Nano Today
Multifunctional bioreactive-nanoconstructs for sensitive and accurate MRI of cerebrospinal fluid pathology and intervention of Alzheimer’s disease
Introduction
Alzheimer’s disease (AD) is the most common cause of dementia affecting 40–50 million people worldwide, including ∼5.8 million Americans [1]. Unfortunately the standard of care treatments for AD only manage symptoms, and currently there is no curative or approved disease-modifying therapeutics for AD [2,3]. Despite tremendous efforts and expenditures so far, most clinical trials over the past decades have failed to achieve meaningful clinical benefits for AD [1,[3], [4], [5], [6]]. Many contributing factors to such failures have been postulated to include variable origins and rates of disease progression, inappropriate therapeutic targets and patient selection, late intervention, and suboptimal dosing [1,2,4,7]. Moreover, presence of the blood-brain barrier (BBB) limits efficient transport of therapeutic agents into neocortex and deeper structures [3,5,8]. The late intervention of the disease is largely due to the lack of a sensitive and accurate detection method for early stage AD [6,9]. Neurobiological changes in AD can occur much earlier than the cognitive decline diagnosed by clinical methods. For example, changes in cerebrospinal fluid (CSF) biomarkers, e.g. hyperphosphorylated tau (p-tau) and amyloid β-42 (Aβ42) occur 15–20 years prior to the clinical onset of AD [10,11]. However, clinical diagnosis of AD is conducted in accordance with exclusion criteria by clinical symptoms (e.g. impaired memory and cognitive function) [1,10,12]. The diagnostic sensitivity and specificity of these methods are relatively low and are only definitive when identifying AD at its irreversible stages [2,10,12,13]. Therefore, for early detection and complementary imaging-based diagnosis of AD, fluid biomarkers (e.g. CSF Aβ42 and p-tau) are measured. However CSF sampling is labor intensive, costly, and invasive; it requires lumbar puncture at L3-L5 which carries some risk of CNS injury infection. Therefore analysis of CSF biomarkers by this method is not broadly practiced clinically [10,11].
Various imaging techniques have been applied to detect AD non-invasively including positron emission tomography (PET), PET/computed tomography (CT), single-photon emission computed tomography (SPECT) scans and magnetic resonance imaging (MRI) [[14], [15], [16]]. PET scan measures metabolic changes using radiolabeled fluorodeoxyglucose ([18F] FDG), or Aβ plaques by amyloid tracers such as Pittsburgh Compound-B (PiB). However, PET scan requires use of expensive radioactive probes of limited availability [15,17]. In addition, high levels of Aβ plaques found in later stages of AD may not correlate well with AD dementia [17,18]. By contrast MRI is a powerful tool for clinical assessment of patients with suspected AD by measuring the volume and structural changes in the brain [15,19]. Structural MRI can determine atrophy of medial temporal lobe, hippocampal, entorhinal cortex and subiculum, as potential early indicators of future AD dementia risk [20,21].
To enhance MRI signal, gadolinium (Gd) based contrast agents are commonly used clinically. However application of Gd-based contrast agents have been linked to significant nephrogenic systemic fibrosis in some patients, particularly those with severe renal dysfunction [22]. Studies have also shown deposition of Gd in the brain and bones can occur over an extended periods [23,24]. Moreover the majority of current MRI contrast agents possess poor selectivity and limited ability to penetrate the BBB for early and accurate detection of CNS/CSF biomarkers in AD [5,8,25,26].
Increasing evidence has shown that early events in AD are associated with hypoxia and oxidative stress [27,28]. Neuropathology such as neuro-inflammation induced by reactive oxygen species (ROS), e.g. H2O2, reportedly occur prior to overt amyloid plaque accumulation and cognitive AD progression [29]. In addition, ROS, Aβ oligomers and fibrils are known to activate microglia and astrocytes triggering the secretion of pro-inflammatory cytokines that promote disease progression [30]. To circumvent ROS-induced AD progression, various antioxidants and anti-inflammatory drugs have been investigated [31], but with limited success [1,5,8,32].
In light of early signs of oxidative stress, inflammation and neurotoxicity of soluble Aβ oligomers in AD brains, we developed an anti-Aβ antibody (aAβ)-conjugated, brain-targeted (BT), and ROS-activatable (RA) MnO2 nanoparticle (MnO2-NPs)-containing nanoconstruct (NC) (aAβ-BTRA-NC) (Fig. 1a). The multifunctional aAβ-BTRA-NCs can effectively cross the BBB, selectively accumulate in Aβ- and ROS-affected brain tissue (Fig. 1b), react with or break down endogenous H2O2 to produce O2 [33,34], and release paramagnetic manganese ions (Mn2+) in situ enhancing local MRI contrast (Fig. 1c). Meantime, the multifunctional polymer can simultaneously bind with soluble Aβ oligomers or Aβ plaques via the conjugated aAβ and complex with free Mn2+ ions via the carboxylic groups, thereby enhancing MRI contrast in relation to the level and location of ROS and Aβ pertinent to disease stage (Fig. 1c). Owing to the consumption of H2O2 and production of O2, the aAβ-BTRA-NC is expected to reduce local oxidative stress and hypoxia that are detrimental to neurons.
