In Vitro Study of Receptor-Mediated Silica Nanoparticles Delivery across Blood-Brain Barrier

Transcription

1 Supporting Information In Vitro Study of Receptor-Mediated Silica Nanoparticles Delivery across Blood-Brain Barrier Yang Song, Dan Du, Lei Li, Jun Xu, Prashanta Dutta *,, and Yuehe Lin *, School of Mechanical and Materials Engineering, Washington State University, Pullman, Washington 99164, United States Department of Integrative Physiology and Neuroscience, Washington State University, 1815 Ferdinand s Lane, Pullman, WA 99164, USA Corresponding Author * yuehe.lin@wsu.edu. * prashanta@wsu.edu. S-1

2 Experimental section Calculation of mole concentration of Si NPs. The density of Si NPs is around 2.2 g cm -3.We can calculate the single-particle weight of Si NPs with 25 nm, 50nm and 100 nm in size by equation M Si = 4 3 πr3 ρ, and then got their concentrations by equation m C m =. Here the M Si, r, and ρ are the single-particle weight, radius and density of M Si N A V Si NPs, and the NA is the Avogadro constant. The m is the weight of the Si NPs and the V is the volume of solution. Cytotoxicity test. The cytotoxicity of PSi NPs and PSi-Lf NPs for bend.3 vascular endothelial cells in vitro was performed by the standard MTT assay. The bend.3 vascular endothelial cells were seeded into the 96-well plate at the density of 1.0x10 5 per ml for 24 hours (37 C, 5% CO 2 ). Then the cells were removed with Dulbecco's Modified Eagle Medium (DMEM) containing the Si NPs or Si-Lf NPs at different doses for another 24 hrs. Then, the DMEM in the 96 well plates was removed, and the mixture of 180 μl of 1 PBS and 20 μl of MTT solution (5 mg ml -1 ) was added. Then, 150 µl Dimethyl sulfoxide (DMSO) was injected into each well. Finally, the absorption could be recorded at the wavelength of 490 nm. In Vitro BBB Model. In order to investigate on the BBB model, the BBB kit (RBT-24H) was used as the in vitro blood brain barrier model (polyethylene terephthalate) which cultured with primary wistar rat barin capillary endothelial cells, brain pericytes and astrocytes (PharmaCo-Cell Co. Ltd, Nagasaki, Japan, This BBB kit is stored at -80 C and can be used during one month. According to S-2

3 protocol, the BBB kit has to be thawed and activated before use. The medium was de-frozen in 37 C water bath. Then quickly add the medium to brain-side chamber and blood-side chamber, respectively. Next the BBB kit is placed in the incubator and the medium is changed after one day. After 4 days, the kit is activated functionally. Immunofluorescence. The vascular endothelial cells on the in vitro BBB model were fixed with 4% paraformaldehyde for 15 min at room temperature. Then the cells were treated with PBS containing 0.1% Triton X-100 for 10 min followed by washing with PBS to permeabilize the cells. Next, cells were treated with 3% BSA in PBS for 1 h to block the nonspecific binding of antibodies. After washing with PBS for three times, the cells were incubated with rabbit polyclonal antibodies against Claudin-5 ( , Invitrogen, CA, USA), Occludin ( , Invitrogen), ZO-1 ( , Invitrogen) and N-cadherin ( , Invitrogen) for 1 h. Then the cells were incubated with AlexaFluor 594-labeled anti-igg (Invitrogen) for 1 h after washing with PBS. The stained cells were washed with PBS without Mg 2+ and Ca 2+ and mounted on cover glass for imaging by using CLSM. Permeability assay. The permeability assay was measured to evaluate the effects of PSi NPs on the integrity of the BBB. After incubated with various PSi NPs, 200 μl of NaF (10 μg ml -1 ) was added to the upper chamber. In addition, the medium of lower chamber was replaced with 1 ml of PBS t. Then the wells were incubated at 37 ºC. After 30min, the solution of the lower chamber was collected and transferred into microplate S-3

4 and measured with microplate reader. Finally, the permeability coefficient (Papp) was calculated as equation: Papp = V C i A t C f where C i and C f represent the original and collective tracer concentration, respectively. V is chamber volume and A is surface area. S-4

5 Figure S1. Representative pictures of VECs, pericytes and astrocytes. Scale bar is 20 μm. S-5

6 Figure S2. Formation of tight junctions in the in vitro BBB model. The formation of tight junctions was monitored by the expression of Claudin-5, which is a transmembrane protein reported to be present in tight junctions. S-6

7 Figure S3. Co-immunostaining of N-cadherin (red) and actin filaments (green) in endothelial cells. S-7

8 Figure S4. Cellular cytotoxicity of the PSi NPs and PSi-Lf NPs. S-8

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10 Figure S5. Intracellular localization of PSi-Lf NPs with Rab11 in the in vitro BBB model. The in vitro blood brain barrier model was exposed to fluorescently labeled (Alexa488) transferrin for 30 min and the location of the intracellular transferrin (green) observed. Sorting endosome (SEs) colocalization was observed, though not completely absent (an example is shown within the circle), suggesting that SEs does predominately follow the transcytosis pathway. (a) On internalization, most PSi-Lf NPs were pulsed to bond and fused with EEs. Transferrin co-localizes with some of these structures, a common location being particularly large vesicles. (b) Small vesicles containing PSi-Lf NPs then emerge from apical side and then move downward, constituting dynamic endosomes. Furthermore, the formation of what looks like a static channel connecting the apical and basolateral side of the barrier occurs progressively with time. (c) It is clear that substantial portion of the Si-Lf NPs were make excursions across the BBB approaching the basal side and end up at transwell membrane pores, with defusing from SEs so that the particles travel through the pores. S-10

11 Figure S6. Assessment of BBB permeability determined by NaF. PSi NPs and PSi-Lf NPs of 25 nm, 50 nm and 100 nm and negative control (NC). were added to the in vitro BBB model. After 24 h, NaF was added, and NaF that had passed through BBB was measured. S-11

12 Table S1. Surface Charges of Si NPs and PSi NPs Zeta potential (mv) diameters Si NPs PSi NPs 25 nm -35.2± ± nm -38.8± ± nm -41.7± ±2.7 S-12

13 Table S2. The amount of Lf in different PSi NPs diameters 25 nm 50 nm 100 nm Lf number per NPs Surface area ratio 1:4:16 area density of Lf 16.2:17.3:17.8 S-13

14 Table S3. The amount of Lf in different PSi NPs Initial NP concentration at Final NPs concentration the apical side (µm) at the basolateral side (µm) Transport efficacy (%) PSi NPs SiNPs-Lf PSi NPs PSi NPs-Lf PSi NPs PSi NPs-Lf S-14