Richard J. Browning

Electrohydrodynamic encapsulation of cisplatin in poly (lactic-co-glycolic acid) nanoparticles for controlled drug delivery

Targeted delivery of potent, toxic chemotherapy drugs, such as cisplatin, is a significant area of research in cancer treatment. In this study, cisplatin was successfully encapsulated with high efficiency (>70%) in poly (lactic-co-glycolic acid) polymeric nanoparticles by using electrohydrodynamic atomization (EHDA) where applied voltage and solution flow rate as well as the concentration of cisplatin and polymer were varied to control the size of the particles. Thus, nanoparticles were produced with three different drug:polymer ratios (2.5, 5 and 10wt% cisplatin). It was shown that smaller nanoparticles were produced with 10wt% cisplatin. Furthermore, these demonstrated the best sustained release (smallest burst release). By fitting the experimental data with various kinetic models it was concluded that the release is dependent upon the particle morphology and the drug concentration. Thus, these particles have significant potential for cisplatin delivery with controlled dosage and release period that are crucial chemotherapy parameters.

Influence of needle gauge on in vivo ultrasound and microbubble-mediated gene transfection

Ultrasound and microbubble-mediated gene transfection are potential tools for safe, site-selective gene therapy. However, preclinical trials have demonstrated a low transfection efficiency that has hindered the progression of the technique to clinical application. In this paper it is shown that simple changes to the method of intravenous injection can lead to an increase in transfection efficiency when using 6-MHz diagnostic ultrasound and the ultrasound contrast agent, SonoVue. By using needles of progressively smaller gauge, i.e., larger internal diameter (ID), from 29 G (ID 0.184 mm) to 25 G (ID 0.31 mm), the transfection of a luciferase plasmid (pGL4.13) was significantly increased threefold in heart-targeted female CD1 mice. In vitro work indicated that the concentration and size distribution of SonoVue were affected by increasing needle gauge. These results suggest that the process of systemic delivery alters the bubble population and adversely affects transfection. This is exacerbated by using high-gauge needles. These findings demonstrate that the needle with the largest possible ID should be used for systemic delivery of microbubbles and genetic material.

Drug Delivery Strategies for Platinum-Based Chemotherapy

Few chemotherapeutics have had such an impact on cancer management as cis-diamminedichloridoplatinum(II) (CDDP), also known as cisplatin. The first member of the platinum-based drug family, CDDPs potent toxicity in disrupting DNA replication has led to its widespread use in multidrug therapies, with particular benefit in patients with testicular cancers. However, CDDP also produces significant side effects that limit the maximum systemic dose. Various strategies have been developed to address this challenge including encapsulation within micro- or nanocarriers and the use of external stimuli such as ultrasound to promote uptake and release. The aim of this review is to look at these strategies and recent scientific and clinical developments.

Effect of albumin and dextrose concentration on ultrasound and microbubble mediated gene transfection in vivo

Ultrasound and microbubble mediated gene transfection has great potential for site-selective, safe gene delivery. Albumin-based microbubbles have shown the greatest transfection efficiency but have not been optimised specifically for this purpose. Additionally, few studies have highlighted desirable properties for transfection specific microbubbles. In this article, microbubbles were made with 2% or 5% (w/v) albumin and 20% or 40% (w/v) dextrose solutions, yielding four distinct bubble types. These were acoustically characterised and their efficiency in transfecting a luciferase plasmid (pGL4.13) into female, CD1 mice myocardia was measured. For either albumin concentration, increasing the dextrose concentration increased scattering, attenuation and resistance to ultrasound, resulting in significantly increased transfection. A significant interaction was noted between albumin and dextrose; 2% albumin bubbles made with 20% dextrose showed the least transfection but the most transfection with 40% dextrose. This trend was seen for both nonlinear scattering and attenuation behaviour but not for resistance to ultrasound or total scatter. We have determined that the attenuation behaviour is an important microbubble characteristic for effective gene transfection using ultrasound. Microbubble behaviour can also be simply controlled by altering the initial ingredients used during manufacture.

