Harnessing Exosomes for the Development of Brain Drug Delivery

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Harnessing Exosomes for the Development of Brain Drug Delivery Systems Mengna Zheng, Meng Huang, Xinyi Ma, Hong-zhuan Chen, and Xiaoling Gao Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00085 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 12, 2019

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Bioconjugate Chemistry

Harnessing Exosomes for the Development of Brain Drug Delivery Systems Mengna Zheng,§, # Meng Huang,§, # Xinyi Ma,§ Hongzhuan Chen§,‡, Xiaoling Gao*,§,‡ §Department

of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine,

Shanghai 200025, China ‡Shanghai

Universities Collaborative Innovation Center for Translational Medicine, Shanghai 200025, China

ABSTRACT

Brain drug delivery is one of the most important bottlenecks in the development of drugs for the central nervous system. Cumulating evidence has emerged that extracellular vesicles (EVs) play a key role in intercellular communication. Exosomes, a sub-group of EVs, have received the most attention due to their capability in mediating the horizontal transfer of their bioactive inclusions to neighboring and distant cells, and thus specifically regulating the physiological and pathological functions of the recipient cells. This native and unique signaling mechanism confers exosomes with great potential to be developed into an effective, precise and safe drug delivery system. Here, we provide an overview into the challenges of brain drug delivery, the function of exosomes in the brain under physiological and pathological conditions, and discuss how these natural vesicles could be harnessed for brain drug delivery and for the therapy of brain diseases.

Introduction The blood-brain barrier (BBB), which is composed of brain capillary endothelial cells (BCECs), basal lamina, pericytes and astrocytes end-feet, provides an important physical barrier that protects brain from potential detrimental substances in the blood circulation.1 However, this physiological protective barrier also significantly impedes most of the potential drugs from entering into the central nervous system (CNS), representing a key challenge to the brain drug delivery. Over 98% of small molecule drugs and almost 100% proteins, peptides and genes cannot penetrate the BBB.2 Hence, there is an urgent need to develop new therapeutic modalities that can overcome BBB and improve efficacy. Extracellular vesicles (EVs) are membrane-contained vesicles released by cells ranging from prokaryotes to eukaryotes. These vesicles can be found in most body fluids such as blood, urine, breast milk, saliva, and cerebrospinal fluid (CSF). Based on their biogenesis mechanism, EVs are currently classified into three types : 1) Apoptotic bodies (50 nm-5 μm), which are

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released when plasma membrane blebbing occurs during apoptosis; 2) Microvesicles (50-1000 nm), that are produced by outward budding and fission of the plasma membrane; 3) Exosomes (30-150 nm), which are originated from the inward budding of multivesicular bodies (MVB), forming intraluminal vesicles (ILVs), and are released into extracellular space upon fusion of MVBs with plasma membrane. Among these various types of EVs, exosome has attracted the most attention in recent decades. Exosomes, initially thought as cellular garbage cans acting to discard unwanted molecular components, were now found contain rich cargos and play significant biological roles. According to the latest database of exosomes (http://www.exocarta.org/), 9769 proteins, 3408 mRNAs and 2838 microRNAs (microRNA, miRNA, miR) have been found in exosomes. Protein and RNA in exosomes are differentially expressed under physiological and pathological conditions, and some proteins and RNA are specifically expressed in exosomes. Besides, exosomes also contain a variety of lipid molecules. In addition to participating in a variety of biological processes, these lipids also play a role in maintaining the stability of exosomes and protecting the exosomal contents from degradation by extracellular enzymes.3 In the past few years, accumulating evidences demonstrated that these vesicles were key participants in intercellular communication. Exosomes exert their intercellular signaling function through different ways (Figure 1):4 1) Firstly, they may directly contact and activate target cells via their surface ligands. For example, it has been demonstrated that antigen-presenting exosomes derived from dendritic cells provoke T cell-mediated immune responses in vivo.5 2) Secondly, exosomes may transfer surface receptors or cytosolic proteins to target cells. For instance, exosomes could transmit chemoresistance by transfer membrane Ephrin type-A receptor 2 (EphA2) from chemoresistant cells.6 3) Thirdly, exosomes could horizontally transfer genetic material, such as miRNA and mRNA between cells.7 The last two mechanisms depend on membrane fusion or cellular internalization following the interaction between exosomes and the specific cellular surface ligands such as heparin sulfate proteoglycans (HSPGs) in the recipient cells.8 The target cells of exosomes are organ-specific. For example, integrins on exosomes from tumor cells determine organotropic metastasis: the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was related to liver metastasis.9


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Figure 1. The major three steps of exosomes uptake by acceptor cells. (1) exosomes or other EVs subtypes are targeted to the acceptor cell through docking on specific molecules such as membrane-exposed proteins, sugars or lipids. (2) exosomes that enter the acceptor cell through specific endocytosis or through unspecific macropinocytosis or micropinocytosis, which may then be targeted to endosomes. (3) Internalized EVs release their content through the indirect route of endosomal escape. This might involve membrane fusion through unknown molecular mechanisms, which could resemble those used by viruses harboring the fusogenic protein VSVG. Once internalized, EVs might also be recycled and re-secreted or targeted to the lysosome for degradation. (3′) An alternative, direct route of content delivery to the acceptor cell could involve EVs fusion with the plasma membrane and cargo release directly into the cytosol. SNARE, soluble N-ethylmaleimide-sensitive fusion attachment protein receptor. Reprinted with permission from Ref.4 As natural nanovesicles, compared with other drug delivery systems, exosomes possess obvious advantages.10 Firstly, the small size (30~150 nm) enables efficient penetration of exosomes across the biological barriers including the BBB. Secondly, the natural proteins and lipid components on the exosomal membrane enable efficient uptake of exosomes into their target cells. Thirdly, the biological endogenous property of exosomes ensures their high biocompability, stability, low immunogenicity and low inherent toxicity (Figure 2). In contrast, the synthetic nanocarriers, even the well-studied pegylated nanoparticles, such as PEG-PLGA nanoparticles, induced the production of antibodies and exhibited short blood circulation half-lives after multiple dosing.11,12 Therefore, the unique endogenous characteristics confer exosomes as a promising drug-delivery system.



