| 摘要: | Background
There is a need for efficient intranasal drug delivery systems (DDS) capable to by-pass the blood brain barrier for treating brain disorders, like glioblastoma. A multitude of synthetic DDS are under development and a few introduced to the market. At the same time, there is a growing interest in natural drug carrier systems, one of the most prominent examples being extracellular vesicles (EVs), which should exhibit good biocompatibility and low immune response. Furthermore, the membrane of EVs exposes receptors from the mother cells that can exert specific targeting to pathological tissues or cells. Clinical-grade platelets represent a potentially valuable source of EVs (p-EVs) for DDS but much work is still needed to understand the optimal population of p-EVs that could diffuse into the brain safely and target glioblastoma.
Aims
Develop and assess p-EVs as a delivery system to the brain glioblastoma sites.
Material and Methods
Therapeutic-grade human platelet concentrates (PC) were centrifuged to pellet the platelets. Platelets were resuspended in platelet additive solution (PAS) with 6% Dimethyl sulfoxide (DMSO) for frozen storage. p-EVs were generated using 2 different methods: freeze/thaw treatment activation (3 cycles at -80°C/1hr, 37°C/3 min; p-EV-F) or sonication (40 kHz; 30 min; p-EV-S). Natural platelet microparticle (NPMP) a reference p-EV preparation available in our laboratory and isolated from the PC, were also used. The number and size distribution of the NPMP and p-EVs were characterized by nanoparticle tracking analysis (NTA) and Dynamic light scattering (DLS). The labeling procedure for NPMP and p-EVs was performed using the Protein Labeling Kit - Alexa Fluor 568 (AF568). These labeled NPMP and p-EVs were administered intranasally to normal mice. The mice were sacrificed at different time-points 4 hours and 7 hours, after intranasal administration, and the brain was perfused and carefully isolated. Brain slices with 30 μm thickness were cut along the sagittal or coronal axis according to the experimental requirements to compare their diffusion into the brain. Orthotopic xenograft mice are implanted with the U373MG cell line to establish GBM mice, monitored using amplified Magnetic Resonance Imaging (aMRI), also received intranasal administration of the labeled p-EVs and coronal sections were prepared after sacrifice for hematoxylin-Eosin stain and fluorescence imaging. Imaging was performed with a slide scanner (ImageXpress? Pico) to track concussion in mice and visualize whether the intranasally delivered labeled NPMP and p-EVs could target the tumor site.
Results
Based on previous experimental results, sonication (S) and freeze/thaw (F) were selected to generate p-EVs and compare to NPMPs. The mean size and mode size of NPMP and p-EVs was 155-200 nm and 110-150 nm, respectively, by NTA. We selected NPMP as our p-EV model to perform preliminary brain diffusion studies. The brain images results showed red fluorescent AF568-NPMP was able to enter and diffuse into the brain of normal mice within 4 hours and 7 hours upon intranasal administration. In comparison with the control group (PBS), the results indicated that both AF568-p-EV-S and AF568-p-EV-F were also able to reach the brain of normal mice within 4 hours and 7 hours via intranasal delivery. The diffusion patterns of these two p-EVs were different, and changed over time in normal mice. H&E staining of GBM mice revealed that only one mouse in each of the NPMP and p-EV-S groups exhibited increased hematoxylin staining. No increased hematoxylin staining was found in the p-EV-F group, like in the controls. Brain diffusion studies in GBM mice revealed red fluorescence of intranasal labeled NPMP surrounding areas at the tumor cell implants with increased density of blue fluorescent nuclei.
Conclusions
Our study revealed that NPMP, p-EV-S and p-EV-F can all diffuse within the brain upon intranasal delivery but with apparently different diffusion patterns. In an orthotopic xenograft mouse model, NPMP was found to aggregate around cells with increased nucleus density, suggesting that it may have the potential for specific diffusion and retention to GBM site. It remains necessary to improve the tumor model to confirm any targeting potential of NPMP, p-EV-S and p-EV-F in GBM. Future experiments will be needed to further delineate the ability of p-EVs to serve as drug carriers for the treatment of brain tumors. |
