To increase the solubility of FU in DMF, FU was dissolved in DI water, treated with Dowex®

50WX2 ion-exchange resin, and then an excess amount of TBAB was dropwise added to the
solution (Miao et al., 2013; Yue et al., 2013).
FU in DMF was dissolved in DI water to increase its solubility.

After removing the water, FU-TBAB salt (500 mg) was dissolved in anhydrous DMF with a
successive addition of succinic anhydride (1 g) and DMAP (100 mg). After the solution was
stirred for 24 hours to activate the OH groups of FU, the product was purified by precipitation
using cold diethyl ether. Modified FU was dissolved in DMSO and EDC (200 mg), NHS (100
mg), Dox (50 mg), and TEA (50 μl) were added successively to the stirring solution. After 24
hours, the solution mixture was subjected to dialysis over DMSO (MWCO = 3,500 Da) for 3
days to remove unreacted Dox and reagents. The purified FU-Dox conjugate was obtained after
precipitation using cold diethyl ether and dried under a vacuum. Proton nuclear magnetic
resonance (1H NMR) spectroscopy was used to examine the succinylation of FU and the
formation of FU-Dox conjugate. The UV-vis absorbance spectra of FU-Dox conjugate and free
Dox in DMSO were recorded using a UV plate reader. The Dox content was determined by
dissolving 10 mg of the dried FU-Dox sample in1 ml of DMSO and calculating the Dox
concentration using a UV absorbance calibration curve at 480 nm. To form NPs, 1 mg of DSPE-
mPEG was first dissolved in THF with the concentration of 10 mg/ml, and then it was dropwise
added to the stirring water. Amphiphilic FU-Dox conjugate (10 mg) was dispersed in the
solution of DSPE-mPEG in water using sonication for 10 minutes. The final FU-Dox
nanoparticles were filtered using ultra-filtration (3,000 g, 15 min, MWCO 100 kDa) to remove
residual THF, resuspended in 1 ml PBS and Dox loading was determined using UV
measurement at 480 nm. The size and zeta potential of the NPs were measured using dynamic
light scattering (DLS).
μl of FU-Dox NPs (0.1 mg/ml) in water was added onto carbon-coated copper grids. After
removing the excess water by a filter paper, the grid was immersed in a staining solution (1%
(w/v) uranyl acetate) for at least 2 minutes and dried under the fume hood (Zhou, Kennell, Jafari
et al., 2017).
The stability of the NPs was studied in PBS buffer solution at pH 7.4 and 5.2 for up to 3 days at
37 ℃ by measuring the size of the NPs using DLS. To evaluate the stability of the NPs in serum,
an absorbance-based approach was used to detect the formation of aggregates by measuring the
UV absorbance at 560 nm in PBS solutions with 10 and 50% FBS at pH 7.4 and 5.2.

The release kinetics of the FU-Dox NPs was measured by an indirect method of dialysis in PBS
solution of pH 7.4 and pH 5.2. After transferring the FU-Dox NPs solution into a dialysis
membrane tubing with the MWCO of 3.5 kDa, the tubing was immersed in a 200-ml PBS
solution. The concentration of Dox inside the tubing was calculated using UV absorbance at 480
nm after removing 50 μl of the solution at different time points (1, 3, 6, 18, 24, 48, and 72 h)
(Zhou, Kennell, Lee, Leung, & Tarapore, 2017)