Figure 2. The advantages of exosomes as drug delivery systems. (1) The small size (30~150 nm) enables exosomes to cross the biological barriers. (2) The natural proteins and lipid components on the exosomal membrane enable efficient uptake of exosomes into target cells. (3) The biological endogenous property of exosomes ensures biocompability, stability, low immunogenicity and low inherent toxicity. In recent years, there are many innovative attempts to exploit exosome as a novel drug delivery system for CNS diseases, among which genetic manipulating the progenitor cells to produce BBB penetrating exosomes is of particular interest.13 Here we provide an overview into exosomes mediated drug delivery and discuss how these natural vesicles could be harnessed for brain drug delivery and the therapy of brain diseases.

1.

Challenges of brain drug delivery Brain is one of the most challenging organs for drug delivery. The importance of the brain requires that drug delivery

systems designed for the treatment of brain diseases meet three requirements: safety, effectiveness, and precision.

1.1 The challenges to cross the BBB The major obstacle for the efficient and precise brain drug delivery is the BBB. On the one hand, BBB confers the CNS as a relatively privileged microenvironment and protects it from periphery substances which may disturb neuronal functions. On the other hand, it makes the translocation of drugs from blood into brain parenchyma extremely difficult.14 First of all, the tight junctions of BBB significantly reduce permeation of ions and other hydrophilic substances through the intercellular space, forming the ‘physical barrier’. Secondly, the membrane efflux pumps, such as P-glycoprotein in BCECs, remove metabolic wastes and other exotic substances from the brain parenchyma to the blood, forming the ‘transport barrier’. Thirdly, extracellular and intracellular enzymes in brain parenchyma, metabolize most toxic compounds, providing the ‘enzymatic barrier’.15 All these barriers contribute to the low penetration efficiency of drugs and drug delivery systems into the brain. With the elucidation of the structure and molecular components of BBB, especially at the intraluminal side, several noninvasive approaches have been developed to allow drug delivery across the BBB, among which lipid-mediated drug transport, carrier-mediated transcytosis, receptor-mediated transcytosis, adsorptive-mediated transcytosis and intranasal administration are the most-widely used appraoches.16 The carrier-mediated transcytosis, receptor-mediated transcytosis


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and adsorptive-mediated transcytosis mechanisms, naturally serve to transport various physiological requirements such as glucose and amino acids that are incapable of diffusing through the cell membrane to the brain, have been utilized to design the brain-targeted nanoparticulated drug delivery systems.16 Not only polymers, lipids but also inorganic materialsbased nanoparticles have been utilized for brain drug delivery. However, the issue of relatively low penetration efficiency still remains to be resolved.

1.2 The safety issues Neurons communicate through the combination of chemical signals (neurotransmitters and modulators) and electrical signals. To insure precise communication, a stable internal environment must be maintained. Any acute disturbance, caused by accumulated toxics or immunological activation in brain parenchyma, may lead to severe pathological changes of brain functions.17 Thus, biocompatibility, biodegradability and low immunogenicity are the fundamental requirements for brain drug delivery systems. However, up to now, the toxicity of various nanoparticulate drug delivery systems to the CNS and how are they cleared from the brain have not been fully elucidated. Exosomes, the natural nanostructure, may provide a drug delivery nanoplatform with optimal biocompatibility, biodegradability and low immunogenicity.

2.

Exosomes in the brain under physiological and pathological conditions. Accumulated studies showed that exosomes function as a fundamental medium for the communication between brain

and periphery and within the CNS, and play important roles in brain homeostasis, neuronal-glial metabolic exchange and neuronal plasticity.18 Exosomes secretion has been observed from neurons,19 oligodendrocytes,20 microglia,21 astrocytes,22 brain endothelial cells,23 and pericytes.24 Exosomes released by these cells contribute to the formation of complex interconnected networks that underlie the physiology and pathology of the CNS (Figure 3).

Figure 3. Intercellular communication of exosomes between the periphery and the CNS. Peripheral circulating exosomes can enter the CNS through receptors on BBB and release their contents for functions. In turn, the exosomes secreted by various cells in the brain can also enter the periphery freely. In the CNS, exosomes also mediate communication between



neuron and glia cells. Under physiological conditions, exosomes deliver active proteins and small RNAs to mediate normal CNS function; under pathological conditions, exosomes transmit inflammatory cytokines, misfolded proteins to promote the development of diseases.