Section snippets
Synthesis of BTRA-NCs and aAβ-BTRA-NCs
A brain targeted polymer (BTP) was synthesized by grafting polymerization of poly(methacrylic acid) and polysorbate 80 (PS 80) onto starch [35]. Anti-Aβ antibody 4G8 conjugated-BTP (aAβ-BTP) was prepared by EDC/ N-hydroxysuccinimide (NHS) coupling of 4G8 with the BTP. Briefly, 500 mg of purified BTP, 100 mg of EDC, and 100 mg of NHS were dissolved in 10 mL of distilled de-ionized water (DDIW, Milli-Q water, MilliPore Canada Ltd, Etobicoke, ON, CA) and allowed to react for 2 h at room
Results and discussion
The novel multifunctional polymer-lipid-inorganic hybrid NC (i.e., BTRA-NC) were prepared using a facile and easily scaled-up “one-pot” synthesis method. The inorganic MnO2-NPs were generated by reducing KMnO4 with PVA in an aqueous medium, to which melted lipid was introduced to form a MnO2-NPs-lipid crude emulsion. The hydrophobic interaction between BTP and the lipid domain led to the formation of the NCs upon cooling with a monolithic-matrix structure by self-assembly (Figs. 1a, S1). The
Conclusions
In summary, we have developed a novel multifunctional and ROS-responsive nanotheranostic system, aAβ-BTRA-NC, and demonstrated its ability to enable sensitive detection of early AD by non-invasive MRI of CSF pathology. The aAβ-BTRA-NC effectively crossed the BBB, selectively accumulated in the disease affected areas and significantly enhanced T1-wt MR signals in the CSF, cortex, hippocampus areas of TgCRND8 AD mouse brains, where overexpressed oxidative stress and Aβ pathology occur.
Author contributions
X.Y.W, C.H, T.A, J.T.H., and A.Z.A. conceived the hypotheses, designed the experiments, interpreted results, and wrote the paper. C.H, T.A, J.T.H., A.Z.A., L.Y.L, and P.C. performed the experiments. W.D.F. performed the MRI data acquisition and analysis and edited the manuscript. E.K. and P.E.F performed the experiments related to generating TgCRND8 mouse model. P.E.F. and A.M.R. designed the experiments and wrote the paper.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
This work is supported by the Weston Brain Institute Transformational Grant to X.Y.W, J.T.H. and P.E.F., University of Toronto Connaught Innovation Award, Killam Research Fellowship by the Canada Council for the Arts, and Equipment Grants from the Natural Sciences and Engineering Research Council of Canada to X.Y.W. The authors also thank the University of Toronto Connaught International Scholarship for Doctoral Students to T.A., the Centre for Collaborative Drug Research Graduate Student
Dr. Chunsheng He received his PhD degree on Chemo-Biological Technology and Engineering from the East China University of Science and Technology, Shanghai, China. He then worked at the University of Toronto as a postdoctoral fellow from 2012 to 2017. After that, he works as a Research Associate and project leader in Dr. Xiao Yu Wu’s lab, University of Toronto. His research focuses on the development of novel polymer-lipid based nano formulations for targeting and locoregional delivery of
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Dr. Chunsheng He received his PhD degree on Chemo-Biological Technology and Engineering from the East China University of Science and Technology, Shanghai, China. He then worked at the University of Toronto as a postdoctoral fellow from 2012 to 2017. After that, he works as a Research Associate and project leader in Dr. Xiao Yu Wu’s lab, University of Toronto. His research focuses on the development of novel polymer-lipid based nano formulations for targeting and locoregional delivery of anticancer drugs, polypeptides, proteins, antibodies, and hybrid MnO2 nanoparticles for the treatment and diagnosis of cancer and central nervous system diseases.
Taksim Ahmed is a PhD Candidate under the supervision of Professor Wu at the University of Toronto. He received B. Pharmacy degree from Jahangirnagar University, Bangladesh in 2009. Afterwards, he worked in ACI Pharmaceuticals LTD, Bangladesh (2009−10). He received his Master’s in Pharmacy degree from the College of Pharmacy, Chosun University, S. Korea (2012) and second M.Sc. degree at the School of Pharmacy, University of Waterloo, Canada (2015). He is a recipient of Connaught International Scholarship for Doctoral Students. His current research is focused on developing nanoparticulate drug delivery systems for the treatment of cancer and central nervous system diseases.
