase in erythropoietin signaling. In this study, we show that J2E erythroid cells lacking Lyn have impaired signaling and reduced levels of transcription factors STAT5a, EKLF and GATA-1. Since mice lacking STAT5, EKLF or GATA-1 have red cell abnormalities, this study also examined the erythroid compartment of Lyn−/− mice. Significantly, STAT5, EKLF and GATA-1 levels were appreciably lower in Lyn−/− erythroblasts, and the phenotype of Lyn−/− animals was remarkably similar to GATA-1low animals. Although young adult Lyn-deficient mice had normal hematocrits, older mice developed anemia. Grossly enlarged erythroblasts and florid erythrophagocytosis were detected in the bone marrow of mice lacking Lyn. Markedly elevated erythroid progenitors and precursor levels were observed in the spleens, but not bone marrow, of Lyn−/− animals indicating that extramedullary erythropoiesis was occurring. These data indicate that Lyn−/− mice display extramedullary stress erythropoiesis to compensate for intrinsic and extrinsic erythroid defects.

Thermosensitive, near-infrared-labeled nanoparticles for topotecan delivery to tumors.

Liposomal nanoparticles have proven to be versatile systems for drug delivery. However, the progress in clinic has been slower and less efficient than expected. This suggests a need for further development using carefully designed chemical components to improve usefulness under clinical conditions and maximize therapeutic effect. For cancer chemotherapy, PEGylated liposomes were the first nanomedicine to reach the market and have been used clinically for several years. Approaches toward targeted drug delivery using next generation thermally triggered nanoparticles are now in clinical trials. However, clinically tested thermosensitive liposomes (TSLs) lack the markers that allow tumor labeling and improved imaging for tissue specific applied hyperthermia. Here we describe the development of optically labeled TSLs for image guidance drug delivery and proof-of-concept results for their application in the treatment of murine xenograft tumors using the anticancer drug topotecan. These labeled TSLs also allow the simultaneous, real-time diagnostic imaging of nanoparticle biodistribution using a near-infrared (NIR; 750-950 nm) fluorophore coupled to a lipidic component of the lipid bilayer. When combined with multispectral fluorescence analysis, this allows for specific and high sensitivity tracking of the nanoparticles in vivo. The application of NIR fluorescence-labeled TSLs could have a transformative effect on future cancer chemotherapy.

Cytotoxicity of polycations: Relationship of molecular weight and the hydrolytic theory of the mechanism of toxicity

The mechanism of polycation cytotoxicity and the relationship to polymer molecular weight is poorly understood. To gain an insight into this important phenomenon a range of newly synthesised uniform (near monodisperse) linear polyethylenimines, commercially available poly(l-lysine)s and two commonly used PEI-based transfectants (broad 22kDa linear and 25kDa branched) were tested for their cytotoxicity against the A549 human lung carcinoma cell line. Cell membrane damage assays (LDH release) and cell viability assays (MTT) showed a strong relationship to dose and polymer molecular weight, and increasing incubation times revealed that even supposedly non-toxic low molecular weight polymers still damage cell membranes. The newly proposed mechanism of cell membrane damage is acid catalysed hydrolysis of lipidic phosphoester bonds, which was supported by observations of the hydrolysis of DOPC liposomes.

Multiple catalytic activities of Escherichia coli lysyl-tRNA synthetase (LysU) are dissected by site-directed mutagenesis

The heat‐inducible lysyl‐tRNA synthetase from Escherichia coli (LysU; EC6/1/1/6.html) converts ATP to diadenosine tri‐ and tetraphosphates (Ap3A/Ap4A) in the presence of l‐lysine/Mg2+/Zn2+. To understand LysU in more detail, 26 mutants were prepared: six of E264, four of R269 and sixteen mutants by alanine‐scanning of the inner shell/motif 2 loop. In the presence of glycerol and absence of exogenously added Zn2+/l‐lysine, we unexpectedly found that E264K catalysed the production of glycerol‐3‐phosphate, powered by ATP turnover to ADP. E264Q and E264N are also capable of this activity, but all three show little formation of Ap4A/Ap3A under normal conditions (additional Zn2+/l‐lysine/Mg2+). By contrast, wild‐type LysU has a weaker glycerol kinase‐like capability in the absence of Zn2+ and is dominated by Ap4A/Ap3A synthesis in its presence. Kinetic and isothermal titration calorimetry results suggest that E264 is a crucial residue for Zn2+ promotion of Ap4A/Ap3A synthesis. This is consistent with the hypothesis that E264 provides an anchor point for a Zn2+ ion complexed to the active site, with simultaneous coordination to the enzyme bound lysyl‐adenylate intermediate and secondary substrate ATP/ADP. The glycerol kinase‐like activity is uncovered on disruption of this specific coordination.

