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Intranasal Delivery of Mesenchymal Stem Cell Derived Exosomes Loaded with Phosphatase and Tensin Homolog siRNA Repairs Complete Spinal Cord Injury Shaowei Guo, Nisim Perets, Oshra Betzer, Shahar Ben-Shaul, Anton Sheinin, Izhak Michaelevski, Rachela Popovtzer, Daniel Offen, and Shulamit Levenberg ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b01892 • Publication Date (Web): 27 Aug 2019 Downloaded from pubs.acs.org on August 27, 2019
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Intranasal Delivery of Mesenchymal Stem Cell Derived Exosomes Loaded with Phosphatase and Tensin Homolog siRNA Repairs Complete Spinal Cord Injury Shaowei Guo, Nisim Perets, Oshra Betzer, Shahar Ben-Shaul, Anton Sheinin, †,‡
§
†
⊥
Izhak Michaelevski, Rachela Popovtzer, Daniel Offen, Shulamit Levenberg* ∥
†
⊥
§,∇
§
†
Department of Biomedical Engineering, Technion-Israel Institute of Technology,
Haifa 3200003, Israel. ‡
The First Affiliated Hospital, Shantou University Medical College, Shantou
515041, China. §
Sagol School of Neuroscience, and Sackler School of Medicine, Department of ∇
Human Molecular Genetics and Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel. ⊥
Faculty of Engineering and the Institute of Nanotechnology & Advanced Materials,
Bar-Ilan University, Ramat Gan 5290002, Israel. ∥
Department of Molecular Biology, Ariel University, Ariel 40700, Israel.
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* Corresponding author. Email:
[email protected] ABSTRACT Individuals with spinal cord injury (SCI) usually suffer from permanent neurological deficits, while spontaneous recovery and therapeutic efficacy are limited. Here, we demonstrate that when given intranasally, exosomes derived from mesenchymal stem cells (MSC-Exo) could pass the blood brain barrier, and migrate to the injured spinal cord area. Furthermore, MSC-Exo loaded with phosphatase and tensin homolog small interfering RNA (ExoPTEN) could attenuate the expression of PTEN in the injured spinal cord region following intranasal administrations. In addition, the loaded MSC-Exo considerably enhanced axonal growth and neovascularization, while reducing microgliosis and astrogliosis. The intranasal ExoPTEN therapy could also partly improve structural and electrophysiological function, and most importantly, significantly elicited functional recovery in rats with complete SCI. The results imply that intranasal ExoPTEN may be used clinically to promote recovery for SCI individuals.
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KEYWORDS: intranasal, exosome, PTEN siRNA, targeted delivery, functional
recovery, complete spinal cord injury
Spinal cord injury (SCI) is a debilitating disease without effective treatment currently. Axonal growth and functional recovery following SCI is limited, because 1
of poor innate regenerative capacity of adult central nervous system neurons, and 2,
hostile injury environment comprising inflammation, 4, 5
inhibitors, 7
3
myelin-associated 6,
glial scar components including chondroitin sulphate proteoglycans, 8
and compromised blood supply. Despite previous attempts to treat SCI via
targeting extrinsic mechanisms controlling axonal regeneration, yet success has been limited in complete SCI. Extracellular inhibitory molecule removal, 10, 11
neurotrophic factor delivery,
9
12
or permissive substrate grafting all failed to elicit
robust regeneration of injured axons and substantial functional recovery. These studies suggest the necessity of targeting the diminished intrinsic regenerative ability of neurons. As one of the major intrinsic impediments to axonal growth,
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PTEN is expressed in neurons and regenerating axons, and plays a vital role in controlling the regeneration of corticospinal neurons via downregulating 13-16
cytoplasmic mammalian target of rapamycin (mTOR) activity.
This mTOR
pathway is intensely inhibited in axotomized adult neurons, restricting protein synthesis to sustain axonal growth.
17
Exosomes have emerged as promising nanocarriers for drug delivery and targeted 18
therapy, as alternatives to stem cell therapy.
Exosomes are natural membrane
vesicles (50-150 nm) of endosomal origin, secreted by various cells including mesenchymal stem cells (MSCs).
19, 20
Exosomes carry proteins, lipids and genetic
materials reflective of their cell origins, which facilitate intercellular communication and induce a multitude of biological effects, locally or distally.
19, 21-23
Exosomes
derived from MSCs (MSC-Exo) have been extensively characterized regarding 24-27
their proteins, lipids and RNA profiles.
MSC-derived exosomes have been
examined to support regeneration in the context of numerous diseases
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28
such as
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autism,
29
stroke,
30-32
33, 34
35
traumatic brain injury,
28, 36
Alzheimer’s disease.
and
When intranasally administered, exosomes can pass the
blood brain barrier (BBB),
29, 35, 37-39
35, 38
delivered intravenously.
Parkinson’s disease
and are better retained in injury sites than when
Moreover, exosomes are loadable with an array of 35, 37, 40-42
therapeutic cargos for specific diseases.
As natural delivery vehicles,
exosomes advance the development of RNAi-based therapeutics in CNS diseases, as RNAi delivery is hampered by the blood brain barrier, susceptibility to nuclease 43, 44
degradation, and lack of cell-specific targeting.
As such, intranasal exosome
administration has broad potentials and offers an alternative to cell transplantation in treating SCI.
This study presents intranasal administrations of MSC-derived exosomes loaded with PTEN-siRNA (ExoPTEN), which targeted the spinal cord lesion in rats with complete spinal cord injury and enabled significant functional recovery. The results
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have significant clinical therapeutic application for SCI and other neurological diseases with neuroinflammation.
RESULTS AND DISCUSSIONS Intranasal MSC-Exo Cross the BBB, and Migrate to Spinal Cord Isolated MSC-Exo were of mean size 111±64 nm and at a concentration of 40.43x108 particles/ml (Figure S1D-E). MSC-Exo visualized with Cryo-TEM, showed a typical sphere shape (Figure S1B). In order to examine whether MSCExo can cross the BBB, we labeled MSC-Exo with gold nanoparticles (GNPs), an established contrast agent for CT imaging and cell tracking, 38
recently published protocol.
39, 45, 46
according to a
Cryo-TEM imaging of the MSC-Exo loaded with
GNPs indicated GNP uptake into the exosomes (Figure S1A-C). Although recent studies have showed that intranasally delivered exosomes can surmount the BBB 37-39, 47
and reach pathological sites in the brain,
patterns of exosome migration
within the whole central nervous system as well as bio-distribution in the body,
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neural cell affinity in the lesion, and mechanism of migration have not been fully characterized. To examine whether MSC-Exo can pass the BBB following intranasal (IN) administration, GNP-loaded MSC-Exo were IN administered three hours after complete spinal cord transection. Micro-CT scanning 24 h postadministration (Figure 1A) demonstrated significant GNP accumulation in the spinal cord lesion area, but not in the brain (Figure 1B, upper panel). In contrast, in healthy controls, the GNPs were localized mainly in the brain and olfactory bulbs (Figure 1B, lower panel). Inductively coupled plasma (ICP) assessments confirmed that the amount of GNPs in the T10 spinal segment area, was significantly higher in the injured rats than healthy controls (Figure 1C, one-way ANOVA F (3, 8) = 27.5, p