Electrohydrodynamic fabrication of core-shell PLGA nanoparticles with controlled release of cisplatin for enhanced cancer treatment

Increasing the clinical efficacy of toxic chemotherapy drugs such as cisplatin (CDDP), via targeted drug delivery, is a key area of research in cancer treatment. In this study, CDDP-loaded poly(lactic-co-glycolic acid) (PLGA) polymeric nanoparticles (NPs) were successfully prepared using electrohydrodynamic atomization (EHDA). The configuration was varied to control the distribution of CDDP within the particles, and high encapsulation efficiency (>70%) of the drug was achieved. NPs were produced with either a core–shell (CS) or a matrix (uniform) structure. It was shown that CS NPs had the most sustained release of the 2 formulations, demonstrating a slower linear release post initial “burst” and longer duration. The role of particle architecture on the rate of drug release in vitro was confirmed by fitting the experimental data with various kinetic models. This indicated that the release process was a simple diffusion mechanism. The CS NPs were effectively internalized into the endolysosomal compartments of cancer cells and demonstrated an increased cytotoxic efficacy (concentration of a drug that gives half maximal response [EC50] reaching 6.2 µM) compared to free drug (EC50 =9 µM) and uniform CDDP-distributed NPs (EC50 =7.6 µM) in vitro. Thus, these experiments indicate that engineering the structure of PLGA NPs can be exploited to control both the dosage and the release characteristics for improved clinical chemotherapy treatment.

Characterization of Contrast Agent Microbubbles for Ultrasound Imaging and Therapy Research

The high efficiency with which gas microbubbles can scatter ultrasound compared with the surrounding blood pool or tissues has led to their widespread employment as contrast agents in ultrasound imaging. In recent years, their applications have been extended to include super-resolution imaging and the stimulation of localized bio-effects for therapy. The growing exploitation of contrast agents in ultrasound and in particular these recent developments have amplified the need to characterize and fully understand microbubble behavior. The aim in doing so is to more fully exploit their utility for both diagnostic imaging and potential future therapeutic applications. This paper presents the key characteristics of microbubbles that determine their efficacy in diagnostic and therapeutic applications and the corresponding techniques for their measurement. In each case, we have presented information regarding the methods available and their respective strengths and limitations, with the aim of presenting information relevant to the selection of appropriate characterization methods. First, we examine methods for determining the physical properties of microbubble suspensions and then techniques for acoustic characterization of both suspensions and single microbubbles. The next section covers characterization of microbubbles as therapeutic agents, including as drug carriers for which detailed understanding of their surface characteristics and drug loading capacity is required. Finally, we discuss the attempts that have been made to allow comparison across the methods employed by various groups to characterize and describe their microbubble suspensions and promote wider discussion and comparison of microbubble behavior.

Enhanced gene transfection in vivo using magnetic localisation of ultrasound contrast agents: Preliminary results

In previous work we demonstrated that microbubble mediated gene delivery can be enhanced in vitro through simultaneous exposure of cells to ultrasound and magnetic fields in the presence of magnetically loaded microbubble ultrasound contrast agents. The aim of this preliminary study was to investigate the feasibility of the technique for in vivo applications. Phospholipid coated microbubbles loaded with a hydrocarbon suspension of magnetic nanoparticles were prepared through sonication and sized using optical microscopy (concentration 1.4 × 108 bubbles/ml). Plasmid pGL4.13, which encodes for firefly luciferase, was prepared at a concentration of 4 μg/μl in endotoxin-free water. A Siemens Acuson Sequioa clinical imaging system with a 26 mm linear array transducer (15L8) was used throughout the investigation. 20, 6–8 week old CD1 female mice were injected with of 150 μl of microbubble suspension and 50 μl plasmid intra-venously through the tail vein. Mice were anaesthetized using isoflurance and imaged with the transducer above the left lung (14 MHz, 0.06 MI) to locate the thoracic region. Immediately following injection, a NdFeB permanent magnet was positioned over the right lung and the acoustic output was increased (H7MHz, 1.7 MI, focal depth 7.5 mm). Exposure to ultrasound and/or magnetic field was maintained for two minutes. 20 mice were exposed to ultrasound and magnetic field, two to ultrasound only and two to magnetic field only. On the third day post treatment, luciferase substrate (D-luciferin) was administered through intra-peritoneal injection and allowed to catalyse the transfected substrate for 10 minutes before animals were sacrificed and their organs recovered for individual bioluminescence imaging (IVIS 100, Xenogen) and quantification (Living Image Software, Xenogen). Animals treated with both ultrasound and the magnetic field showed transfection in the right lung, while no animals showed transfection in the contralateral organs. Of the 20 mice treated, 17 showed transfection at a level greater than for ultrasound alone and 12 greater than that of magnetic field alone. The results of this preliminary study indicate that microbubbles which include magnetic nanoparticles within their shells may be used to control the location of transfection in vivo. Further work is required to improve microbubble formulations and magnetic array design to allow more accurate targeting of transfection.