2.1 Exosomes for the maintenance of healthy brain environment Neuronal exosomes act as trans-synaptic carriers for those signaling proteins involved in synaptic plasticity. For instance, Korkut et al.25, 26 showed that in Drosophila, the Wnt-family signaling protein Wingless was sorted into exosomes for transsynaptic transport, and the inhibition of this trafficking impaired the formation of synaptic boutons. Neuronal exosomes also contain mRNA of activity-regulated cytoskeleton-associated protein (Arc). This exosomal Arc mRNA could be translated into Arc proteins in recipient neurons, and act as a regulator of synaptic plasticity.27, 28 Neuronal exosomes maintain the brain vascular integrity by transferring miR-132 into the vascular endothelial cells, leading to up-regulation of vascular endothelial cadherin (VE-cadherin), an important adherens junction protein.29 Disruption of neuronal miR-132 expression or inhibition of exosome secretion appeared to cause intracerebral hemorrhage in zebrafish.29 Neuronal exosomes can also transfer cargoes to glia cells and modulate their function. Morel, L. et al.30 found that the uptake of neuronal miR-124a-loaded exosomes into astrocytes resulted in increased protein expression of glutamate transporter-1 (GLT1), which further regulated extracellular glutamate levels and modulated synaptic activation. Glial cells also provide support and feedback for synaptic activity of neurons by secreting exosomes. For instance, oligodendrocytes secrete exosomes in response to the neurotransmitter glutamate, and these exosomes encounter different fate when enter different cells. When internalized by neurons, oligodendrocytes-derived exosomes enhance the neuronal stress tolerance, increase neuronal firing rate and alter gene expression of several plasticity-related proteins, such as VGF nerve growth factor inducible (Vgf) and brain-derived neurotrophic factor (Bdnf).31, 32 In contrast, when internalized by microglia, these oligodendrocyte-derived exosomes were subjected to degradation.33

2.2 Exosomes as mediators in the pathological conditions Accumulating evidences emerge that exosomes significantly contribute to the development of several neuropathology (Figure 4).18 For example, in infectious diseases, human immunodeficiency virus (HIV)-infected cells propagate susceptibility by releasing exosomes that packaged with trans-activation response element (TAR) miRNA to the recipient cells such as astrocytes.34 Exosomes are also involved in the propagation of inflammation across the BBB and throughout the CNS. The cerebral endothelial cells could be initially activated by systemic inflammation, and then activated their neighboring cells via exosome serection.18, 35 Microglia-derived exosomes were also found to play a pathogenic role in traumatic brain injury (TBI). Following intracortical injection in naïve animals, both circulating exosomes and microglia obtained from TBI animals induced neuroinflammation.36, 37 These pathogenic effects of microglia-derived exosomes could be derived from the pro-inflammatory factors such as TNF-α, IL-1β and miR-155 that entrapped in the exosomes.37 In neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease, aggregation of misfolded proteins is the key pathological change. The formation of exosomes in selected areas of the CNS plays active roles in the pathogenesis and aggravation of these diseases.38 Exosomes not only contribute to the spreading of toxic protein aggregates, but also affect the aggregation process and the clearance of the aggregates.38 For instance, exosomes are involved in the pathogenesis of AD by affecting the accumulation of extracellular amyloid-β (Aβ), the major hallmark of AD. Rajendran et al.39 demonstrated that the cleavage of amyloid precursor protein (APP) by β-secretase occurs


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in early endosomes followed by routing Aβ to MVBs, from where it is released in association with exosomes. In addition, exosomal proteins such as Alix and flotillin-1 are found to accumulate around amyloid plaques in the brains of AD patients, supporting the relevance between exosomes and Aβ spreading.39 Furthermore, Hirohide et al.40 demonstrated that microglia used exosomes to spread aggregated tau, another hallmark of AD, and inhibition of microglial exosome synthesis significantly reduced tau propagation both in vitro and in vivo. Similarly, exosomes are also involved in the spreading of aggregated α-synuclein (α-Syn), the major hallmark of PD.41, 42 In addition to neurons, other cell types including astrocytes can also internalize the α-Syn deposits released by pathologic neurons.43

Figure 4. The contribution of exosomes in mediating mechanisms to neuroinflammation and neuronal survival in the context of CNS injury. Neuronal injury or stress triggers the release of ATP and other danger signals, which can cause release of proinflammatory exosomes from glia cells. Proinflammatory exosomes released from microglia and astrocyte can lead to a decrease in synaptic strength upon neuronal uptake, and these exosomes can also cross the BBB to interact with peripheral targets such as liver to cause acute cytokine response (ACR). Exosomes derived from oligodendrocytes also contain RNA and protein cargo that confer neuroprotection during ischemic stress. IL-1β, interleukin-1β; PrP, prion protein; SOD1, superoxide dismutase 1. Reprinted with permission from Ref 18.

3. Exosomes for Brain Drug Delivery: Challenges and Solutions 3.1 The strategies to overcome the BBB The existence of BBB is the major limiting factor for efficient delivery of therapeutics into the CNS. In terms of overcoming the BBB, exosomes have significant advantages due to their small size and endogenous properties. Emerging evidences found that exosomes can cross or bypass the BBB with or without surface modification both in vivo and in vitro.



