Supplementary Materials

Nanoparticle passage though porcine jejunal

mucus: Microfluidics and rheology

Sourav Bhattacharjee,a,b,‡* Eugene Mahon,a,‡ Sabine M. Harrison,c,‡

Jim McGetrick,b Mohankumar Muniyappa,d Stephen D. Carrington,b David J. Braydena,b

aConway Institute of Biomolecular and Biomedical Research, University College Dublin

(UCD), Belfield, Dublin 4, Ireland

bSchool of Veterinary Medicine, University College Dublin (UCD), Belfield, Dublin 4,

Ireland

cSchool of Agriculture and Food Science, University College Dublin (UCD), Belfield, Dublin

4, Ireland

dNational Institute for Bioprocessing Research and Training (NIBRT), University College

Dublin (UCD), Belfield, Dublin 4, Ireland

‡These authors contributed equally

*Corresponding author: sourav.bhattacharjee@ucd.ie; +353 1 716 6271

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S1: Relevant literature on mucin/mucus-NP interaction

1. Pereira de Sousa I, Steiner C, Schmutzler M, Wilcox MD, Veldhuis GJ, Pearson JP, et al.

Mucus permeating carriers: formulation and characterization of highly densely charged

nanoparticles. Eur J Pharm Biopharm 2015;97,Part A:273-9.

2. Oh S, Wilcox M, Pearson JP, Borrós S. Optimal design for studying mucoadhesive

polymers interaction with gastric mucin using a quartz crystal microbalance with

dissipation (QCM-D): Comparison of two different mucin origins. Eur J Pharm Biopharm

2015;96:477-83.

3. Pearson JP, Chater PI, Wilcox MD. The properties of the mucus barrier, a unique gel –

how can nanoparticles cross it? Ther Del 2016;7(4):229-244

4. Liu S, Jones L, Gu FX. Development of mucoadhesive drug delivery system using

phenylboronic acid functionalized poly(D,L-lactide)-b-Dextran nanoparticles. Macromol

Biosci 2012;12(12):1622-6.

5. Maisel K, Reddy M, Xu Q, Chattopadhyay S, Cone R, Ensign LM, et al. Nanoparticles

coated with high molecular weight PEG penetrate mucus and provide uniform vaginal

and colorectal distribution in vivo. Nanomedicine (Lond.) 2016;11(11):1337-43.

6. Thomsen TB, Li L, Howard KA. Mucus barrier-triggered disassembly of siRNA

nanocarriers. Nanoscale 2014;6(21):12547-54.

7. das Neves J, Rocha CMR, Gonçalves MP, Carrier RL, Amiji M, Bahia MF, et al.

Interactions of microbicide nanoparticles with a simulated vaginal fluid. Mol Pharm

2012;9(11):3347-56.

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S2: Methods

Materials

TEOS (tetraethylorthoslicate, catalogue no. 86578), APTMS ((3-aminopropyl)-

trimethoxysilane; catalogue no. 281778), FITC (fluorescein isothiocyanate, Isomer I) (catalogue no.

F7250) were obtained from Sigma-Aldrich (Ireland). Fresh porcine jejunal mucus samples were

harvested and maintained at 4°C under a 5% CO2/95 % N2 mixture to prevent oxidation. Uncoated

micro-slides with sterilized microchannels (l×b×h=17 mm×1 mm×0.1 mm; 1.7 µl) of rectangular

cross sections were purchased from Ibidi GmbH (µ-Slide VI0.1, Cat. No. 80661).

