Multifunctional exosome-mimetics for focused anti-glioblastoma remedy by manipulating protein corona | Journal of Nanobiotechnology


Cell tradition and animals

bEnd.3 Mind-derived Endothelial cells, U87-MG/U87-MG-luc human glioblastoma cells and RAW264.7 macrophages have been maintained in Dulbecco’s modified Eagle’s minimal important medium supplemented with 10% fetal bovine serum (FBS) (VivoCell, Shanghai, China). Cells have been incubated at 37 °C in humidified air with 5% CO2. Animals have been obtained from SJA Laboratory Animal Co., Ltd (Hunan, China). Animal research have been permitted by the Division of Laboratory Animals of Central South College.

Preparation, characterization and drug loading of nanoformulations

U87-MG cells-derived exosomes have been ready as described beforehand [34]. Briefly, the supernatant of U87-MG cells tradition was collected (48 h after incubation), and differentially centrifuged and filtered by a 0.2 μm filter adopted by ultracentrifugation at 110,000 × g for 70 min and washed with phosphate-buffered saline (PBS). Cell membrane proteins of U87-MG cells have been extracted utilizing a Mem-PER™ Plus Membrane Protein Extraction Package (Thermo Fisher Scientific, USA) in accordance with manufactures’ directions. Liposomes (Lipo) have been synthesized by the skinny layer evaporation (TLE) adopted by the extrusion methodology. Briefly, 10 mg of DMPC, 2 mg of DSPE-PEG2000 and 1 mg of ldl cholesterol have been dissolved in methanol (9 mL). The solvent was evaporated utilizing a rotatory evaporator (N-1300, EYELA, Japan) to type a skinny movie. Movies have been hydrated with water for 30 min. Lipid suspension was extruded by 200-nm cellulose acetate membranes (Whatman, USA) for 15 occasions utilizing Avanti Mini-Extruder (Avanti Polar Lipids, USA). For the development of EM, lipid movies have been hydrated with 100 μL of PBS containing 70 μg of membrane proteins. Ang (TFFYGGSRGKRNNFKTEEY) was obtained from Ruixi Organic Expertise Co., Ltd. (Xi’an, China). Ang-conjugated Lipo or EM have been developed by introducing DSPE-PEG2000-Angiopep-2 to the lipids. Ang on DSPE-PEG2000 was characterised by 1H-NMR.

Measurement distribution, polydispersed index (PDI) and zeta potentials of Lipo, Ang-Lipo, Ang-EM and U87-MG cells-derived exosomes have been analyzed utilizing Zetasizer (ZS90, Malvern, UK). Additionally, nanoparticle monitoring evaluation (NTA) was carried out to evaluate the dimensions distribution of U87-MG cells-derived exosomes (Nanosight NS300, Malvern, UK). Morphology was noticed by transmission electron microscopy (TEM). Photos have been captured utilizing a Tecnai G2 Spirit TWIN Electron Microscope (FEI, USA). Protein profiles of Ang-EM and exosomes have been in contrast by Coomassie sensible blue staining (Beyotime, China). The presence of protein markers CD47 (ab175388, Abcam, UK), CD9 (ab92726, Abcam, UK) and CD63 (ab216130, Abcam, UK) have been detected by way of western blotting and analyzed utilizing a gel imaging system (ChemiDoc™ Contact, Bio-Rad, USA).

Drug-loaded formulations have been obtained by add 1 mg of docetaxel (DTX) to the lipids (13 mg) earlier than skinny layer formation. Free medication have been eliminated by ultrafiltration (100 kDa). The dimensions distributions, zeta potentials, drug-loading capability and encapsulation effectivity of nanoparticles after loading DTX have been measured. The quantity of DTX loaded into nanoparticles have been measured by HPLC (HA-20 T, Shimadzu, Japan). The discharge profile of DTX-loaded nanoparticles below shaking (100 rpm) was evaluated utilizing an ultrafiltration tube with 10 kDa cutoff (Millipore) towards PBS.

Mobile uptake and in vitro BBB transport

Lipo, Ang-Lipo and Ang-EM have been labeled with fluorescent dye DiL (Yeason, China). Briefly, 4 μL of DiL (1 mg/mL) was added to the lipids dissolved in methanol as described above. Unbounded DiL was eliminated by utilizing a ten kDa ultrafiltration tube. bEnd.3 cells and U87-MG cells in 24-well plate have been handled with DiL-labeled nanoparticles for 3 h adopted by fixing with paraformaldehyde and nuclei staining with DAPI (Beyotime, China). Mobile internalization was noticed utilizing Olympus IX73 fluorescence microscope (Olympus, Japan).

The in vitro BBB mannequin was developed as beforehand described [9]. Briefly, cell tradition inserts (Corning, NY, USA), pre-coated with human plasma fibronectin (50 μg/mL) for 1 h, have been put into 24-well plates. Then, 1 × 104 bEnd.3 cells have been seeded on every higher chamber insert and cultured with 0.5 ml of medium. After the cell confluency within the higher chamber reached 80%, the underside chamber was seeded with 1 × 104 U87-MG cells crammed with 1.5 ml of tradition medium. To judge the transcytosis, DiL-labeled nanoparticles have been added to the higher chamber of the in vitro BBB mannequin. Cells within the decrease chamber have been imaged by fluorescence microscope at completely different time factors to look at the uptake of nanoparticles.

