Tristan D. Clemons

Multimodal analysis of PEI-mediated endocytosis of nanoparticles in neural cells.

Polymer nanoparticles are widely used as a highly generalizable tool to entrap a range of different drugs for controlled or site-specific release. However, despite numerous studies examining the kinetics of controlled release, the biological behavior of such nanoparticles remains poorly understood, particularly with respect to endocytosis and intracellular trafficking. We synthesized polyethylenimine-decorated polymer nanospheres (ca. 100-250 nm) of the type commonly used for drug release and used correlated electron microscopy, fluorescence spectroscopy and microscopy, and relaxometry to track endocytosis in neural cells. These capabilities provide insight into how polyethylenimine mediates the entry of nanoparticles into neural cells and show that polymer nanosphere uptake involves three distinct steps, namely, plasma membrane attachment, fluid-phase as well as clathrin- and caveolin-independent endocytosis, and progressive accumulation in membrane-bound intracellular vesicles. These findings provide detailed insight into how the intracellular delivery of nanoparticles is mediated by polyethylenimine, which is presently the most commonly used nonviral gene transfer agent. This fundamental knowledge may also assist in the preparation of next-generation nonviral vectors.

In vivo Imaging and Biodistribution of Multimodal Polymeric Nanoparticles Delivered to the Optic Nerve

The use of nanoparticles for targeted delivery of therapeutic agents to sites of injury or disease in the central nervous system (CNS) holds great promise. However, the biodistribution of nanoparticles following in vivo administration is often unknown, and concerns have been raised regarding potential toxicity. Using poly(glycidyl methacrylate) (PGMA) nanoparticles coated with polyethylenimine (PEI) and containing superparamagnetic iron oxide nanoparticles as a magnetic resonance imaging (MRI) contrast agent and rhodamine B as a fluorophore, whole animal MRI and fluorescence analyses are used to demonstrate that these nanoparticles (NP) remain close to the site of injection into a partial injury of the optic nerve, a CNS white matter tract. In addition, some of these NP enter axons and are transported to parent neuronal somata. NP also remain in the eye following intravitreal injection, a non-injury model. Considerable infiltration of activated microglia/macrophages occurs in both models. Using magnetic concentration and fluorescence visualization of tissue homogenates, no dissemination of the NP into peripheral tissues is observed. Histopathological analysis reveals no toxicity in organs other than at the injection sites. Multifunctional nanoparticles may be a useful mechanism to deliver therapeutic agents to the injury site and somata of injured CNS neurons and thus may be of therapeutic value following brain or spinal cord trauma.

Nanoparticle-Mediated Dual Delivery of an Antioxidant and a Peptide against the L-Type Ca2+ Channel Enables Simultaneous Reduction of Cardiac Ischemia-Reperfusion Injury

Increased reactive oxygen species (ROS) production and elevated intracellular Ca(2+) following cardiac ischemia-reperfusion injury are key mediators of cell death and the development of cardiac hypertrophy. The L-type Ca(2+) channel is the main route for calcium influx in cardiac myocytes. Activation of the L-type Ca(2+) channel leads to a further increase in mitochondrial ROS production and metabolism. We have previously shown that the application of a peptide derived against the alpha-interacting domain of the L-type Ca(2+) channel (AID) decreases myocardial injury post reperfusion. Herein, we examine the efficacy of simultaneous delivery of the AID peptide in combination with the potent antioxidants curcumin or resveratrol using multifunctional poly(glycidyl methacrylate) (PGMA) nanoparticles. We highlight that drug loading and dissolution are important parameters that have to be taken into account when designing novel combinatorial therapies following cardiac ischemia-reperfusion injury. In the case of resveratrol low loading capacity and fast release rates hinder its applicability as an effective candidate for simultaneous therapy. However, in the case of curcumin, high loading capacity and sustained release rates enable its effective simultaneous delivery in combination with the AID peptide. Simultaneous delivery of the AID peptide with curcumin allowed for effective attenuation of the L-type Ca(2+) channel-activated increases in superoxide (assessed as changes in DHE fluorescence; Empty NP = 53.1 ± 7.6%; NP-C-AID = 7.32 ± 3.57%) and mitochondrial membrane potential (assessed as changes in JC-1 fluorescence; Empty NP = 19.8 ± 2.8%; NP-C-AID=13.05 ± 1.78%). We demonstrate in isolated rat hearts exposed to ischemia followed by reperfusion, that curcumin and the AID peptide in combination effectively reduce muscle damage, decrease oxidative stress and superoxide production in cardiac myocytes.