Focused ultrasound induced hyperthermia accelerates and increases the uptake of anti-HER-2 antibodies in a xenograft model

Image guided drug delivery has gained significant attention during the last few years. Labelling nanoparticles or macromolecules and monitoring their fate in the body provides information that can be used to modulate their biodistribution and improve their pharmacokinetics. In this study we label antibodies and monitor their distribution in the tumours post intravenous injection. Using Focused Ultrasound (FUS, a non-invasive method of hyperthermia) we increase the tumour temperature to 42°C for a short period of time (3-5min) and we observe an increased accumulation of labelled antibody. Repetition of focused ultrasound induced hyperthermic treatment increased still further the accumulation of the antibodies in the tumour. This treatment also augmented the accumulation of other macromolecules non-specific to the tumour, such as IgG and albumin. These effects may be used to enhance the therapeutic efficiency of antibodies and/or targeted nanoparticles.

CHAPTER 1:Image Guided Focused Ultrasound as a New Method of Targeted Drug Delivery

The field of image guided drug delivery has attracted significant interest for researchers from various disciplines. Imaging is used to guide ultrasound to mediate drug delivery improving drug disposition and achieve tissue or organ specific delivery. Targeting drug delivery can be largely beneficial for diseases usually treated with cytotoxic drugs such as chemotherapy or drugs that may affect healthy functions of organs or cells. The term “focal” drug delivery has been introduced to describe the focal targeting of drugs in specific regions with the help of imaging. An example of this method is the use of imaging and a novel non-invasive technique named focused ultrasound (FUS) in combination with Magnetic Resonance Imaging (MRI). The increased temperature induced by FUS (hyperthermia) can improve blood supply in tissues and therefore improve drug distribution. FUS has also been studied for effects on physiological barrier permeability such as the blood brain barrier (BBB). FUS has been utilised in combination with imaging and theranostics, such as labelled liposomes that respond to temperature increase. This strategy aims to trigger nanoparticles to release their cargo locally when hyperthermia is induced by FUS. MRI guided FUS drug delivery can improve drug bioavailability at targeted tissues and therefore improve the therapeutic profiles of drugs. This strategy can be translated to the clinic as MRgFUS is an established clinically approved approach. However, more basic research is required to understand its physiological mechanisms.

Image guided focused ultrasound delivery of macromolecular drugs in tumours

Drug delivery using focused ultrasound (focal drug delivery) has attracted significant interest during the last few years. Focused Ultrasound (FUS) can induce local tissue hyperthermia, increasing blood flow and vascular permeability, which then enhances the uptake of therapeutics by target tissues. Although a number of studies have focused on nanoparticles (e.g. thermosensitive liposomes) little work has been done on the effects of FUS on the uptake of macromolecular drugs into tumours. In this study we aim to understand the effect of FUS-induced hyperthermia on tumour vascular permeability, as measured by the increased uptake of labelled macromolecules in the tumour. Using optical imaging we monitor the labelled macromolecules tumour localisation in real time in both short (minutes to hours) and long (weeks) term.

