Engineered extracellular vesicles for ischemic stroke treatment

Dear Editor, Nanotechnology-based therapeutic strategies have been proven effective in diseases including cancer, infection, inflammation, etc.1Machtakova M. Thérien-Aubin H. Landfester K. Polymer nano-systems for the encapsulation and delivery of active biomacromolecular therapeutic agents.Chem. Soc. Rev. 2022; 51: 128-152Crossref PubMed Google Scholar However, the application of nanotechnology is greatly restricted in the treatment of central nervous system (CNS) disorders due to physiological CNS barriers. For example, the blood-brain barrier (BBB) can be the “Maginot line” for pharmacologically active molecules, blocking them out of the CNS. Besides, the neuroinflammation and non-directional differentiation of neural stem cells (NSCs) during neurological disorders could also affect neuronal activity and even produce fibrotic scars, thus hindering the CNS regeneration process.2Dorrier C.E. Aran D. Haenelt E.A. et al.CNS fibroblasts form a fibrotic scar in response to immune cell infiltration.Nat. Neurosci. 2021; 24: 234-244Crossref PubMed Scopus (63) Google Scholar Synthetic nanocarriers, including liposomes, micelles, and nanoparticles, can be engineered through PEGylation and functional surface ligands to overcome the BBB and achieve CNS-targeted drug delivery.3Terstappen G.C. Meyer A.H. Bell R.D. Zhang W. Strategies for delivering therapeutics across the blood-brain barrier.Nat. Rev. Drug Discov. 2021; 20: 362-383Crossref PubMed Scopus (190) Google Scholar,4Ruan H. Yao S. Wang S. et al.Stapled RAP12 peptide ligand of LRP1 for micelles-based multifunctional glioma-targeted drug delivery.Chem. Eng. J. 2021; 403: 126296Crossref Scopus (16) Google Scholar However, repeated administrations of synthetic nanoparticles may trigger unexpected immune responses and increase the blood clearance rate of nanocarriers. Inspired by nature, we focus on the naturally evolved biologic nanoparticles, such as extracellular vesicles (EVs), which are cell-secreted membrane-derived signaling nanoparticles, to address these issues. EVs contain biologically active molecules like lipids, DNA, RNAs, and proteins as natural intercellular signals, so they could serve as natural nanomedicine carriers.5Kalluri R. LeBleu V.S. The biology, function, and biomedical applications of exosomes.Science. 2020; 367: eaau6977Crossref PubMed Scopus (2950) Google Scholar More importantly, EVs also demonstrate the ability to cross the BBB. Combined with the advantages of low immunogenicity and good biocompatibility, EVs are a promising class of biological nanocarriers that can be engineered to improve the treatment of neurological disorders.6Zhang Z.G. Buller B. Chopp M. Exosomes - beyond stem cells for restorative therapy in stroke and neurological injury.Nat. Rev. Neurol. 2019; 15: 193-203Crossref PubMed Scopus (279) Google Scholar Taking ischemic stroke as an example, due to the limited number of NSCs in the injured area and the restricted neuronal differentiation efficiency, the neural regeneration in the cerebral ischemic region is inhibited.7Faiz M. Sachewsky N. Gascón S. et al.Adult neural stem cells from the subventricular zone give rise to reactive astrocytes in the cortex after stroke.Cell Stem Cell. 2015; 17: 624-634Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar Thus, a biologically engineered nanomedicine that can transmit NSC recruitment signals and activate their neuronal differentiation ability was designed for ischemic stroke treatment. First, the M2 microglia-derived EVs were used as the core. Then stromal cell-derived factor 1 (SDF-1) was covalently grafted on the surface of EVs through copper-free click chemistry to emit NSC recruitment signals (SDF-1-EVs). When intravenously injected, SDF-1-EVs could cross the BBB and accumulate in the ischemic region (Figure 1A). The immunofluorescent assay (Figure 1B) showed that FITC-labeled SDF-1 could co-localize with Dil-red-labeled EVs, suggesting that SDF-1 was successfully modified on EVs. Besides, the morphology of SDF-1-EVs was in a cup shape with an approximate 120 nm diameter, identical to typical EVs (Figures 1C and 1D). Transwell experiments were carried out to verify the effect of SDF-1-EVs on the recruitment of NSCs. The results revealed that adding SDF-1-EVs in the lower chamber significantly induced NSCs in the upper chamber membrane to migrate to the opposite membrane compared with the control and unmodified EVs groups (Figures 1E and 1F). Additionally, in vitro NSC differentiation assays demonstrated (Figures 1G–1I) that SDF-1-EVs can effectively promote neuronal differentiation (Tuj1+ cells) of NSCs while inhibiting astrocytes (GFAP+) generation. Moreover, the induction effect was similar to the unmodified EVs group, indicating that the modification of SDF-1 did not affect their activity on NSC differentiation while conferring the migratory effect of EVs on NSCs. Next, a transient middle cerebral artery occlusion (tMCAO) mouse model was constructed to simulate clinical ischemic stroke. PBS, EVs, and SDF-1-EVs were intravenously injected respectively. 