Cytotoxicity and cell cycle assay

To evaluate the cytotoxicity of DTX@Ang-EM, U87-MG cells have been seeded into 96-well plates at a density of 5 × 103 cells per properly in a single day after which handled with free DTX or DTX nanoformulation for twenty-four h. Cell viability was assessed by CCK8 assay (NCM biotech, China) by measuring the absorbance at 450 nm utilizing an Infinite F50 microplate reader (Tecan, Switzerland). For cell cycle assay, U87-MG cells have been seeded into 6-well at a density of 1 × 104 per properly and handled with free DTX or DTX nanoformulation at an equal DTX dose (5 μg/mL) for twenty-four h. Cell cycle assay equipment (Beyotime, China) was used in accordance with the producer’s directions. Cell cycles after therapy have been analyzed utilizing circulation cytometry (BD Biosciences, USA).

3D tumor spheroid

U87-MG cells have been cultured and embedded in Matrigel (BD, USA) to type spheroids. Stay spheroids have been imaged. The tumor spheroids have been incubated with completely different drug-loaded formulations for 48 h. Photos of spheroids have been imaged to look at measurement. Additionally, tumor spheroids have been incubated with DiL-labeled nanoparticles for 4 hours and washed with PBS. Nuclei have been stained utilizing Hoechst 33258. Z-stack scanning photographs have been taken utilizing confocal microscopy (Leica TCS SP8 X, Leica, Germany) to look at the penetration of nanoparticles into intact and reside spheroids. Spheroids have been incubated with drug-loaded nanoparticles at equal DTX focus (10 μg/mL) for 2 days and imaged below microscope to look at the in vitro antitumor results.

Protein corona formation and evaluation

To type protein corona in vitro, nanoparticles have been incubated straight with FBS at 37℃ for 1h [35]. Nanoparticles have been separated from extra proteins by centrifugation (15000 g × 15 min) adopted by washing with PBS for twice on the similar centrifugation situation. Measurement distribution, PDI and zeta potentials for nanoparticles earlier than and after protein corona formation have been in contrast. TEM was carried out to look at morphology of protein corona on nanoparticles. BCA assay and SDS-page evaluation of profiles of protein corona on nanoparticles have been additionally carried out.

Proteins have been characterised by mass spectrometry (MS). Briefly, protein samples have been ready by SDT lysis as beforehand described [36]. Samples have been analysed on a nanoElute (Bruker, Bremen, Germany) coupled to a timsTOF Professional (Bruker, Bremen, Germany) geared up with a CaptiveSpray supply. Peptides have been separated on a 25 cm × 75 μm analytical column, 1.6 μm C18 beads with a packed emitter tip (IonOpticks, Australia). The column temperature was maintained at 50 °C utilizing an built-in column oven (Sonation GmbH, Germany). The column was equilibrated utilizing 4 column volumes earlier than loading pattern in 100% buffer A (99.9% MilliQ water, 0.1% FA) (Each steps carried out at 800 bar). Samples have been separated at 300 nl/min utilizing a linear gradient as follows: 3% buffer B for 3 min, 3–28% buffer B for 70 min, 28–38% buffer B for 7 min,38–100% buffer B for five min, maintain in 100% buffer B for five min.

The timsTOF Professional (Bruker, Bremen, Germany) was operated in PASEF mode. Mass Vary 100 to 1700 m/z, 1/K0 Begin 0.6 Vs/cm2 Finish 1.6 Vs/cm2, Ramp time 100 ms, Lock Obligation Cycle to 100%, Capillary Voltage 1500 V, Dry Gasoline 3 l/min, Dry Temp 180 °C, PASEF settings: 10 MS/MS scans (complete cycle time 1.16 s), cost vary 0–5, energetic exclusion for 0.4 min, Scheduling Goal depth 20,000, Depth threshold 2500, CID collision vitality was 42 eV. The MS information have been analysed by label-free quantification utilizing MaxQuant software program model Proteins have been recognized by data-dependent acquisition based mostly on the Uniprot_Bovine_46766_20210308 peptides database.

To judge the consequences of protein corona formation on mobile uptake of nanoparticles, nanoparticles have been labeled by DiL and incubated with FBS to type PC after which incubated with U87-MG cells and Raw264.7 cells to visualise the mobile uptake.


In situ U87-MG GBM mannequin was developed as beforehand described with modification [37]. Mice have been intracranially implanted with 2 × 106 U87-MG cells utilizing a ten μl microsyringe pump. Tumor-bearing mice have been administrated with DiR-labeled nanoparticles or free DiR by way of i.v. injection. 4, 8 and 24 h post-injection, fluorescence was measured utilizing IVIS spectrum (PerkinElmer, USA). Ex vivo biodistribution in brains and different main organs was additionally inspected.

In vivo antitumor research

Mice with GBM have been handled with PBS, DTX, DTX@Lipo, DTX@ANG-Lipo or DTX@ANG-EM (5 mg/kg DTX) 4 occasions with an interval of three days. Bioluminescence photographs have been obtained by IVIS Spectrum (PerkinElmer, USA). After intervention, mice have been sacrificed, blood samples and tumor and main organs have been excised and weight. Main organs have been fastened in 4% PFA and stained with H&E. Tumors have been stained by TUNEL to look at cell loss of life. Plasma ranges of ALT, AST, BUN, Cr have been measured utilizing assay kits (Rayto, China). To look at the tumor penetration of nanoparticles, GBM-bearing mice got Dio-labeled Lipo, Ang-Lipo or Ang-EM for one injection, mind tissues have been collected, fastened, sliced and stained with DAPI and noticed below fluorescence microscope.

Statistical evaluation

Information have been introduced because the imply ± SD. A two-tailed Pupil’s t-test was utilized to check the statistical significance of the distinction between two teams, one-way evaluation of variance (ANOVA) was utilized to check the statistical significance of distinction amongst three or extra teams. The statistical significance was set at * P < 0.05, ** P < 0.01 and ***P < 0.001.


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