Multimodal and multifunctional stealth polymer nanospheres for sustained drug delivery

We report the preparation of fluorescent and magnetic PMMA nanospheres, and a corresponding PEGylated ‘stealth’ analogue prepared using a block copolymer. The nanospheres contain encapsulated magnetite nanoparticles and fluorescent BODIPY dyes, including a new such dye with pH-sensitive fluorescent emission. The new dye could potentially be used as an indicator of the immediate physiological environment. The nanospheres were non-toxic at up to 500 μg ml−1 in PC12 cells. Lomerizine, a lipophilic calcium channel blocker, was also encapsulated in the nanospheres and displayed sustained, pH-dependent release characteristics. The nanospheres may be of use to release lomerizine and other water-insoluble drugs at central nervous system injury sites.

RNA Interference Using c-Myc–Conjugated Nanoparticles Suppresses Breast and Colorectal Cancer Models

In this article, we report the development and preclinical validation of combinatorial therapy for treatment of cancers using RNA interference (RNAi). RNAi technology is an attractive approach to silence genes responsible for disease onset and progression. Currently, the critical challenge facing the clinical success of RNAi technology is in the difficulty of delivery of RNAi inducers, due to low transfection efficiency, difficulties of integration into host DNA and unstable expression. Using the macromolecule polyglycidal methacrylate (PGMA) as a platform to graft multiple polyethyleneimine (PEI) chains, we demonstrate effective delivery of small oligos (anti-miRs and mimics) and larger DNAs (encoding shRNAs) in a wide variety of cancer cell lines by successful silencing/activation of their respective target genes. Furthermore, the effectiveness of this therapy was validated for in vivo tumor suppression using two transgenic mouse models; first, tumor growth arrest and increased animal survival was seen in mice bearing Brca2/p53-mutant mammary tumors following daily intratumoral treatment with nanoparticles conjugated to c-Myc shRNA. Second, oral delivery of the conjugate to an Apc-deficient crypt progenitor colon cancer model increased animal survival and returned intestinal tissue to a non–wnt-deregulated state. This study demonstrates, through careful design of nonviral nanoparticles and appropriate selection of therapeutic gene targets, that RNAi technology can be made an affordable and amenable therapy for cancer. Mol Cancer Ther; 14(5); 1259–69. ©2015 AACR.

Examining Efficacy of “TAT-less” Delivery of a Peptide against the L-Type Calcium Channel in Cardiac Ischemia–Reperfusion Injury

Increased calcium influx through the L-type Ca(2+) channel or overexpression of the alpha subunit of the channel induces cardiac hypertrophy. Cardiac hypertrophy results from increased oxidative stress and alterations in cell calcium levels following ischemia-reperfusion injury and is an independent risk factor for increased morbidity and mortality. We find that decreasing the movement of the auxiliary beta subunit with a peptide derived against the alpha-interacting domain (AID) of the channel attenuates ischemia-reperfusion injury. We compared the efficacy of delivering the AID peptide using a trans-activator of transcription (TAT) sequence with that of the peptide complexed to multifunctional polymeric nanoparticles. The AID-tethered nanoparticles perfused through the myocardium more diffusely and associated with cardiac myocytes more rapidly than the TAT-labeled peptide but had similar effects on intracellular calcium levels. The AID-complexed nanoparticles resulted in a similar reduction in release of creatine kinase and lactate dehydrogenase after ischemia-reperfusion to the TAT-labeled peptide. Since nanoparticle delivery also holds the potential for dual drug delivery, we conclude that AID-complexed nanoparticles may provide an effective platform for peptide delivery in cardiac ischemia-reperfusion injuries.