14 days post-stroke, brains were taken for histological analysis (Figure 1J). Compared with PBS and EVs groups, the significantly higher fluorescent signal of DCX-positive cells in the SDF-1-EVs group indicated that an increased number of immature neurons were migrating toward the ischemic area (Figures 1K and 1N), demonstrating that SDF-1-EVs could efficiently enhance NSC migration and its neuronal differentiation. Moreover, the brain atrophy volume obviously decreased under treatment with SDF-1-EVs (Figures 1L and 1M). The above results all revealed that the biologically engineered SDF-1-EVs exhibited an enhanced therapeutic effect on brain injury. Though EVs can overcome BBB to treat neurological diseases, intravenous administration of EVs is still prone to be quickly cleared by the mononuclear phagocyte system, which is an obstacle to achieving therapeutic drug concentration. Therefore, it is expected to utilize the tissue engineering technique to construct a nanomedicine niche that could serve as the NSC signal transmitter in the specific area of the brain to recruit NSCs and direct their differentiation. Hydrogel microspheres (MSs) prepared using microfluidic techniques are excellent nanomedicine carriers due to their superior injectability, quality controllability, and sustained release.8Zhao Z. Wang Z. Li G. et al.Injectable microfluidic hydrogel microspheres for cell and drug delivery.Adv. Funct. Mater. 2021; 31: 2103339Crossref Scopus (87) Google Scholar By loading SDF-1-EVs into the MSs, the SDF-1-EVs niche ([email protected]) was constructed and stereotactically injected into the intracerebral cavity of the tMCAO model rat. This [email protected] is expected to overcome obstacles of EVs blood clearance and achieve the long-term repair of stroke (Figure 2A). The MSs have a diameter around 200 μm, and they could effectively encapsulate Dil-red-labeled S-EVs (Figure 2B), and the loading efficiency was about 80%. The in vitro release results (Figure 2C) exhibited that S-EVs in [email protected] were slowly released and reached about 46% after 15 days. Besides, [email protected] was co-incubated with NSC in vitro to assess the cytotoxicity of [email protected] It was found that a MS itself had no toxic effect on cell proliferation and exhibited good biocompatibility (Figure 2D). Interestingly, [email protected] was able to promote NSC proliferation, which may be attributed to the function of SDF-1. SDF-1 could promote NSC proliferation in a dose-dependent manner. Also, the growth factors like endothelial growth factor and basic fibroblast growth factor would further enhance the proliferative effect of SDF-1. In vivo studies were performed on the tMCAO model rat. The brain atrophy volume significantly decreased in the [email protected] group compared with the PBS and S-EVs groups, suggesting the importance of MS incorporation (Figures 2E and 2F). Overall, we constructed two different types of engineered EVs as signal transmitters for the recruitment and differentiation induction of NSCs and successfully achieved neural regeneration in the experimental rodent stroke model. We explored the possibility of biologically engineered EVs for neurological disorders treatment using both systemic and local delivery. Systemic therapy exhibits superior clinical compliance, but with the burdens of repeated administration and rapid blood clearance. Although the addition of sustained-release MSs overcomes these defects, the immunogenicity and biocompatibility of MS degradation products remain to be further investigated. Nevertheless, these kinds of engineered EVs therapies, together with related advanced biotechnologies or by itself, show great potential for clinical translation in the diagnosis, monitoring, treatment, and care of not only CNS diseases, but also cardiovascular disease, bone and joint regeneration, cancer, wound healing, and immune disorders.9Zheng D. Ruan H. Chen W. et al.Advances in extracellular vesicle functionalization strategies for tissue regeneration.Bioact. Mater. 2022; https://doi.org/10.1016/j.bioactmat.2022.07.022Crossref Scopus (4) Google Scholar,10Meng F. Li L. Zhang Z. et al.Biosynthetic neoantigen displayed on bacteria derived vesicles elicit systemic antitumour immunity.J. Extracell. Vesicles. 2022; 11: 12289Crossref PubMed Scopus (2) Google Scholar This research was supported by grants from the National Key R&D Program of China (2019YFA0112000), Zhejiang Provincial Natural Science Foundation of China (No. LQ21H300009), National Natural Science Foundation of China (81930051, 82003658, 82202785), and GuangCi Professorship Program of Ruijin Hospital Shanghai Jiao Tong University School of Medicine. The authors declare no competing interests.