Exosomes as drug delivery vehicles for Parkinson's disease therapy

Abstract

Exosomes are naturally occurring nanosized vesicles that have attracted considerable attention as drug delivery vehicles in the past few years. Exosomes are comprised of natural lipid bilayers with the abundance of adhesive proteins that readily interact with cellular membranes. We posit that exosomes secreted by monocytes and macrophages can provide an unprecedented opportunity to avoid entrapment in mononuclear phagocytes (as a part of the host immune system), and at the same time enhance delivery of incorporated drugs to target cells ultimately increasing drug therapeutic efficacy. In light of this, we developed a new exosomal-based delivery system for a potent antioxidant, catalase, to treat Parkinson's disease (PD). Catalase was loaded into exosomes ex vivo using different methods: the incubation at room temperature, permeabilization with saponin, freeze-thaw cycles, sonication, or extrusion. The size of the obtained catalase-loaded exosomes (exoCAT) was in the range of 100-200nm. A reformation of exosomes upon sonication and extrusion, or permeabilization with saponin resulted in high loading efficiency, sustained release, and catalase preservation against proteases degradation. Exosomes were readily taken up by neuronal cells in vitro. A considerable amount of exosomes was detected in PD mouse brain following intranasal administration. ExoCAT provided significant neuroprotective effects in in vitro and in vivo models of PD. Overall, exosome-based catalase formulations have a potential to be a versatile strategy to treat inflammatory and neurodegenerative disorders.

Keywords: Blood–brain barrier; Catalase; Exosomes; Neuroinflammation; Oxidative stress; Parkinson's disease.

Figure 1. Characterization of exoCAT

Exosomes released from Raw 264.7 macrophages were loaded with catalase by different techniques and examined by: (A) western blot, (B) catalase 10 enzymatic activity; and (C) catalase release. ExoCAT morphology was examined by AFM (D).

Figure 2. Preservation of catalase enzymatic activity in exoCAT

ExoCAT obtained by sonication demonstrated the best protection of catalase.

Figure 3. Accumulation of exoCAT in PC12 cells, and therapeutic effects of exoCAT in in vitro models of oxidative stress

The exoCAT uptake in PC12 cells was examined by spectrofluorimetry (A), and confocal microscopy (B). The bar: 20 µm. The neuroprotection by exoCAT formulations was evaluated in the cell pre-incubated with 6-OHDA (C); (1) catalase, (2) empty exosomes, catalase loaded into exosomes by: (3) incubation at RT, (4) saponin permeabilization, (5) freeze/thaw cycles, (6) sonication, (7) extrusion. The ability to decrease levels of ROS produced in activated macrophages (pre-incubated with LPS and TNF-α) by exoCAT was evaluated by Ampex Red assay in vitro (D).

Figure 4. Biodistribution of DIL-labeled exosomes in mouse brain

Exosomes were administered to mice with 6-OHDA-induced brain inflammation through: intranasal (A, B), or intravenous (C) routs; and compared to PBS-injected controls (D). The bar: 40 µm.

Figure 5. Anti-inflammatory effects of exoCAT in PD mouse model

The intranasal administration of exoCAT significantly decreased microglial activation (D, E) in 6-OHDA-intoxicated mice compared to those intoxicated with 6-OHDA and then treated with PBS (C). Catalase alone did not decrease inflammation in PD mice (F). Empty exosomes did not alter the microglial status in healthy animals (B) compared to healthy controls (A). The anti-inflammatory effects of the described exosomal formulations were quantified by the amount of activated microglial cells (G).

Figure 6. Neuroprotective effects of exoCAT in PD mouse model

The i.n. administration of exoCAT protected DA neurons (D, E) in 6-OHDA-intoxicated mice compared to those intoxicated with 6-OHDA and then treated with PBS (C). Catalase alone was not efficient in this model (F). Empty exosomes did not affect the number of DA neurons in healthy animals (B) compared to healthy controls (A) indicating the absence of cytotoxic effects of the exosomal carriers. The neuroprotective effects of the described exosomal formulations were quantified by the amount of DA neurons in the SNpc (G).

Figure 7. Co-localization of exosomes with different cells in the mouse brain with inflammation

Exosomes released by BMM were labeled with DIL (red). C57BL/6 mice were intoxicated with 6-OHDA, and then i.n. injected with fluorescently-labeled exosomes. Four hours later, mice were sacrificed, perfused, and brain slides were subjected for confocal examinations. Brain slides were stained with antibodies to different cell types and then secondary ab 594 (green). Nucleus was stained with DAPI (blue). Bar: 10 µm.

Figure 8. The flow of exoCAT formulations in clinic