Coherency image analysis to quantify collagen architecture: implications in scar assessment

An important histological difference between normal, uninjured dermis and scar tissue such as that found in keloid scars is the pattern (morphological architecture) in which the collagen is deposited and arranged. In the uninjured dermis, collagen bundle architecture appears randomly organized (or in a basket weave formation), whereas in pathological conditions such as keloid scar tissue, collagen bundles are often found in whorls or in a hypotrophic scar collagen is more densely packed in a parallel configuration. In the case of skin, a scar disables the dermis, leaving it weaker, stiff and with a loss of optimal functionality. The absence of objective and quantifiable assessments of collagen orientation is a major bottleneck in monitoring progression of scar therapeutics. In this article, a novel quantitative approach for analyzing collagen orientation is reported. The methodology is demonstrated using collagen produced by cells in a model scar environment and examines collagen remodeling post-TGFβ stimulation in vitro. The method is shown to be reliable and effective in identifying significant coherency differences in the collagen deposited by human keloid scar cells. The technique is also compared for analysing collagen architecture in rat sections of normal, scarred skin and tendon tissue. Results demonstrate that the proposed computational method provides a fast and robust way of analyzing collagen orientation in a manner surpassing existing methods. This study establishes this methodology as a preliminary means of monitoring in vitro and in tissue treatment modalities which are expected to alter collagen morphology.

Prepare, Do, Review: a model used to reduce the negative feelings towards laboratory classes in an introductory chemistry undergraduate unit

Student feelings towards the laboratory component of an introductory chemistry unit were evaluated in an action research study, over a three-year period at the University of Western Australia. In 2013 we found that the percentage of students with negative feelings towards the laboratory increased over the duration of a semester. In 2014 we developed and introduced the use of pre-laboratory online activities, which the students found to be helpful in preparing them for the laboratory. However, there was no change in trend of negative feelings towards laboratory classes from 2013 to 2014. In 2015 we introduced the Prepare, Do, Review model and found that there was a reduction in the percentage of students with negative feelings towards laboratory classes compared with previous years. The Prepare, Do, Review model allows students more time to process the information given in the laboratory. We believe that this model could apply to laboratory programs in any discipline.

Regulation of collagen expression using nanoparticle mediated inhibition of TGF-β activation

PXS64 is a stable mannose-6-phosphate (M6P) analogue which has been shown to be an effective anti-fibrotic agent by inhibiting the activation of latent TGF-β1. However PXS64 is insoluble in physiological conditions. Herein, we report a multifunctional poly(glycidyl methacrylate) (PGMA) polymeric nanoparticle system for intracellular delivery of PXS64 in human primary dermal scar fibroblasts. We demonstrate the efficacy of this anti-fibrotic platform by monitoring the expression of collagen production using an in vitro scar model.

A comparison of haemolytic responses in fore-foot and rear-foot distance runners

ABSTRACT This study examined the haemolytic effects of an interval-based running task in fore-foot and rear-foot striking runners. Nineteen male distance runners (10 fore-foot, 9 rear-foot) completed 8 × 3 min repeats at 90% vVO2peak on a motorised treadmill. Pre- and post-exercise venous blood samples were analysed for serum haptoglobin to quantify the haemolytic response to running. Vertical ground reaction forces were also captured via a force plate beneath the treadmill belt. Haptoglobin levels were significantly decreased following exercise (P = 0.001) in both groups (but not between groups), suggesting that the running task created a haemolytic stress. The ground reaction force data showed strong effect sizes for a greater peak force (d = 1.20) and impulse (d = 1.37) in fore-foot runners, and a greater rate of force development (d = 2.74) in rear-foot runners. The lack of difference in haptoglobin response between groups may be explained by the trend for fore-foot runners to experience greater peak force and impulse during the stance phase of their running gait, potentially negating any impact of the greater rate of force development occurring from the rear-foot runners’ heel strike. Neither type of runner (fore-foot or rear-foot) appears more susceptible to technique-related foot-strike haemolysis.