Nanoparticle use for the study of exosome transport to the brain through the lymphatic pathway
Authors:   , Ramos Zaldívar Héctor M. 
Published: September 2023

Introduction: Exosomes are extracellular vesicles with a size of 50-150 nm that have been associated with the transportation of various biological contents and with intercellular communication. Given their role in metastasis, understanding exosome tissue distribution is critical to cancer pathophysiology. The exact routes and mechanisms of exosome distribution from peripheral organs to the central nervous system (CNS) remain unknown. A possible route is through the recently discovered brain lymphatic system, due to its connection with the deep cervical lymph nodes and its morphological characteristics. Hypothesis: Metastatic cell-derived exosomes are transported from the deep cervical lymph nodes to the central nervous system through the meningeal lymphatic vessels. Objective: To develop nanoparticle-loaded exosomes derived from a metastatic cell line and administer these exosomes via the cervical and meningeal lymphatic system to evaluate their arrival to the central nervous system. Methodology: Superparamagnetic iron oxide nanoparticles (SPIONs) (mean size 8.3 ± 2.9 nm and Zeta potential 36.8 ± 5.44 mV) were prepared by chemical coprecipitation of ferric and ferrous chlorides. Exosomes (41.77 ± 1.64 nm and -10.8 ± 2.49 mV) were isolated from the melanoma B16F10 cell line through the Exo-Spin column protocol and loaded with SPIONs through electroporation. Gold nanorods (11.25 ± 0.57 nm and 45.4 ± 7.62 mV) were prepared and functionalized with polyethylene glycol. Chinese ink nanoparticles (61.62 ± 4.84 nm and -6.34 ± 0.63 mV) were also used. C57BL/6 mice were used to evaluate the anterograde and retrograde route of the lymphatic meningeal system with post-mortem and in-vivo procedures. All animal procedures were approved by the Ethical Animal Committee of our institution. Mice were anesthetized with isoflurane. To evaluate the anterograde nanoparticle flow we injected 10 µL of each nanoparticle solution in the cisterna magna (3 animals per condition). To evaluate the retrograde nanoparticle flow we injected 10 µl of each nanoparticle solution (SPIONs 3200 μg/mL; exosomes + SPIONs 1.67 x 1011 particles/mL; gold nanorods 1.71 x 1014 particles/mL; Chinese ink 10%) in the deep cervical lymph node (3 animals per condition). The animals were euthanized after 30 min post injection. The head and neck were fixed with 4% paraformaldehyde for histological analysis and post-mortem MRI imaging. Results: Anterograde pathway: Both SPIONs and SPION-loaded exosomes showed hypointense signals of cervical lymphatic structures after intracerebroventricular injections through the cisterna magna in the T2w and T2* MRI images. Gold Enhancement technique confirmed anterograde flow of both gold nanorods and Chinese ink nanoparticles by cervical lymphatic staining. Macroscopically, cisterna magna injections showed staining of deep cervical lymph nodes within the first minute after the administration of Evans Blue dye and Chinese ink. Retrograde pathway: Both SPIONs and SPION-loaded exosomes revealed hypointense signals in the brain ventricles and parenchyma in MRI T2w image and T2* map, after 30 min of deep cervical lymphatic injection. Gold Enhancement staining showed histological confirmation of the arrival of gold nanorods and Chinese ink nanoparticles to the brain parenchyma from the cervical injections. Macroscopically, deep cervical lymph node injections with Evans Blue and Chinese ink showed staining of the meninges and brain parenchyma. Nanoparticles colocalized with the stain of meningeal lymphatic vessels using anti-LYVE-1. Discussion: The cervical and meningeal lymphatic system can transport nanoparticles not only in the classically described lymphatic drainage towards the thorax but can also serve as an access gate to the brain. This newly discovered mechanism for the meningeal lymphatic pathway could be exploited in the theranostic field of nanomedicine to deliver drugs for the treatment of various neurological diseases and the developing of diagnostic contrast media. The understanding of cancer exosome distribution through the cervical and meningeal lymphatic system will aid in a more profound comprehension of brain metastasis pathophysiology.