HEK293 cells were seeded in 225-cm
2 flasks (Corning). When the cells reached approximately 70-80% confluence, they were co-transfected with plasmids encoding Lamp2b-RVG and MOR siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cell culture medium was then harvested 48 h after transfection, and the exosomes loaded with MOR siRNA were harvested from the medium using an exosome isolation kit (Invitrogen) according to the manufacturer’s instructions. The resulting pellet was then resuspended in PBS.
For transmission electron microscopy analysis, the exosome samples were prepared as described above. Briefly, the exosome pellet was placed in a droplet of 2.5% glutaraldehyde in PBS buffer and fixed overnight at 4 °C. The exosome samples were rinsed 3 times in PBS for 10 min each and then fixed in 1% osmium tetroxide for 60 min at room temperature. Then, the samples were embedded in 10% gelatine, fixed in glutaraldehyde at 4 °C and cut into small blocks (less than 1 mm
3). The samples were dehydrated in increasing concentrations of alcohol. Then, the samples were placed in propylene oxide and infiltrated with increasing concentrations of Quetol-812 epoxy resin mixed with propylene oxide for 3 h per step. Finally, the samples were embedded in pure, fresh Quetol-812 epoxy resin and polymerised at 35 °C for 12 h, 45 °C for 12 h, and 60 °C for 24 h. Ultrathin sections were cut using a Leica UC6 ultra-microtome and stained with uranyl acetate for 10 min and lead citrate for 5 min at room temperature. The samples were then observed with a transmission electron microscope (JEM-1010) at a voltage of 80 kV.
For confocal microscopy analysis, exosomes (100 μg) loaded with fluorescence-labelled siRNA were incubated with the Neuro2A cells (10
6 cells). After 6 hours, the cells were washed, fixed and observed under a confocal microscope (FV 1000; Olympus, Tokyo). Pictures were taken under the following conditions: objective lens: PLAPON 60 × O; NA: 1.42; scan mode: XY; excitation wavelength: 405 nm for Hoechst 33342 and 555 nm for Alexa Fluor 555; and image size: 1024 × 1024 pixels.
Exosomes (100 μg) loaded with MOR siRNAs were incubated with Neuro2A cells (10
6 cells). After 24 h incubation, the recipient cells were collected for total RNA extraction and subsequent quantitative RT-PCR analysis of MOR siRNA and MOR mRNA, and for total protein isolation and subsequent western blotting analysis of MOR protein.
In CPP test, researchers place an animal in a distinctively designed chamber and inject or infuse it with the substance being studied (e.g., morphine in this study). Once trained to associate the chamber with the test agent, the animal is placed in an anteroom connecting a neutral chamber and the drug-associated chamber. The animal indicates its preferred chamber by spending more time there; its choice reveals the animal’s preference for or aversion to the test substance.
Here, C57BL/6J mice (n=6 for each group) were purchased from the Model Animal Research Centre (MARC) of Nanjing University (Nanjing, China). The animals received humane care according to the guidelines prepared by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. A two-chamber CPP apparatus (Yishu Software Technology Co. Ltd., Shanghai, China) was used in this study. The two chambers were identical in size but differed in color and floor texture. The two distinct chambers were linked by a smaller intermediate compartment with a shutter on each side that allowed for control of access to either side of the chamber. One chamber had white walls with a barred floor and was illuminated, while the other had black walls with a dotted floor and was not illuminated.
Pre-conditioning (Day 1): Mice were allowed free access to both chambers for 30 min, and the number of crossings and the time spent in each chamber were recorded. As recommended by the manufacturer's instructions, animals with fewer than 20 crossings or less than 300 s spent in one chamber were excluded.
Conditioning (Days 2–11): On days 2, 4, 6, 8 and 10, mice received a morphine injection (10 mg/kg) before being placed in the white chamber for 30 min. On days 3, 5, 7, 9 and 11, mice received a saline injection (10 ml/kg) before being placed in the black chamber for 30 min. On day 12, all mice were allowed free access to both chambers for 30 min for the CPP test.
Extinction (Days 13-25): All mice were kept in their cages without any treatment.
Exosome injection (Days 26-32): Before exosome injection, all mice underwent a CPP test. On days 26, 28, 30 and 32, mice were injected intravenously with saline, 200 µg exosomes loaded with MOR siRNA, or 200 µg RVG exosomes loaded with MOR siRNA, respectively.
Reinstatement (Days 33-34): On day 33, before being placed in the white chamber for 30 min, all mice received morphine (10 mg/kg). On day 34, all mice underwent a 30 min CPP test.
C57BL/6J Mice (n=3 for each group) were randomly divided into control group and test group. Mice in the control group received saline injection and mice in the test group received exosome injection (200 μg RVG exosomes loaded with MOR siRNA). After two days, mice were subjected to two trials during which they were plunged individually and forced to swim in a tank (height 30 cm, length 50 cm, width 40 cm) filled with 15 cm of water maintained at 25 °C. The first training trial lasted 15 min and was performed one day before testing. Then, after 24 h, a second trial was performed and lasted for 10 min. The time that the test animal spent in the second trial without making any movements beyond those required to keep its head above water was measured and further analyzed. Both of two trials were videoed.
