大鼠雾化仪

DNP. Any unentrapped DNP was removed by filtration through a
0.22 μm filter membrane (Millipore, St. Louis, MO, USA), followed
by gel filtration through Sepharose (CL-4B, Solarbio, China).
GPQ-EL-DNP was prepared using a membrane extrusion method.
L-929 cell-derived exosomes were harvested by ultracentrifugation.23
Briefly, L-929 cells were cultured in exosome-free medium for 48 h,
and the culture medium was centrifuged at 300g for 10 min, 2000g for
20 min, and 10000g for 30 min at 4 °C to remove cells, cell debris,
and microvesicles. Finally, the supernatant was centrifuged at 120000g
for 60 min to obtain a precipitate consisting of exosomes. The pellets
were rinsed and ultracentrifuged, then resuspended in sterile PBS.
The protein concentration of the exosomes was determined by BCA
assay, and the concentration was adjusted to 200 μg/mL. Exosome
markers, CD63 and HSP70, were detected by Western blotting
analysis. A 1 mL solution containing mixed exosomes and GPQ-L-
DNP (protein and lipid weight ratio of 1:5) was sonicated and then
extruded 10 times through a 200 nm polycarbonate membrane (Mini-
Extruder, Avanti Polar Lipoids). The successful synthesis of GPQ-EL-
DNP was further confirmed by FTIR (Nicolet 6700, Thermo Electron
Co., Waltham, MA, USA). Coumarin-6-loaded liposomes, DiD-
loaded liposomes, and SIM-loaded liposomes were prepared using the
same method, except 2% coumarin-6, DiD (ThermoFisher, Waltham,
MA, USA), or 5 mg of SIM was added in chloroform to prepare the
corresponding nanoparticles. The fluorescence intensity of coumarin-
6 and DiD showed a linear relationship with the concentration
(Figure S7A,B). L-DNP/SIM and GPQ-EL-DNP/SIM were prepared
with 3 mg of DNP and 1.5 mg of SIM dissolved in chloroform.
The particle size, polydispersity index, and zeta potential of the
exosomes and liposomes were determined by dynamic light scattering
using a Zetasizer Nano-ZS90 (Malvern Instruments, Malvern, UK),
and the morphology was observed using a transmission electron
microscope (TEM) (JEM-1400, JEOL, Tokyo, Japan). The
encapsulation efficiencies of DNP and SIM in nanoparticles were
quantified using HPLC (LC-20A, Shimadzu, Tokyo, Japan), with
ultraviolet detection at 260 nm for DNP and at 238 nm for SIM. The
mobile phase consisted of acetonitrile and 1% acetate acid (95:5, v/v)
at a flow rate of 1.0 mL/min at room temperature.
The membrane fusion of exosomes and GPQ-liposomes was
verified by FRET. FRET liposomes containing 2% fluorescence-
labeled phospholipids in a lipid layer involved equal amounts of the
electron donor PE-NBD (λex/λem = 488/525 nm) and the electron
acceptor PE-RhB (λex/λem = 560/583 nm). Before and after fusion
with exosomes, the emission spectra of the FRET liposomes were
depicted using a fluorescence spectrometer (F-4600, Hitachi, Tokyo,
Japan) and ranged from 500 to 700 nm at an excitation wavelength of
488 nm. The FRET efficiency was calculated by the following
equation: % FRET efficiency = Fa/(Fa + Fd) × 100%. Fa indicates the
emission fluorescence of RhB, and Fd indicates the emission
fluorescence of NBD.
MMP-9 Responsiveness of GPQ-EL. In vitro release testing of
GPQ-EL was performed using a dialysis membrane diffusion
technique. Coumarin-6-loaded GPQ-EL in medium with or without
MMP-9 (5 nM) was added to a dialysis bag (molecular weight cutoff,
3.5 kDa) and then placed in HEPES (pH 7.4) supplemented with
0.3% sodium dodecyl sulfate maintained at 37 °C with constant
stirring at 100 rpm. At predetermined time intervals, the fluorescence
intensity of coumarin-6 outside the dialysis bag was measured by
fluorescence spectroscopy, and the fresh dissolution medium was
replenished.
The responsiveness of MMP-9 was also verified in L-929 cells with
high MMP-9 expression. L-929 cells (5 × 105/well) were seeded in a
six-well plate containing glass coverslips. After 24 h of incubation,
glucose (22 mM) and homocysteine (100 μM) were added for an
addition 6 h of incubation to stimulate MMP-9 expression, and then
FRET liposomes were added. After incubation at 37 °C for 4 h, the
cells were subjected to a confocal laser scanning microscopy (TCS
SP8, Leica, Wetzlar, Germany) for detection in the NBD (525−550
nm) and RhB (560−600 nm) channels at an excitation wavelength of
488 and 560 nm, respectively.