Synthesis of fluorescent SiNP

N-1-(3-trimethoxysilylpropyl)-Nʹ-fluorescyl thiourea (APTMS) solution was prepared as a

fluorescent conjugate by dissolving 4 mg fluorescein isothiocyanate (FITC, ~0.001 mmol) in 2 ml

anhydrous ethanol followed by addition of 20 μl APTMS (11× molar excess). The light-sensitive

mixture was mixed at room temperature for 4 h. In the case of 50 nm SiNP, 38 g ethanol (0.83 mmol)

was added to 1.82 g of 28% aqueous ammonia, rapidly stirred and equilibrated in a water bath at

25°C. 1 ml of FITC-APTMS conjugate was then added followed by addition of 1.9 ml TEOS (1.78

g/0.038 mmol). The reaction was stirred for a further 20 h in a sealed tube. SiNP (~50 nm) were

washed by repeated centrifugation (14000 rpm for 30 min) followed by re-suspension in ethanol (×2)

and then in water (×2) using bath sonication. Finally, the re-suspension volume was adjusted to obtain

a 15 mg/ml suspension of SiNP in water. SiNP concentrations were then measured by vacuum drying

the 3×250 ml aliquots at 60°C for 5 h. The synthetic process was modified by using different ratios of

ingredients to prepare SiNP 100 nm diameter SiNP (Supplementary Materials S3). To synthesize

~200 nm diameter SiNP, a 12.5 ml suspension of ~100 nm SiNP was aliquoted to which 37.5 ml

methanol plus 4.41 g (0.1 mol) aqueous ammonia (water content: 2.55 g) were added and the

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temperature was equilibrated to 40°C while stirring at 600 rpm. A 2 ml aliquot of TEOS (1.87 g/0.008

mmol) was then added to the mixture and allowed to react for 3 h. As before, NPs were then washed

by centrifugation at 14000 rpm for 10 min followed with re-suspension in ethanol (×2) and water (×2)

using bath sonication. In order to prepare ~10 nm diameter SiNP, the dye-conjugate solution was

prepared as described above. 500 µl dye-conjugate was then added to 30 ml water at 70°C containing

1 mg/ml (0.32 mmol) tetramethylammonium hydroxide (TMAOH) with rapid stirring. 400 µl of

TMOS (tetramethyl orthosilicate) was then injected into the mixture and slowly stirred at 200 rpm for

1 min, followed by addition of 1.8 g TEOS (0.008 mmol). The reaction mixture was sealed and

allowed to react for 24 h. SiNP were then washed and collected as above. In order to synthesize

different mPEGylated (~1000 and ~2000 Da) SiNP, mPEG1000/2000-silanes (2-[methoxy

(polyetheleneoxy)propyl]-trimethoxysilane) were used (Gelest Ltd., Catalog No. SIM 6492.73). The

silanes were added (100 µl/ml for ~10 nm SiNP, and 2 µl/ml for the remaining ~50, 100 and 200 nm

SiNP) to an aqueous suspension of SiNP (10 mg/ml) and allowed to react at 70°C for 24 h before

being washed and re-suspended in water. To synthesize aminated SiNP, NP were re-suspended (10

mg/ml) in TMAOH solution (5 mg/ml). APTMS (0.5 wt. % final concentration) was then added and

the reaction mixture was stirred for 2 h at 70°C. Aminated SiNP were then washed four times with

water. SiNP were characterized by dynamic light scattering (DLS) and zeta potential (ZP) using a

Malvern Zetasizer®, differential centrifugal sedimentation (DCS), and imaged by Transmission

Electron Microscopy (TEM) (FEI Tecnai 120; 300 kV) (Supplementary Materials S4). The grafted

surface ligands on the NPs were confirmed as presented by 1H NMR (Supplementary Materials S6).

Image analysis and plotting of the data

The images of fluorescence signals originating from fluorescent SiNP from microchannels

were analysed with ImageJ® software after converting it into 8-bit grayscale image without

enhancement. In short, microchannel areas were cropped from the figures and scales were set from the

“Set Scale” option under the “Analyze” tool based on the length of the microchannel (17 mm). The

area of the microchannel was selected with the rectangular selection tool to mark it as ROI (region of

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interest). In the “Set Measurements” option, the area (A), standard deviation (SD), integrated density

and mean grey value boxes were selected and measurements were run. Similar measurements from at

least three areas from the dark background without fluorescence were also carried out as background.

