An anti-CD19-exosome supply system navigates the blood–mind barrier for focusing on of central nervous system lymphoma | Journal of Nanobiotechnology

March 6, 2025

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Cell tradition and preparation

hAMSCs had been extracted from human adipose tissue. SU-DHL-8, HEK-293T, hCMEC/D3, HA, HT22 and HK-2 cell strains had been bought from the American Sort Tradition Assortment. The cell strains had been authenticated by way of quick tandem repeat profiling performed by the cell line characterization core (Genetic Testing Biotechnology Company, China).

Preparation of anti-CD19 modified hAMSCs

For the genetic modification of anti-CD19 to hAMSCs, anti-CD19 lentivirus encoding a CAR comprising an FMC63-derived CD19-specific scFv fused to a modified CD8α-hinge spacer, a CD28 transmembrane area, and a CD3ζ signaling area, with GFP labeling (Further file 1 Determine S1A) had been added to the cells to permit exact enumeration of transduced hAMSCs by move cytometry. Then polybrene with a closing focus of 8 μg/mL was added. After 48 hours of tradition, non-modified hAMSCs served as management cells to ascertain the brink for the hAMSC inhabitants utilizing a Sony automated move cytometer with FITC-GFP emission filters (Further file 1 Determine S1E). The FITC-GFP-labeled hAMSCs had been then sorted and cultured.

Isolation and characterization of exosomes

Exosomes had been remoted from the hAMSCs tradition medium by a differential centrifugation methodology. In short, the collected cell-conditioned medium was centrifuged at 300×g for five min, 2000×g for 15 min, and 10,000×g for 30 min. After filtration with a 0.22 μm filter, the filtrate was centrifuged at 100,000×g for two h at 4 °C. The pellet was resuspended in PBS and subsequently centrifugated at 100,000×g for one more 2 h. The pellets had been resuspended with PBS and analyzed utilizing a BCA Protein Assay package (Beyotime Biotechnology) to quantify the protein content material of exosomes for subsequent experiments.

Western blot

The exosomes had been lysed in RIPA lysis buffer (Yeasen), and equal quantities of protein had been separated by SDS-PAGE gels and transferred to PVDF membranes (Merck Millipore, MA, USA). The blots had been blocked with 5% BSA Tween-20 (0.1%) PBS buffer (T-PBS, pH 7.4) for 1 h at room temperature. The membranes had been incubated with major antibodies at 4 °C in a single day, adopted by incubation with HRP-linked secondary antibodies at 37 °C for 1 h. Antibodies had been: anti-CD63 (proteintech, 25682); anti-CD81 (proteintech, 66866); anti-TSG1013 (proteintech, 67381); anti-Albumin (proteintech, 16475).

Dynamic mild scattering (DLS)

A ten µL aliquot from the resuspended exosome pattern was diluted in 990 µL of PBS and blended effectively. The dimensions distribution and Zeta potential of exosome had been analyzed utilizing the Zetasizer Nano ZS90 (Malvern, UK). Three determinations per pattern had been taken at room temperature, the ensuing knowledge had been analyzed with the Malvern software program (Zetasizer Ver. 7.11).

Transmission electron microscopy (TEM)

10uL exosome suspension was dropped onto Formvar-carbon-coated EM grids (Alliance Biosystems, Japan) and stained with 2% phosphotungstic acid. The ensuing samples had been examined with a Tecnai G2 spirit BioTwin electron microscope (FEI, USA).

Colloidal gold immunoelectron microscopy

Purified exosomes had been positioned on Formvar-carbon-coated EM grids and glued with paraformaldehyde, blocked, adopted by incubation with a 10-nm protein gold conjugate (BBI Options, Cardiff, UK) for 20 min to label the anti-CD19. Every staining step was adopted by three TBST washes after which staining with 1% glutaraldehyde. Then, grids had been air-dried and visualized utilizing a transmission electron microscope. Anti-CD19 on the floor of anti-CD19-Exo exosomes was particularly labeled with gold nanoparticles, whereas no staining was noticed on unmodified exosomes.

Circulate cytometry

At the very least 10,000 occasions had been acquired utilizing totally different emission filters, utilizing a FACSVerse Circulate Cytometer (BD, USA). The gating technique used to establish viable cells and the brink willpower for the experimental group cell populations, based mostly on management cells, are offered in Further File 1 Determine S1D, E and Determine S2A–D.