2.3. In vitro cell tests

To check the feasibility of targeting P-selectin for breast cancer, real-time polymerase chain
reaction (RT-PCR) was used to screen the expression of P-selectin on the two well-known breast
cancer cell lines, MDA-MB-231 and MDA-MB-468. MDA-MB-231 and MDA-MB-468 cells
were cultured in ATCC-formulated Leibovitz's L-15 Medium supplemented with 10% FBS and
1% PS in an incubator with humidified atmospheric air at 37 °C. Cells were cultured in 6-well
plates with a seeding density of 200 × 103 cells/well. After two days, the total RNA was extracted
using the mirVana TM isolation kit (Ambion®) by following the manufacturer’s protocol. The
total RNA was reverse transcribed to cDNA using the SuperScript® First-Strand Synthesis
system (Invitrogen) and oligo(dT) primers. The cDNA was amplified using PCR Master Mix
(2X) (Thermo Fisher Scientific). The primers specifically used for the amplification of P-selectin
(SELP) expression with an amplicon size of 497 bps were as follows: upstream primer, 5'-AGT
GGC AGC ATG GAC TTA TC-3'; and downstream primer, 5'-CAA GTT CTC CAC ACT CTC
TCA C-3'. In addition, GAPDH, as the housekeeping gene, was amplified with an amplicon size
of 100 bps with upstream and downstream primers, 5'-ACAGCGACACCCACTCCTC-3', and 5'-
TAGCCAAATTCGTTGTCATACCAG-3', respectively. The PCR products were visualized
using a digital camera under ultraviolet light after running on 2% E-Gel™ EX agarose gel pre-
stained with SYBR Gold DNA stain (Thermo Fisher Scientific).
Relative P-selectin protein expression was quantified using western blot analysis. Lysates of the
MDA-MB-231, MDA-MB-468, and MCF-12A cells were prepared using
Radioimmunoprecipitation (RIPA) cell lysis buffer (Abcam, Cambridge) (Xu, Orkwis, DeVine,
& Harris, 2020). Cells were cultured in 100 mm dishes with the seeding density of 2 × 106. After
two days, they were washed with PBS and 0.5 ml of RIPA buffer was added. After 5 minutes of
incubation on ice, cells were scrapped, sonicated, and centrifuged for 10 minutes. The
supernatant was collected, and the protein concentration was measured by a bicinchoninic acid
(BCA) protein assay kit (Thermo Fisher Scientific) using an iMark microplate reader (Bio‐Rad).
10 μg of the total protein was mixed with 10% DL‐Dithiothreitol and 50% 2x Laemmli buffer
(Bio‐Rad) and denatured at 96 °C for 5 min. Denatured samples containing an equal amount of
total protein were resolved by 8% or 10% Sodium dodecyl sulfate (SDS)-acrylamide (SDS:
VWR, 0227, 40% Acrylamide solution: Thermo Fisher Scientific, bis-acrylamide: Thermo
Fisher Scientific) gel followed by transfer to nitrocellulose membrane using a Trans-Blot Turbo
system (Bio-Rad). The membrane was incubated with 3% bovine serum albumin (BSA) in Tris-
buffered saline with 0.1% Tween-20 (TBST) before incubation at 4 °C overnight in rabbit anti-P-
selectin antibody (Thermo Fisher Scientific) or rabbit anti-GAPDH antibody (Cell Signaling,
Beverly, MA) at a 1:1,000 dilution. The membrane was rinsed five times with TBST before
incubation with anti-rabbit IgG, HRP-linked Antibody (Cell Signaling, Beverly, MA) at room
temperature for 1 hour. The membrane was then washed with TBST and incubated with Pierce
ECL Plus Western Blotting substrate (Thermo Fisher Scientific) for an hour at room temperature
and imaged using a ChemiDoc MP imaging system (Bio-Rad). Western Blot images were
analyzed using Image Lab software (Bio-Rad, version 6.0.1).
P-selectin expression on MDA-MB-231 and MDA-MB-468 cells was visualized using
immunofluorescence (IF) imaging. Cells were grown on coverslips in 12-well plates with the
seeding density of 100 × 103 cells per well. After one day of incubation at 37 °C, cells were fixed
with 4% paraformaldehyde for 15 min and washed few times with PBS. Then cells were
permeabilized using 0.1% Triton X-100 in PBS at room temperature for 15 min and rinsed three
times with PBS. Cells were incubated with P-selectin recombinant rabbit monoclonal antibody
(Thermo Fisher Scientific) overnight at 4 °C. After washing the cells with PBS, Alexa Fluor 488-
conjugated goat anti-rabbit secondary antibody (Invitrogen™) was added and incubated for 45
min at room temperature. Finally, cells were washed with PBS and mounted using 10 μl of a
mounting medium containing DAPI (Abcam, Cambridge, USA) for staining the nuclei and
visualized using a fluorescence microscopy.
The cell uptake distribution of FU-Dox NPs was visualized using fluorescence images. MDA-
MB-231 and MDA-MB-468 cells were cultured on coverslips in 12-well plates with the seeding
density of 100 × 103 cells per well. On the following day, the cells were incubated with the free
form of Dox or FU-Dox NPs with an equivalent Dox concentration of 0.2 μg/ml for 30 minutes
or 6 hours, respectively. Then the cells were washed with PBS two times and they were fixed
using 4% (wt/vol) solution of formaldehyde in PBS for 15 minutes. Finally, the cells were
mounted using 10 μl of the same mounting medium with DAPI used for IF imaging.
The impact of P-selectin inhibitor on FU-Dox NPs uptake was evaluated using flow cytometry
and fluorescent imaging. For the flow cytometry analysis, MDA-MB-231 and MDA-MB-468
cells were cultured in 6-well plates with the seeding density of 300 × 103 cells/well. The next
day, P-selectin inhibitor (KF 38789, Tocris) was added to the cells with the final concentration of
1 μM. After 1 hour of pretreatment with the inhibitor, cells were incubated with FU-Dox NPs
with an equivalent Dox concentration of 0.2 μg/ml for 4 hours. The cells were washed two times
with PBS, trypsinized, and then resuspended in PBS and analyzed for Dox fluorescent intensity
using flow cytometry. Cells treated with FU-Dox NPs without the pre-incubation with P-selectin
inhibitor were used as a positive control, and cells without any treatment were used as a negative
control. For the fluorescent imaging, the cells were cultured on coverslips in 12-well plates with
a seeding density of 100 × 103 cells per well. On the following day, the P-selectin inhibitor was
added at a final concentration of 1 μM. After 1 hour of pretreatment with the inhibitor, the cells
were incubated with FU-Dox NPs with a Dox-equivalent concentration of 0.2 μg/ml for 4 hours.
Then the cells were washed, fixed, and mounted using 10 μl of the mounting medium with
DAPI.
To measure the cytotoxicity of the NPs, cells were cultured in 96-well plates with the seeding
density of 5,000 cells/well. On the following day, cells were treated with the free form of Dox or
FU-Dox NPs with an equivalent Dox concentrations for 48 hours, and then MTS assay
(Promega) was performed according to the protocol provided by the vendor. The cell viability
was calculated by measuring the UV absorbance at 490 nm based on the equation below, where
positive control was the cells without any treatment and negative control was a blank well
containing the MTS reagents and cell culture media.