Abstract 5398: Nanoparticles for image guided focused ultrasound drug delivery

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnThermally triggered, PEGylated drug- liposomes are currently in clinical trials. Conventional thermosensitive liposomes (TSLs) lack the labels that would allow tumor imaging upon intravenous injection and a better mark for tissue specific applied hyperthermia (image guidance). We suggest dual- labelled thermosensitive liposomes for focused ultrasound (FUS) induced hyperthermia, triggered-release and functional delivery of the therapeutic anti-cancer drugs to xenograft tumors in response to thermal stimuli in vivo. In addition to labeling for MR Imaging we introduce the use of near infrared (NIR) fluorophores that allow for real time imaging of nanoparticle behavior in the absence of any significant intrinsic tissue background fluorescence, with good to excellent transmission and reduced scattering throughout tissues. We have synthesized these labels as lipid-probe conjugates and using these labels and thermosensitive lipids we have prepared TSL nanoparticles. N`-XenoLight750-N,N-distearylamidomethylamine (XLA750.DSA) and Gadolinium(III) 2-{4,7-Bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N′-amidomethyl]-1,4,7,10-tetraazacyclododec-1-yl}acetic Acid (Gd.DOTA.DSA) were synthesized and characterized. Both lipids were used to prepare TSL nanoparticles carrying the anticancer drugs at substantial encapsulation efficiency. Nanoparticles were tested for their biophysical characteristics (thermal drug release, stability in plasma, size) in vitro. MRI contrast enhancement ability and Near-IR signal were tested in vitro and in vivo. Nanoparticles carrying drugs were injected intravenously in mice bearing tumors. Nanoparticle kinetics in tumors were assessed with optical imaging and at defined time intervals (post injection), FUS (TIPS Phillips: 1.4MHz) was applied for 3-5 min to induce a small increase in temperature from 37°C to 41°C. Optical imaging revealed both substantial nanoparticle accumulation and drug release immediately after and only in tumors treated with focused ultrasound induced (focal) hyperthermia. Tumors treated with both nanoparticles and FUS post injection showed substantial growth inhibition compared to tumors treated with nanoparticles only. The applications of these multifunctional nanoparticles with short and mild hyperthermia could have a transformative effect on cancer chemotherapy.nnCitation Format: Maya Thanou, Michael Wright, Miguel Centelles. Nanoparticles for image guided focused ultrasound drug delivery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5398. doi:10.1158/1538-7445.AM2014-5398

Zinc oxide nanoparticles as contrast‐enhancing agents for microwave imaging

PURPOSEnMicrowave imaging/sensing is an emerging technology that shows potential for healthcare diagnostic applications, particularly in breast cancer detection. This technique estimates the anatomically variant dielectric properties of the breast. Similar to other imaging modalities, nanoparticles (NPs) could potentially be utilized as contrast agents to increase contrast between healthy and malignant tissues.nnnMETHODSnIn this study, aqueous suspensions of NPs such as surface-modified single-walled carbon nanotubes, zinc oxide, and silicon dioxide are studied to assess their potential effective contrast for microwave imaging. Morphology characterization of the NPs has been achieved using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The size and stability of colloidal dispersions have been characterized by dynamic light scattering technique (DLS) and Ultraviolet-visible spectrophotometry (UV-Vis). The dielectric characterization of the aqueous-based colloidal suspensions is recorded over the microwave frequency range between 1 and 4xa0GHz.nnnRESULTSnZinc oxide NP dispersion has shown an increase in the dielectric constant compared to the background medium. Furthermore, PEGylation of ZnO NPs can achieve a valid increase in the dielectric constant compared to water, which was shown to be concentration dependent.nnnCONCLUSIONnThese results suggest that ZnO nanomaterials have the potential to be used in biomedical applications such as breast imaging to improve diagnostic capabilities.

Image-guided thermosensitive liposomes for focused ultrasound drug delivery: Using NIRF-labelled lipids and topotecan to visualise the effects of hyperthermia in tumours

Abstract Image guided drug delivery using imageable thermosensitive liposomes (iTSLs) and high intensity focused ultrasound (FUS or HIFU) has attracted interest as a novel and non‐invasive route to targeted delivery of anti‐cancer therapeutics. FUS‐induced hyperthermia is used as an externally applied “trigger” for the release of a drug cargo from within thermosensitive drug carriers. It is suggested that sub‐ablative hyperthermia significantly modifies the permeability of tumour vasculature and enhances nanoparticle uptake. Here we describe the preparation and use of magnetic resonance imaging (MRI) and near infrared fluorescence (NIRF) labelled thermosensitive liposomes for imaging and tracking of biodistribution and drug release in a murine cancer model. We prepared iTSLs to encapsulate topotecan (Hycamtin®), a chemotherapeutic agent which when released in tumours can be monitored by an increase in its intrinsic drug fluorescence. FUS was applied using feedback via subcutaneously placed fine‐wire thermocouples to maintain and monitor hyperthermic temperatures. iTSL accumulation was detected within tumours using NIRF imaging immediately after liposome administration. Mild FUS‐induced hyperthermia (3 min at 42 °C, 30 min post i.v. administration) greatly enhanced iTSLs uptake. A co‐localised enhancement of topotecan fluorescence emission was also observed immediately after application of FUS indicating rapid triggered drug release. The phenomena of increased iTSL accumulation and concomitant topotecan release appeared to be amplified by a second mild hyperthermia treatment applied one hour after the first. MRI in vivo also confirmed enhanced iTSLs uptake due to the FUS treatments. Our imaging results indicate the effects of hyperthermia on the uptake of carriers and drug. FUS‐induced hyperthermia combined with real time imaging could be used as a tool for tumour targeted drug delivery. Graphical abstract Figure. No caption available.