The synthesis of anti-CD19-Exo-MTX

MTX (500 μg) was added into 1mL of PBS together with anti-CD19-Exo (1 mg) and incubated in a relentless temperature shaking at 37 °C for twenty-four h. The combination was purified utilizing an ultrafiltration tube with molecular weight cut-off of 100 kDa (Millipore) for five occasions (2500 r/min) to take away of unloaded medicine and procure anti-CD19-Exo-MTX. The content material of MTX in exosomes was decided by excessive efficiency liquid chromatography (HPLC; Agilent Applied sciences). Anti-CD19-Exo-MTX was added with DMSO and totally blended, after ultrasound, centrifuging at 16,500×g for 20 min. The supernatant was detected by HPLC. The supernatant was filtered with a 0.22 μm syringe filter and 20 μL aliquots had been transferred into HPLC autosampler vials. The drug loading effectivity and encapsulation effectivity of MTX was calculated utilizing the next equations [27]:

$${textual content{Loading effectivity }} = , left( {textual content{weight of the encapsulated MTX}} proper)/left( {textual content{weight of anti – CD19 – Exo – MTX}} proper) , occasions , 100% ,{textual content{ encapsulation effectivity }} = , left( {textual content{weight of the encapsulated MTX}} proper)/left( {textual content{weight of the overall MTX added}} proper) , occasions , 100% .$$

Exosome labeling and uptake

Exosomes had been labeled utilizing DiR Iodide (Yeasen) and PKH67 (Sigma-Aldrich) in accordance with the producer’s directions. In short, exosomes had been incubated for 30 min with DiR dye at 37 °C, or at room temperature with PKH67 dye for five min, terminated with FBS adopted by centrifugation at 100,000×g for two h at 4 °C. The pellet was resuspended in PBS and subsequently centrifugated at 100,000×g for one more 2 h.

Subsequently, the MTX-loaded exosomes labeled with PKH67 had been incubated with SU-DHL-8 cells for twenty-four h. A few of the cells had been then fastened and stained with β-actin and 4′,6-diamidino-2-phenylindole (DAPI, 40727ES10, Yesen). Photos had been obtained on a confocal microscope. The fluorescence depth of intracellular exosomes was quantitatively decided by FCM to evaluate the mobile uptake.

Intracellular MTX accumulation

To quantify the quantity of MTX accumulation in lymphoma cells, anti-CD19-Exo-MTX was added in and incubated with SU-DHL-8 cells for six, 12 or 24 h, then the cells had been collected to be resuspended in PBS earlier than subjected to cell counting. The counted cells had been disrupted by an ultrasonic methodology. The precipitation was eliminated utilizing a 0.22 μm syringe filter, and the supernatant was measured through the use of HPLC to find out the concentrations of MTX.

Drug launch

The in vitro launch habits of MTX from anti-CD19-Exo-MTX was investigated utilizing a dialysis methodology. MTX was used because the management teams. 1 mL anti-CD19-Exo-MTX (100 μg MTX) in a dialysis bag (3500Da, MWCO), adopted by dialysis in 10mL medium (PH 7.4) for additional incubation at 37 °C with light shaking. 1 mL medium was collected at 0.5, 1, 2, 4, 8, 12, 24 and 48 h, adopted by including 1 mL recent medium. Lastly, the launched quantities of MTX within the collected media had been measured utilizing HPLC.

TEER measurement

To watch cell confluence and the event of tight junctions, transendothelial electrical resistance (TEER) was recorded utilizing a Millicell ERS-2 Voltohm-meter (Millipore, MA, USA) [28]. One electrode was positioned on the luminal facet and the opposite electrode on the abluminal facet with the endothelial layer separating them. The TEER measurements of clean filters (with out cells) had been subtracted from these of filters with cells. Then the ensuing worth was multiplied by the membrane space to acquire the TEER measurements in Ω.cm². The method is as follows: TEER (Ω × cm²) = (resistance of co-cultured cells – resistance of empty Transwell filter) × floor space (cm²).

Permeability assay

The permeability of BBB was evaluated by measuring the clearance of sodium fluorescein (NaFl, 376.3 Da, Sigma-Aldrich), as described beforehand [29]. Briefly, NaFl was added to the higher compartment of Transwel inserts at a focus of 100 μg/mL. Medium samples had been collected from the underside effectively at 30, 60, and 120 min to measure the fluorescence depth, utilizing a Synergy NEO (BioTek). All experiments had been carried out in triplicate for every situation. Transendothelial permeability coefficient (Pe) was calculated as beforehand described by Deli et al. [30, 31].