A versatile method for the preparation of particle-loaded microbubbles for multimodality imaging and targeted drug delivery

Microbubbles are currently in clinical use as ultrasound contrast agents and under active investigation as mediators of ultrasound therapy. To improve the theranostic potential of microbubbles, nanoparticles can be attached to the bubble shell for imaging, targeting and/or enhancement of acoustic response. Existing methods for fabricating particle-loaded bubbles, however, require the use of polymers, oil layers or chemical reactions for particle incorporation; embed/attach the particles that can reduce echogenicity; impair biocompatibility; and/or involve multiple processing steps. Here, we describe a simple method to embed nanoparticles in a phospholipid-coated microbubble formulation that overcomes these limitations. Magnetic nanoparticles are used to demonstrate the method with a range of different microbubble formulations. The size distribution and yield of microbubbles are shown to be unaffected by the addition of the particles. We further show that the microbubbles can be retained against flow using a permanent magnet, can be visualised by both ultrasound and magnetic resonance imaging (MRI) and can be used to transfect SH-SY5Y cells with fluorescent small interfering RNA under the application of a magnetic field and ultrasound field.

Spectral imaging toolbox: segmentation, hyperstack reconstruction, and batch processing of spectral images for the determination of cell and model membrane lipid order.

BackgroundSpectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and model membrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by the generalized polarization (GP) parameter. With the development of commercial microscopes equipped with spectral detectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membrane properties. The existing tools for spectral image processing, however, are insufficient for processing the large data sets afforded by this technological advancement, and are unsuitable for processing images acquired with rapidly internalized fluorescent probes.ResultsHere we present a MATLAB spectral imaging toolbox with the aim of overcoming these limitations. In addition to common operations, such as the calculation of distributions of GP values, generation of pseudo-colored GP maps, and spectral analysis, a key highlight of this tool is reliable membrane segmentation for probes that are rapidly internalized. Furthermore, handling for hyperstacks, 3D reconstruction and batch processing facilitates analysis of data sets generated by time series, z-stack, and area scan microscope operations. Finally, the object size distribution is determined, which can provide insight into the mechanisms underlying changes in membrane properties and is desirable for e.g. studies involving model membranes and surfactant coated particles. Analysis is demonstrated for cell membranes, cell-derived vesicles, model membranes, and microbubbles with environmentally-sensitive probes Laurdan, carboxyl-modified Laurdan (C-Laurdan), Di-4-ANEPPDHQ, and Di-4-AN(F)EPPTEA (FE), for quantification of the local lateral density of lipids or lipid packing.ConclusionsThe Spectral Imaging Toolbox is a powerful tool for the segmentation and processing of large spectral imaging datasets with a reliable method for membrane segmentation and no ability in programming required. The Spectral Imaging Toolbox can be downloaded from https://uk.mathworks.com/matlabcentral/fileexchange/62617-spectral-imaging-toolbox.

Ultrasound enhanced delivery of cisplatin loaded nanoparticles

Cisplatin forms the basis for many chemotherapy regimens, however the maximum permissible dose is limited by its systemic toxicity. Nanoencapsulation of drugs has been shown to reduce off-target side effects and can potentially improve treatment burden on patients. However, uptake of nanoformulations at tumor sites is minimal without some form of active delivery. We have developed a submicron, polymeric nanoparticle based on biocompatible and degradable poly(lactic-co-glycolic acid) (PLGA) capable of encapsulating cisplatin and which can be bound to the surface of a phospholipid coated microbubble. The acoustic behavior and stability of the resulting nanoparticle loaded microbubbles will be compared with those of unloaded microbubbles. Results will also be presented on the extravasation of particles in a tissue mimicking phantom using a novel long working distance confocal microscope that enables particle distributions to be measured in situ and in real time.