3.1.1 Natural exosomes to cross the BBB Different types of natural exosomes can adhere to or be uptake by different cells. This native targeting ability of exosomes depends on the components of exosomes as well as the physiologic state of the recipient cells. For instance, integrins in tumor exosomes determined organotropic metastasis.9 Cell surface receptors such as HSPGs mediated cancer cell exosomes internalization and their functional activity.8 These mechanism could also enable the penetration of exosome across the BBB. Increasing studies demonstrated that certain types of natural exosomes can cross the BBB under experimental conditions. The interaction between exosomes surface ligands and receptors on brain endothelial cells was proposed as the major mechanism. For example, Yuan et al. 44 showed that the naïve macrophage (Mφ) exosomes can cross the BBB through the interaction between the integrin lymphocyte function-associated antigen 1 (LFA-1) on exosomes and 1) intercellular adhesion molecule 1 (ICAM-1) on BBB, 2) and the carbohydrate-binding C-type lectin receptors on brain microvessel endothelial cells. Furthermore, they showed that upregulation of ICAM-1, a common process in inflammation, promotes the uptake of Mφ exosomes in the BBB cells, which indicated that the entry of exosomes into brain can be enhanced in pathological conditions. Pharmacokinetic data showed that the brain accumulation of Mφ exosomes in inflamed brain 0.538 ± 0.315 %ID/g, 5.8-fold greater than in the healthy brain. Qu et al.45 found that blood exosomes exhibited natural brain targeting ability through the transferrin-transferrin receptor interaction. Thus, they developed a biocompatible platform based on blood exosomes for delivering dopamine across the BBB for PD therapy. Claire et al.46 demonstrated that luciferase-carrying exosomes crossed the brain microvascular endothelial cells (BMEC) monolayer through transcytosis under stroke-like, inflamed conditions (TNF-α activated) but not under normal conditions. Beside the BBB-crossing entry pathway, Grapp et al.47 demonstrated that exosomes can also get access to the brain by crossing the cerebrospinal fluid-brain barrier in choroid plexus. They found that the folate receptor-α (FRα)-containing exosomes generated from choroid plexus epithelial cells can cross the ependymal cell layer and distribute into the brain parenchyma, where they can be taken up by astrocytes and neurons. In contrast, FRα-negative exosomes hardly cross the ependymal cell layer and are majorly taken up by microglia. This study provided a potential pathway that can be utilized for the cell-type specific brain drug delivery.

3.1.2 Modified exosomes to cross the BBB Receptor-mediated transcytosis is a naturally existed strategy that can be used to promote exosomes to overcome the BBB. The receptors on the BBB, such as low-density lipoprotein receptor (LDLR), can be utilized to facilitate brain penetration. For example, for the treatment of glioblastoma multiforme, Ye et al.48 developed methotrexate (MTX)-loaded exosomes that functionalized with therapeutic [Lys-Leu-Ala (KLA)] and targeted [low-density lipoprotein (LDL)] peptide. Both ex vivo and in vivo imaging experiments demonstrated that peptide LDL promoted exosomes extravasation across the BBB and drug accumulation at the glioma sites. Another genius modification of exosomes is to transfect the progenitor cells with the fusion gene of brain targeted peptide-encoding gene and the exosomal proteins marker-encoding gene, to generate exosomes enriched with the fusion protein and possessing the ability to cross the BBB. The pioneer study in this area is led by Alvarez et al.13 In order to reduce immunogenicity, they used self-derived dendritic cells for exosome production. Brain targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the rabies virus glycoprotein (RVG) peptide, which can target neurons through acetylcholinergic receptor (Figure 5). Intravenously injection of these modified exosomes delivered GAPDH siRNA to neurons, microglia and oligodendrocytes in the brain, and resulted in a specific gene knockdown. Hung et al.49 used a similar method to enhance the targeted delivery of exosomes to


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neuroblastoma cells. Ohno et al.50 fused GE11 peptide (amino-acid sequence YHWYGYTPQNVI), binds specifically to epidermal growth factor receptor (EGFR), to the transmembrane domain of platelet-derived growth factor receptor, to target tumor cells expressing EGFR. Collectively, these work support the concept of engineering exosomes for tissue and cell-type specific targeted drug delivery to enhance BBB penetration. In addition, for diseases such as brain tumors, Gao’s review provides us with a better idea of double-targeted delivery,51 which can be reasonably used for exosome brain delivery.

Figure 5. Schematic representation of production, harvest and re-administration of targeted self-exosomes for neurontargeting gene delivery. Plasmids encoding the exosomal targeting protein-Lamp2b fused with brain targeting peptide-RVG were transfected into the dendritic cells 4 d before exosome purification. Reprinted with permission from Ref.13

3.1.3 Exosomes to bypass the BBB through intranasal administration Intranasal administration, a noninvasive drug delivery technique, provides another option to allow exosomes cross the BBB and reach the lesions within the CNS. It has been suggested that intranasally applied drugs were transported into the brain mainly via both the olfactory and the trigeminal nerve pathways.52 Many studies have tested the possibility of exosomes brain delivery following intranasal administration. For example, Haney et al.53 found that a considerable amount of catalase-loaded exosomes reached the PD mouse brain following intranasal administration. Exosomes derived from human bone marrow derived mesenchymal stem cells can also reach the hippocampus and enter neurons and microglia in a status epilepticus model mouse within 6 h after intranasal administration.54 Zhuang et al.55 showed that mouse lymphoma cell line-derived exosomes led to rapid brain delivery of the encapsulated drugs after intranasal administration. The accumulation of drugs reached peak concentrations at 1 h after intranasal administration. Although the feasibility of intranasal administration of exosomes for brain drug delivery has been well demonstrated, the specific mechanisms still remain to be elucidated.