Investigation of the mechanism of blood-brain barrier penetration in vitro

Every group of medicine was added to the mannequin and the MTX focus within the mannequin was decided at time factors (1, 2, 4, 6, 8, 12, 24 and 48 h) to assessing the permeability of medicine throughout the BBB. To find out the integrity of the endothelial monolayer, 10 kDa CY5-dextran, and 70 kDa fluorescein isothiocyanate (FITC)-dextran had been added to the higher chamber of the Transwell filters (100 μg/mL), and the fluorescence depth within the media of the decrease chamber was measured after 2 h utilizing a Synergy NEO (BioTek).

On this mannequin, the cells had been fed with endothelial progress media supplemented with 8-(4-chlorophenylthio)-adenosine 3′,5′-cyclic monophosphate (8-CPT-cAMP, 50 nM, HY-134299 MCE) and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro20-1724, 17.5 nM, E0346 selleck). After 48 h, affirmation of elevated expression of ZO-1 protein in endothelial cell tight junctions by way of modifications in permeability measured by confocal microscopy and dextran diffusion.

To guage the transport pathway of anti-CD19-Exo-MTX utilizing this mannequin with SU-DHL-8 cells had been cultured within the decrease chamber, filters had been pretreated with Dynasore (100 μM, S9849 selleck) and 5-(N-Ethyl-N-isopropyl)- Amiloride (EIPA) (100 μM, S9849 selleck) for 30 min previous to including the Dir-labeled anti-CD19-Exo-MTX after which incubated for twenty-four h at 37 °C. The fluorescence depth of intracellular exosomes within the decrease chamber was quantitatively decided by FCM to evaluate permeability of anti-CD19-Exo-MTX.

In vivo mannequin of CNSL

Animals had been housed and maintained in accordance with the institutional tips for the usage of laboratory animals and after buying permission from the ethics committee of Soochow College for animal experimentation. SU-DHL-8 cells (1 × 10⁶ cells per mouse) had been subcutaneously inoculated into the left axilla, and lymphoma tumor tissues had been harvested to organize tumor tissue suspensions after two weeks. The mouse mannequin of CNSL was established by intracranial positioning method. After anesthesia, animals had been fastened into the stereotactic headframe, disinfected, reduce alongside the midline of the mouse’s head with the size of about 5 mm and 1.5 mm bur gap was drilled 1 mm anterior to the coronal suture on the left hemisphere and a pair of mm lateral from the midline. 4 μL SU-DHL-8 cell tissue suspension (1 × 10⁶ cells per mouse) which from NOD-scid mice was injected into the left frontal lobe of the mind. Bone wax crammed the cranium gap and the incision was sutured. Tumor dimension and site had been monitored by T₂-weighted Magnetic resonance imaging (MRI) on an MRS 3000 scanner (3T).

Antitumor efficacy of anti-CD19-Exo-MTX in CNS lymphoma mouse mannequin

Six-week-old feminine NOD-scid mice had been bought from a biocytogen firm (Beijing, China), and had been acclimated one week previous to tumor cell inoculation. CNSL mice fashions had been established, and assigned every mouse a quantity, then used a random quantity desk to allocate 5 mice to every group: (1) Management group: intravenous (i.v.) injection of 100 μL PBS, (2) MTX group: i.v. injection of 5 mg/kg MTX (100 μL), (3) Exo-MTX group: i.v. injection of 100 μL Exo-MTX (equal to five mg/kg MTX), (4) anti-CD19-Exo-MTX group: i.v. injection of 100 μL anti-CD19-Exo-MTX (equal to five mg/kg MTX). Formulations for the totally different therapy teams had been administered each two days, for a complete of seven doses. Through the analysis, the mice had been monitored every day for physique weight and tumor quantity by Magnetic resonance imaging (MRI, MRS-3031, UK) on day 7 and 14.

Focused distribution of anti-CD19-Exo-MTX in vivo

DiR-labeled Exo-MTX and anti-CD19-Exo-MTX (equal to five mg/kg MTX) had been administrated into the CNSL mice through tail vein injection. The fluorescence distribution was photographed by the IVIS Lumina III optical imaging system (USA) at 1, 6, 12, 24, and 48 h post-administration. The mice had been euthanized at 48 h-post administration and their brains and the opposite main organs had been collected to investigate the biodistribution of DiR-labeled Exo-MTX and anti-CD19-Exo-MTX utilizing the IVIS imaging system.

