Extracellular Vesicles Mediate the Intercellular Exchange of Nanoparticles

Abstract To exert their therapeutic effects, nanoparticles (NPs) often need to travel into the tissues composed of multilayered cells. Accumulative evidence has revealed the crucial role of transcellular transport route (entry into one cell, exocytosis, and re‐entry into another) in this process. While NP endocytosis and subcellular transport are intensively characterized, the exocytosis and re‐entry steps are poorly understood, which becomes a barrier for NP delivery into complex tissues. Here, the authors term the exocytosis and re‐entry steps together as intercellular exchange. A collagen‐based three‐dimension assay is developed to specifically quantify the intercellular exchange of NPs, and distinguish the contributions of several potential mechanisms. The authors show that NPs can be exocytosed freely or enclosed inside extracellular vesicles (EVs) for re‐entry, while direct cell–cell contact is hardly involved. EVs account for a significant fraction of NP intercellular exchange, and its importance in NP transport is demonstrated in vitro and in vivo. While freely released NPs engage with the same receptors for re‐entry, EV‐enclosed ones bypass this dependence. These studies provide an easy and precise system to investigate the intercellular exchange stage of NP delivery, and shed the first light in the importance of EVs in NP transport between cells and into complex tissues.

T-Dextran was prepared by conjugate TAT peptide with Dextran-tetramethylrhodamine (70,000 MW, lysine fixable, Fisher, D1818). Breifly, proper molar of NHS-PEG2000-Mal (Jenkem) was added into the solution of Dextran-tetramethylrhodamine to conjugated the maleimide on the surface of the Dextran. Then Cysteine-TAT (Lifetein) was then conjugated onto the Dextran through maleimide. Unreacted NHS-PEG2000-Mal and Cysteine-TAT were removed by dialysis with a 50kDa kit.

Optimization of the intercellular exchange assay:
To optimize the conditions for intercellular exchange assay, T-AgNPs was applied to PC-3 / PC3-GFP combinations in the assay. The transfer time (24 h, 48 h), cell number for recipient cells (6×10 4 , 9×10 4 and 1.2×10 5 ), donors to recipients ratio (2:1, 1:1, 1:2), concentration of gap collagen solution (2, 4 and 8 mg/mL), w / wo FBS in medium and the form of donor cells (monolayer or in collagen) was tested to get the optimal conditions. We first tested the transfer time. 6×10 4 PC3-GFP cells were seeded in recipient layer as Next, we optimized the recipient cell number and the ratio of donor cells to recipient cells in the intercellular exchange assay. 6×10 4 (60K in Fig. S3), 9×10 4 (90K in Fig. S3) and 1.2×10 5 (120K in Fig. S3) PC3-GFP cells were seeded in recipient layer as described in Methods, respectively. Then PC-3 cells was added into each well according to the ratio of 2:1, 1:1 and 1:2 (PC-3:PC3-GFP), respectively. After 24 h incubation, the intercellular exchange efficacy was tested and analyzed as described in Methods. As shown in Fig.   S3C, the percentage of AgNP positive recipient cells increased as the ratio of donors to recipient increased regardless which number of recipient cells we were using. We also tried 6:1 (PC-3:PC3-GFP) ratio and no further increase of intercellular exchange efficacy was observed (Data not shown). Besides, 90K recipient cells group showed significant increase of AgNP positive recipient cells in the system than that of 60K group. Further increased the recipient cells to 120K didn't further increase this percentage. We then chose 90K recipient cells with 2:1 ratio of donor : recipient cells as the condition to do the rest of our study.
Whether the concentration of gap collagen solution between donor and recipient cells had any effect on the intercellular exchange was also investigated. The intercellular exchange assay was prepared as described in Methods. 2, 4, 8 mg/mL of collagen solution was used to prepare the gap between donor and recipient cells, respectively. The intercellular exchange efficacy was measured as described in Methods. As demonstrated in Fig. S3C, no significant difference of the intercellular exchange efficacy was observed among three groups. 2 mg/mL of collagen solution was used in our study if otherwise indicated.
Next, the effect of FBS in the medium on the intercellular exchange efficacy was tested.
The assay was carried out in medium w / wo FBS, respectively. The intercellular exchange efficacy was measured as described in Methods. As demonstrated in Fig

Confirmation of no direct cell-cell contact in the intercellular exchange assay:
Intercellular exchange assays with 100 µL of collagen gap (equivalent to 30 µL in 96-well plate) were prepared in 8 Chamber Polystyrene Vessel Tissue Culture Treated Glass Slide (Falcon, cat. no. 354108) with PC3-GFP and NHLF cells as described in Methods.
After 12 h and 24 h incubation, cells were fixed with 500 µL 10% formalin overnight at room temperature. Formalin was removed and 800 µL O.C.T. compound (Tissue-Tek, SAKURA ®️ , cat. no. 4583) was added into the chamber. Then the whole chamber was transferred into -80 °C overnight. The next day, the frozen bulk gel in the chamber was removed and frozen sections of bulk gel was prepared according to standard cryo-section protocol. The chamber slides were then mounted in DAPI-containing mounting medium (Vector Laboratories, Burlingame, CA) with a coverslip and examined using EVOS M5000 microscope (Thermo Fisher Scientific).

Evaluation of cell viability with GW4869 treatment:
CellTiter-Glo® 3D Cell Viability Assay (Promega, catalog #: G9681) was applied to evaluate cell viability in 3D intercellular exchange assay. 3D intercellular exchange assay was prepared as described in Methods. After 24 h incubation, the cell viability was measured according to manufacturer's instruction of CellTiter-Glo® 3D Cell Viability Assay.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, ) was applied to evaluate cell viability with GW4869 treatment. Cells was seeded at the density of 1.5×10 4 cells/well in a 96-well plate. After incubation for 24 h, the medium was changed to GW4869-containing medium at the dosage of 0, 10, 20 and 40 µM and cells were incubated for another 24 h. The medium was then discarded and 100 µL of 0.5 mg/mL MTT in FBS-free medium was added. After incubation at 37°C for 4 h, 150 µL DMSO was added into each well and mixed well by pipetting. The plate was incubated at room temperature for 30 min and then absorbance was measured at 570 nm using a SpectraMax M2 plate reader (Molecular Devices, Inc.).   Cell viability of PC-3 (donors) and PC3-GFP (recipients) in 3D intercellular exchange assay after 24 h incubation with presence or absence of constant etching (x axis) was tested as described in

Evaluation of etching efficiency
Supplementary Methods, respectively. The result was normalized to that of without etching (y axis).