3.2 Strategies of Drug Loading Encapsulation of therapeutic agents (chemicals, RNA, DNA, peptides, proteins or lipids) into nanocarriers may greatly enhance their therapeutic efficacy by maintaining their in vivo integrity and improving their biodistribution. However,



effective drug loading in exosomes has been one of the biggest challenges in the field. Various methods have been developed. It can be generally divided into preloading the agents into the donor cell prior to exosomes formation and loading drugs into the purified exosomes (Figure. 6).

Figure 6. The process of drug loading and surface modification of exosomes as brain drug delivery systems. (1) Preloading the therapeutic agents into the donor cells and modifying exosomes with targeting peptides during their formation; (2) Drug loading and surface modification of the purified exosomes after their extraction; (3) Hybridizing with synthetic vectors to improve drug loading. Drug loading during the biogenesis process of exosomes requires hijacking the endogenous loading machinery of the progenitor cells to produce exosomes that contain specific molecules which have been previously internalized by cells. The internalization can be achieved by incubating progenitor cells with the desired therapeutic agents. Different loading methods have been utilized. The most straightforward method is to directly treat the host cells with the agent needed to be loaded, and then collect and purify the therapeutic exosomes for disease treatment. Such method is mostly used for the loading of small molecules. For instance, mesenchymal stem cells (MSCs) and tumor cells, when incubated with chemotherapeutic substances such as paclitaxel or methotrexate, were able to package these chemotherapeutic agents within the exosomes they secreted. Such chemotherapy drug-loaded exosomes showed a strong anti-proliferative activity.56 In contrast, for gene therapy, electroporation and viral transduction (lentivirus, adenovirus) are the main methods for transfecting donor cells with miRNA and siRNA. By using this preloading technique, MSC-derived exosome has been used to deliver exogenous miRNA mimics and pre-miRNAs to human neural progenitor cells (NPCs) and astrocytes, in which the delivered exogenous miR-124 significantly reduced the expression of the target gene Sox9 and increased neuronal differentiation of NPC.57 Furthermore, the delivered miR-124 increased the expression of glutamate transporters EAAT1 and NPC in NPCs and EAAT2 in astrocytes. Similar methods have also been used for siRNA delivery.58 Although such method of drug loading during the biogenesis of exosomes realize drug loading in exosomes in vitro, because of the complexity of exosomal intracellular biogenesis processes, it is accompanied with low and un-controlled loading efficiency. To improve loading efficiency, a new method, the so-called exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs), was developed.59,

60

By combining blue light-controlled reversible protein-protein interaction


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modules with endogenous processes of exosome biogenesis, Yim et al. successfully loaded cargo proteins into newly generated exosomes. They compared the loading capacity of ex vitro protein-loading via extrusion and EXPLOR technology, and found that the latter approach achieved loading capacity forty times higher than the former one. Furthermore, controlled drug loading can also be achieved by designer exosomes. For example, Kojima et al. realized enhanced exosome production, specific mRNA packaging, and delivery of mRNA into the cytosol of target cells through genetically encoding devices in exosome producer cells.61 In contrast, drug loading into exosomes after its formation is a more common strategy, not only because of the simplicity of the process, but also due to the controlled and relative higher loading. The simplest method is passive loading, by incubating the isolated exosomes with the therapeutic molecules. For example, the incubation of exosomes with the hydrophobic drug curcumin led to efficient loading and delivery of curcumin into the brain.55 Hydrophobically-modified siRNAs (hsiRNAs) were also efficiently loaded into exosomes upon co-incubation, without altering the size distribution or integrity of the vesicles.62 However, simple incubation only achieved acceptable loading efficiency for hydrophobic agents. To achieve a higher loading efficiency for hydrophilic therapeutics, active loading strategies, such as electroporation, have been extensively applied. For example, Alvarez-Erviti et al.13 loaded siRNA into purified exosomes via electroporation without substantial alteration of the physical properties of exosomes. Besides, permeabilization with saponin, freeze-thaw cycles, sonication and extrusion have also been applied for exosomal drug loading. Haney et al.53 compared the loading efficiency of the above methods for loading antioxidant enzyme catalase into monocyte/macrophage-derived exosomes, and found that higher loading efficiencies were achieved through sonication, extrusion and permeabilization with saponin. These active loading processes maintained the integrity of exosomes, and also protected the loaded catalase from degradation in the circulation. Moreover, hybrid nanocarriers, by combining the exosomal endogenous properties and the drug-loading capacity of synthetic vectors, have been developed. For instance, Zheng et al.63 developed a biomimetic metal-organic framework nanosystem with exosomes camouflage for systemic and intracellular delivery of therapeutic proteins. The obtained nanoparticles showed high loading efficiency (up to ∼94%) and loading capacity (up to ∼41%), protected the protein cargos from degradation, alleviated the phagocyte-dependent clearance, enabled targeted delivery to the tumor sites, and promoted intracellular delivery. Moreover, lipid-based nanoparticles have also been used to improve the drug loading capacity of exosomes. For example, Tan et al.64 hybrid exosomes with liposomes, achieving nanoparticles with efficient encapsulation of large plasmids, including the CRISPR/Cas9 expression vectors.