MTX quantification

The MTX options with concentrations of 12.5, 25, 50, 100, and 200 μM had been then added to the HPLC instrument. Chromatographic circumstances: Column: C18 column (Lengthen-C18, 250 × 4.6 mm, 5 µm; Agilent Applied sciences); Cellular section: Water-acetonitrile (90:10); Circulate fee: 1 mL/min; UV detection wavelength: 302 nm; Temperature: 25 °C; Injection quantity: 20 μL.

Pharmacokinetic research

5 mice every group had been sacrificed at 0.5, 1, 2, 4, 6, 8, 12, 24 and 48 h after intravenous administration of MTX and anti-CD19-Exo-MTX at a dose of 5 mg/kg MTX. The blood was collected and centrifuged at 4000 rpm for 10 min to acquire the plasma. About 100 μL of the supernatant plasma was blended with 50 μL of methanol, then 50 μL of trichloroacetic acid was added. The combination was vortexed for five minutes and left to face at 4 °C for 12 h to precipitate the plasma proteins. Then centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was withdrawn and filtered by way of a 0.22-μm polyvinylidene difluoride membrane. MTX ranges in blood had been quantified by first establishing a typical calibration curve utilizing the beforehand described methodology, adopted by measuring the processed blood samples through HPLC.

Blood–mind barrier permeability evaluation in vivo

PBS, MTX, Exo-MTX and anti-CD19-Exo-MTX had been intravenously injected into the CNSL mice on the dose of 5 mg/kg MTX respectively. After 24 h, blood and cerebrospinal fluid (CSF) samples had been collected from totally different therapy teams mice after anesthesia, all mice had been euthanized. After euthanasia, mind tissue was eliminated, weighed, and homogenized in 1.5 mL physiological saline. Then, chloroform and methanol had been added, vortexed for five min, and centrifuged at 3000 rpm for 10 min. The decrease chloroform layer was collected, evaporated in a fume hood, and re-dissolved in 50 μL DMSO. The focus of MTX in blood, CSF and mind tissue had been decided utilizing HPLC.

LC-MS/MS evaluation

The hAMSC-Exo samples had been subjected to ultrasonic lysis, and 5 μL of the pattern was taken for silver staining. After silver staining, the samples had been subjected to enzymatic digestion, and the amount was adjusted to a constant stage with lysis buffer. Dithiothreitol (DTT, Sigma-Aldrich) was then added to a closing focus of 5 mM, and the combination was decreased at 56 °C for 30 min. Iodoacetamide (IAM, Sigma-Aldrich) was subsequently added to a closing focus of 11 mM, and the samples had been incubated in the dead of night at room temperature for 15 min. TEAB (Sigma-Aldrich) was used to dilute urea to make sure the ultimate focus was beneath 2 M. Trypsin (Promega) was added at a 1:50 ratio for in a single day digestion, adopted by a second addition of trypsin at a 1:100 ratio for an additional 4 h of digestion. The peptides had been dissolved in cell section A for liquid chromatography and separated utilizing the ultrahigh-performance liquid chromatography system (EASY-nLC 1200). Cellular section A consisted of water with 0.1% formic acid (Fluka) and a pair of% acetonitrile (ThermoFisher Scientific), whereas cell section B contained water with 0.1% formic acid and 90% acetonitrile. The liquid chromatography gradient was set as follows: 0–68 min, 6–23% B; 68.0–82.0 min, 23–32% B; 82.0–86.0 min, 32–80% B; 86.0–90.0 min, 80% B, with a move fee of 500 nL/min. After separation, the peptides had been launched into the NSI ion supply for ionization and analyzed utilizing the Orbitrap Exploris™ 480 (ThermoFisher Scientific) mass spectrometer. The ion supply voltage was set to 2.3 kV, with FAIMS compensation voltages (CV) of −45 and −65 V. Each dad or mum ions and their secondary fragments had been detected and analyzed utilizing high-resolution Orbitrap detection.

Statistical evaluation

All statistical analyses had been carried out utilizing GraphPad Prism 8.0. All knowledge had been proven as imply ± SEM. For usually distributed knowledge, the importance of imply variations was decided utilizing unpaired two-tailed Pupil’s t-test between two teams or ANOVA adopted by Newman-Keuls a number of comparability check amongst a number of teams. For all assessments, a p-value < 0.05 was thought-about to be statistically vital.

Different detailed assays together with isolation, tradition and identification of hAMSCs, focusing on experiment in vitro, in vitro mannequin of BBB, cell viability assays, the whole blood depend and serum biochemistry assay, H&E and Ki67 staining, TUNEL and Nissl staining can be found within the Further file 2.