Dr. Azhar Z. Abbasi received Master’s degree from Quaid-i-Azam University, Pakistan, in 2006 and Ph.D. degree from Philipps-Universität Marburg, Germany, in 2010. In 2011, he joined Professor Wu at University of Toronto as a Postdoctoral fellow. In 2016, Dr. Abbasi joined pharmaceutical company, Apotex Inc., and worked in Research and Development Department for two years. Currently, he is working as a Research associate and Project leader at Faculty of Pharmacy, University of Toronto. His current research focuses on development of biocompatible and multifunctional polymer/lipid nanoparticle formulations, for drug delivery, imaging and diagnostic of cancer and central nervous system (CNS) diseases.
Lily Yi Li is a Ph.D. student in the laboratory of Professor X.Y. Wu at the University of Toronto. She received her B.Sc. degree (2015) in Pharmaceutical Chemistry and M.Sc. degree (2017) in Pharmaceutical Sciences from University of Toronto, Canada. She started doing research since 2nd year of her undergraduate program, then joined the current lab for her studies. Her research interests include Alzheimer’s research, oncology research, developing and synthesizing bio-environmental responsive and targeting nanomaterials and intelligent drug delivery systems, as well as mathematical modeling and computational simulation for mechanistic studies on drug delivery.
Dr. Warren D. Foltz is the MRI physicist at the STTARR Innovation Centre, where he operates and consults on MRI projects using a 7 T Biospec (Bruker Corporation) and 1.5 T Aera (Siemens Corporation). He has broad experience across quantitative MRI applications, field strengths, nanoparticles, and animal and disease models. He is currently an Assistant Professor in the Faculty of Radiation Oncology of the University Health Network and University of Toronto. He received his Ph.D. from Medical Biophysics at the University of Toronto.
Dr. Ping Cai received his MD and MSc degrees in Medicine in China, conducted postdoctoral research in toxicology at UTMB (TX, US) and in drug toxicity at the University of Toronto, Canada. He has worked in Dr. Wu’s laboratory as a Research Associate since 2011. His research work includes development of animal models of primary tumors and cancer metastases in the lungs and brain, CNS diseases, in vivo studies of toxicity, efficacy, and immune modulation of nanomedicine.
fx7Dr. Erin Knock received her PhD degree, in 2009, from McGill University. She then worked at the University of Cambridge as a postdoctoral scholar from 2009 to 2012. After that, she worked at the University of Toronto as a post-doc from 2012 to 2016 on the development of human stem cell-derived neuronal models of Alzheimer’s Disease. In 2016, Dr. Knock joined the Research and Development Department at STEMCELL Technologies Inc. as a Senior Scientist. Dr Knock’s current research focuses on developing products for primary neural culture and pluripotent stem cell differentiation to neural cell types.
Dr. Paul E. Fraser is a full professor in the Departments of Medical Biophysics at the University of Toronto and Jeno Diener Chair in Neurodegenerative Diseases, Tanz Centre, Canada. His group has been examining the organization and mechanisms by which amyloid-β (Aβ) peptides are assembled into oligomeric and fibrillar structures in Alzheimer disease (AD). Research is also being conducted into the unique family of presenilin proteins since their discovery at the Tanz Centre, and the investigations have been expanded to determine the mechanisms of action for groups of AD at-risk genes identified by genome-wide association studies.
Dr. Andrew M. Rauth is an Emeritus Professor in the Departments of Medical Biophysics and Radiation Oncology at the University of Toronto and Scientist Emeritus at Princess Margaret Cancer Centre, University Health Network Toronto, Ontario, Canada. Research interests are radiation biology, mechanisms of drug action and nanoparticles. He has been a collaborator of Dr. Wu for over 20 years.
Dr. Jeffrey T. Henderson is an Associate Professor in the Faculty of Pharmacy, University of Toronto. He received his Ph.D. in Biochemistry at University of Illinois, Chicago. Following postdoctoral work in the Divisions of Development and Neuroscience, and Molecular Biology and Cancer at the Samuel Lunenfeld Research Institute Mount Sinai Hospital he became an Assistant Scientist at SLRI in Molecular Neuroscience. His laboratory focuses on mechanisms of natural and neuropathologic cell death signaling within the mammalian CNS with the aim of developing small molecular PCD neurotherapeutics. He joined the Faculty of Pharmacy, University of Toronto in 2002.
Dr. Xiao Yu Wu is full professor at the Leslie Dan Faculty of Pharmacy at the University of Toronto, Canada. She received her Ph.D. degree in Chemical Engineering from McMaster University, Canada. After postdoctoral research at the University of Toronto, she joined the Faculty of Pharmacy in 1994. Her research projects are centered on advanced pharmaceutics and drug delivery technologies including blood-brain barrier penetrating nanoparticles for brain cancer and CNS diseases; synergistic drug combination nanomedicine for enhanced chemotherapy; hybrid MnO2 nanoparticles for enhancing cancer therapies and theranostics of Alzheimer’s disease; computer-aided design of controlled release dosage forms; glucose-responsive insulin/glucagon delivery.
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These authors contributed equally.
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Current address: STEMCELL Technologies Inc., 570 West Seventh Avenue, Vancouver, BC, V5Z 1B3, Canada.