3.3 Source, stability and scaling up of exosomes Another important issue with the exosome-derived brain drug delivery system is the choice of exosomal donor cells. For brain drug delivery, the progenitor cells can be derived from both the CNS and the peripheral systems. Exosomes from different sources contain diverse contents and exert different functions. More applications of CNS-derived exosomes are for the delivery of biomolecules from donor cells into recipient cells to complete intercellular communication and mediate the physiological and pathological processes in the CNS. For example, astrocyte-derived exosomes stimulate dendritic arborization of neurons by transporting synapsin I.65 Astrocytes also produce exosomes to regulate neuronal plasticity through miRNAs. For example, astrocyte-derived exosomes carrying miR-26a to modulate neuronal function and brain physiology through the targets such as GSK-3β, PTEN and Wnt5a.66 CNS-derived exosomes are also involved in the process of pathogenesis. For example, glioma cells promote angiogenesis through the release of exosomes containing long non-



coding RNA POU3F3.67 After lipopolysaccharide (LPS) stimulation, exosomal miR-214-5p released from glioblastoma cells regulates the inflammatory response of microglia by targeting CXCR5.68 A close relationship can also be found between the peripheral exosomes and the CNS. Li et al.69 found that serum-derived exosomes from LPS-challenged mice activated microglia in healthy normal mice by increasing expression of proinflammatory cytokine mRNA and inflammation-related microRNAs in the CNS of the recipient mice. In contrast, exosomes derived from serum of young animals promoted oligodendrocyte precursor cells differentiation, and improved remyelination after lysolecithin-induced demyelination in brain slice cultures.70 Such effect was found to be mediated by miR-219, which is abundant in the peripherally exosomes. Beside blood-derived exosomes, MSCs-derived exosomes also play an important role in the CNS, and can be applied for the treatment of brain diseases. For example, MSC-derived exosomes have been used for the management of TBI,71 stroke72 and autism,73 possibly by delivering gene materials such as miR-133b to enhance neurite growth,74 and promote neural plasticity and functional recovery.72 In addition to exchanging their naturally carried cargoes, exosomes can also deliver exogenous biomolecules into the CNS. The choice of donor cells is diverse. Macrophage Raw 264.7,44, 53, 75-77 MSC,57, 78, 79 dendritic cells13 and engineering cells such as 293T cells are the widely used donor cells.58, 60, 80 Secretory cells are one of the best choices of donor cells as these cells can produce more exosomes. Engineered cells, can be easily manipulated, are taken as the optimal donor cells for exogenous nucleic acids delivery. In a word, the source of exosomes is one of the most important factors that determine the brain drug delivery property of exosomes, and needed to be taken into careful consideration. Another consideration for exosomes as drug delivery vectors is stability. One of the major advantages of exosomes as drug delivery carriers is their endogenous nature, which may lead to good biocompatibility and long circulation time. Wiklander et al.81 studied the biodistribution of exosomes from various cell sources, and found that cell origin, exosome dose, and route of administration all influenced its biodistribution. Lai et al.82 determined the distribution and clearance of exosomes in vivo using a multimodal imaging reporter, finding that most exosomes were cleared within 6 hours after intravenous injection. Active uptake by phagocytic cells such as Kupffer cells in the liver and alveolar macrophages in the lung contributed to the fast clearance, and inhibition of such exosome-phagocytic cells interaction slowed the clearance. CD47, a ligand for signal regulatory protein alpha (SIRPα), inhibited phagocytosis and prolonged the systemic circulation of exosomes through interacting with SIRPα and initiating the ‘don’t eat me’ signal.83 For instance, Kamerkar et al.84 found that human foreskin fibroblast-derived exosomes exhibited longer systemic circulation than liposome through such CD47mediated phagocytosis escape. Besides, the introduction of polyethylene glycol (PEGylation) on the surface of exosomes can also significantly increase their circulation time,85 and such technique might be used to enhance exosomes accumulation in the tumor and inflammatory sites. Last but not least, the preclinical and clinical development of exosome technology as a delivery platform requires a large number of exosomes. Currently, the low yield of exosomes generated by conventional methods has hindered the application of exosomes as drug delivery platforms. To solve this problem, new strategies compatible with good manufacturing practices (GMPs) have emerged, such as the combination of microcarrier-based 3D cell culture and tangential flow filtration (TFF), which significantly increased the production of exosomes.86 With technological innovation, researchers may be able to obtain high yield, high purity and even customized exosomes.87 In addition, to achieve better quality control, different exosome characterization methods, both biophysical and molecular, have also been developed to characterize these vesicles. These methods are classified into three types:88, 89 firstly, based on morphological characterization of exosomes, such as scanning electron microscopy, transmission electron microscopy, frozen electron microscopy, and atomic force


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microscopy; secondly, based on particle size, including dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), nano flow cytometry; and thirdly, based on specific molecular characterization of exosomes, such as western blot, immunofluorescence, and flow cytometry. In summary, the source of exosomes and their stability in the body are the key factors that determine their brain delivery efficiency. There are still a lot of research space on which donor cells to select and how to modify exosomes to achieve optimal brain drug delivery. Large-scale preparation and quality control are also key issues that need to be addressed for future application.

4. Applications of Exosomes for Brain Drug Delivery Extensive efforts have been devoted into using exosomes as nanocarriers for brain drug delivery (Table 1). The effect of exosomes from different cell sources, with certain targeted design and various drug loading strategies, on brain drug delivery has been evaluated in animal models of various brain diseases.

Table1. Applications of Exosome for Brain Drug Delivery Diseases

Donor Cell

Loading Drug

Loading Strategy

References

PD

Blood

Dopamine

Incubation

45

Raw 264.7

Catalase

a. Incubation at RT with or

53

without saponin; b. freeze-thaw cycles; c. sonication; d. extrusion genetically-modified

GDNF

Transfection

75

Catalase pDNA

Transfection

76

α-Syn SiRNA

Transfection

90

Raw 264.7 Raw 264.7 Murine

dendritic

cells AD

Dendritic cell

BACE1 siRNA

Electroporation

13

Glioma

Raw264.7

Curcumin

Electroporation

77

BM-MSCs

anti-miR-9

Transfection

91

HEK293T

pCD-UPRT

Transfection

92

MCAO

BM-MSCs

Curcumin

Incubation

79

Ischemia

BM-MSCs

miR-124

Electroporation

93

Neuro-

Raw 264.7 Mϕs

BDNF

Incubation

44

inflammation

EL-4

Curcumin, JSI-124

Incubation

55



morphine

HEK293T

relapse

opioid receptor mu

Transfection

Page 14 of 29

94

(MOR) siRNA

4.1 Neurodegeneration diseases PD is a neurodegenerative disorder characterized with selective degeneration of dopaminergic neurons in the substantia nigra, formation of Lewy bodies composed of fibrillar α-Syn, and the release of neurotoxic reactive oxygen species.95 Exosomes have been used to alleviate the pathological symptoms of PD by loading with small molecules, proteins and gene drugs. For example, Qu et al.45 successfully delivered dopamine to the striatum and substantia nigra through blood exosomes. Zhao et al.75 ameliorated neurodegeneration and neuroinflammation in PD mice by systemic administration of macrophages-derived exosomes carrying glial cell-line derived neurotropic factor. Haney et al.53 loaded the antioxidant catalase into the exosomes released from Raw 264.7 cells, and used it to protect neuron in PD model mice through intranasal administration. They also developed genetically modified macrophages that carry reporter and therapeutic genes to neural cells. The transfected macrophages secreted exosomes to efficiently transferred their contents to contiguous neurons, induced in de novo catalase synthesis in target cells and achieved significant improvements in the motor functions of PD model mice.76 Cooper et al.90 used RVG-modified exosomes to achieve widespread delivery of α-Syn siRNAs to the brain, and significantly reduced α-Syn mRNA and protein levels in both normal and the S129D α-Syn transgenic mice. Besides, exosomes have also been applied for the treatment of AD. Aβ is the major hallmark of AD and is derived from cleavage of APP by β secretase (BACE1) and γ secretase. The therapeutic strategies for treating AD is mainly focused on reducing the production or accelerating the clearance of Aβ. Alvarez-Erviti et al.13 developed brain-targeted siRNA delivery system for AD therapy using exosomes from self-derived dendritic cells. As mentioned above, the targeting was achieved by fusing Lamp2b, an exosomal membrane protein, to the neuron-specific RVG peptide. The therapeutic potential of this exosome-mediated siRNA delivery system was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a potential therapeutic target for AD treatment.13

4.2 Brain cancers Glioblastoma is one of the most infiltrating, aggressive and poorly-treated brain tumors. The majority of the therapeutics on market for the treatment of glioblastoma are ineffective, mainly due to the limited drug penetration to the tumor sites and rapid development of resistance to chemotherapy in glioblastoma cells. Exosomes have also been applied for the delivery of gene drugs for the anti-tumor therapy of brain cancers. Erkan et al.92 encapsulated an in-frame fusion of cytosine deaminase and uracil phosphoribosyltransferase as a suicide therapeutic molecule into the exosomes to cause DNA replication defects and apoptosis in cancer cells by converting 5-fluorocytosine into 5-fluorouracil. Munoz et al.91 used MSCderived exosomes to deliver anti-miR-9 to inhibit the expression of the drug efflux transporter P-glycoprotein to reduce the resistance to temozolomide. More recently, Jia G et al.77 loaded superparamagnetic iron oxide nanoparticles and curcumin into exosomes, and modified the exosomes with neuropilin-1-targeting peptides through click chemistry to achieve gliomatargeted imaging and therapy.

4.3 Ischemic stroke Acute ischemic stroke, caused by brain arterial occlusion, is one of the most common causes of death and disability worldwide. Systemic administration of RVG-exosomes loaded with miR-124 has been developed to treat ischemic injury through strengthening cortical neurogenesis.93 c(RGDyK)-conjugated exosomes loaded with curcumin were found


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efficiently target the ischemic brain areas and suppress the inflammatory response and cellular apoptosis following intravenous administration in a transient mouse middle cerebral artery occlusion mouse model.79

4.4 Neuroinflammation Yuan et al.44 demonstrated that native Mφ exosomes were able to cross the BBB and deliver BDNF to the inflammation brain after intravenous administration. Such exosomes were found to interact with brain microvessel endothelial cells through the interaction between LFA-1 and ICAM-1/the carbohydrate-binding C-type lectin receptors that overexpressed in inflammation. Zhuang et al.55 showed that mouse lymphoma cell line-derived exosomes loaded with curcumin or Stat3 inhibitor JSI-124 led to rapid brain delivery of the encapsulated drugs after intranasal administration. By selectively internalized by microglia in the brain, the curcumin-loaded exosomes induced microglial apoptosis and delayed the progress of experimental autoimmune encephalomyelitis disease, and the JSI-124-loaded exosomes inhibited brain tumor growth in a GL26 tumor model.

4.5 Addiction Drug addiction is a chronic relapsing disease caused by the long-term effects of drugs on the brain. Opioid receptor mu (MOR) is the main target of clinical use of opioid analgesics, and is involved in the main enhancement and addiction of opioids. Liu et al.94 designed an MOR siRNA-loaded, RVG peptide-modified exosomes to treat morphine addiction. The obtained exosomes efficiently delivered MOR siRNA to neurons both in vitro and in vivo, significantly reduced MOR mRNA and protein levels, and strongly inhibited morphine relapse.

CONCLUSIONS AND FUTURE PERSPECTIVES Brain drug delivery has been one the most challenging tasks in the pharmaceutical field. Exosomes, nano-sized vesicles secreted by almost all cell types, play a very important role in intercellular communication, and open up a new path for drug transportation into and within the CNS. As a natural, novel drug delivery system, exosomes possess many advantages over traditional drug carriers. Firstly, the exosomes have a small particle size and is highly permeable to the BBB for the treatment of local diseases. Secondly, the exosomes are endogenous substances that can be isolated from the patient itself, thus greatly reducing the risk of toxicity and immunogenicity. Furthermore, exosomes derived from specific cells contain naturally therapeutic molecules, which may be important for the clarification of pathogenesis mechanisms and the discovery of natural drugs. However, there are still many problems need to be solved. Firstly, we still don’t understand how the exosomes in the brain realize intercellular communication. Secondly, we still don’t know how peripheral exosomes access the brain and deliver their cargoes into the CNS. The clarification of the above mechanisms would pose important impact on the exosomemediated brain drug delivery and brain disease treatment. In addition, the drug loading approaches, source selection, stability and targeting properties of exosomes are the major factors that determine the clinical translation value of exosomes as brain drug delivery systems. How to solve the above problems and how to achieve its maximum clinical potential still requires joint efforts from multidisciplinary.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].



Author Contributions #M.Z.

and M.H. contributed equally. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This contribution was financially supported by National Natural Science Foundation of China (No. 81373351, 81573382, 81722043), National Science and Technology Major Project (2018ZX09734005, 2017ZX09304016), and “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (15SG14).

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Table of content



Figure 1. The major three steps of exosomes uptake by acceptor cells. (1) Exosomes or other EVs subtypes are targeted to the acceptor cells through docking on specific molecules such as membrane-exposed proteins, sugars or lipids. (2) Exosomes that enter the acceptor cells through specific endocytosis or through unspecific macropinocytosis or micropinocytosis, which may then be targeted to endosomes. (3) Internalized EVs release their content through the indirect route of endosomal escape. This might involve membrane fusion through unknown molecular mechanisms, which could resemble those used by viruses harboring the fusogenic protein VSVG. Once internalized, EVs might also be recycled and re-secreted or targeted to the lysosome for degradation. (3′) An alternative, direct route of content delivery to the acceptor cell could involve EVs fusion with the plasma membrane and cargo release directly into the cytosol. SNARE, soluble Nethylmaleimide-sensitive fusion attachment protein receptor. Reprinted with permission from Ref.4


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Figure 2. The advantages of exosomes as drug delivery systems. (1) The small size (30~150 nm) enables exosomes to cross the biological barriers. (2) The natural proteins and lipid components on the exosomal membrane enable efficient uptake of exosomes into target cells. (3) The biological endogenous property of exosomes ensures biocompability, stability, low immunogenicity and low inherent toxicity.



Figure 3. Intercellular communication of exosomes between the periphery and the CNS. Peripheral circulating exosomes can enter the CNS through receptors on BBB and release their contents for functions. In turn, the exosomes secreted by various cells in the brain can also enter the periphery freely. In the CNS, exosomes also mediate communication between neuron and glia cells. Under physiological conditions, exosomes deliver active proteins and small RNAs to mediate normal CNS function; under pathological conditions, exosomes transmit inflammatory cytokines, misfolded proteins to promote the development of diseases.


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Figure 4. The contribution of exosomes in mediating mechanisms to neuroinflammation and neuronal survival in the context of CNS injury. Neuronal injury or stress triggers the release of ATP and other danger signals, which can cause release of proinflammatory exosomes from glia cells. Proinflammatory exosomes released from microglia and astrocyte can lead to a decrease in synaptic strength upon neuronal uptake, and these exosomes can also cross the BBB to interact with peripheral targets such as liver to cause acute cytokine response (ACR). Exosomes derived from oligodendrocytes also contain RNA and protein cargo that confer neuroprotection during ischemic stress. IL-1β, interleukin-1β; PrP, prion protein; SOD1, superoxide dismutase 1. Reprinted with permission from Ref 18.



Figure 5. Schematic representation of production, harvest and re-administration of targeted self-exosomes for neuron-targeting gene delivery. Plasmids encoding the exosomal targeting protein-Lamp2b fused with brain targeting peptide-RVG were transfected into the dendritic cells 4 d before exosome purification. Reprinted with permission from Ref.13


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Figure 6. The process of drug loading and surface modification of exosomes as brain drug delivery systems. (1) Preloading the therapeutic agents into the donor cells and modifying exosomes with targeting peptides during their formation; (2) Drug loading and surface modification of the purified exosomes after their extraction; (3) Hybridizing with synthetic vectors to improve drug loading.


Harnessing Exosomes for the Development of Brain Drug Delivery

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