The CID (corrected integrated density) for the area signifying the microchannel was derived by the

following formula and expressed as an AFU (arbitrary fluorescence unit):

CID = integrated density – (area of microchannel × mean fluorescence of background readings)

CID obtained from microchannels filled with only 100 µg/ml dispersions of SiNP in water

was used as control (100%) while the data for the sixteen SiNP used in this study was expressed as %

to it. As the FITC-content was same for all the SiNP this could be used to quantify the transport of

SiNP through mucus-filled microchannels.

Statistical analysis

All the experiments were repeated thrice independently (n=3) and the data were analyzed and

plotted in OriginPro v9.0 (OriginLab). Results are shown as mean ± SD. *P<0.05, compared to

cationic SiNP for each size (Figures 3 and 4).

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Table S3: Ratios of ingredients used to synthesize SiNP of different diameters

SiNP 50 nm 100 nm

Solvent 38 g (0.8 mol) ethanol 37.8 g (1.18 mol) methanol

TEOS 1.78 g 1.78 g

Aq. ammonia (28%) 1.82 g (0.02 mol) 5.88 g (0.09 mol)

Water ‒ 3.4 g (0.33 mol)

Temperature 25 °C 40 °C

Dye conjugate 1 ml 1 ml

Reaction time 16 h 3 h

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Figure S4: Characterization data of the SiNP by (a) TEM and (b) DCS

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S5: Mucopermeation, bioadhesion and rheology studies with cationic and anionic PSNPs

Well-characterized and monodisperse PSNPs (100 nm) of two variants – cationic (amine-terminated) rhodamine-labelled and anionic (acid-terminated) FITC-labelled – were chosen to perform control experiments. An aqueous dispersion of PSNPs (100 μg/ml) was used for micro-slide experiments. Both relative transport and binding to mucin were measured like the SiNP described within the main manuscript. The data are shown below. In summary, both the PSNPs showed less transport where cationic PSNP transported less than the anionic ones. The cationic PSNP showed comparatively higher binding (~4.5 folds) than anionic PSNPs.

(Left) The relative transported amounts of PSNPs through mucus-filled microchannels; (Right) Relative binding to mucin of PSNPs as determined by micro-slide experiments. The reading for

cationic PSNPs was shown as 100%. Results are shown as mean ± SD (n=3).

Rheological measurements were done with the PSNPs in identical set up to that of the SiNP and the data for both Gˊ and G˝ are shown below.

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Figure S6: 1H NMR in D2O of PEGylated SiNP indicates a saturating surface concentration

of 1 PEG/nm2, determined by integration against a DMSO internal standard following

dissolution in 0.2M NaOD, 0.1M NaCl. a. spectrum for precursor 2-[methoxy

(polyetheleneoxy)propyl]-trimethoxysilane dissolved in D2O b. 1H NMR spectrum for

dissolved PEG functionalized NPs showing matching characteristic PEG signal, which

allows concentration to be determined by integration against the internal standard.

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Figure S7: Conductivity of the exposure medium containing 50 nm SiNP (cationic or

anionic) after t = 0, ¼, ½ and 1 h following injection to microchannels. Conductivity was

measured as S (siemens) and increased over for both types of NP, as measured by Malvern

Zetasizer®. Results are shown as mean ± SD (n=3).

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Supplementary Materials S8

SPR (surface plasmon resonance) experiments

The biotinylation of porcine jejunum mucin (PJM) used EZLink TM biotin–sulfo-NHS-

SS-biotinylation kit (Cat # 21445). 2 mg of PJM and 10 mM Sulfo-NHS-SS-biotin were

dissolved in 1 ml PBS and incubated at room temperature for 30-60 min. Removal of excess

biotin reagent was performed using a Zeba desalt spin column (supplied with biotinylation

kit). All analyses were carried out on a Biacore T100 instrument at a constant temperature

(25ºC) and flow rate (10 µl/min), unless otherwise stated, using PBS as the run buffer. The

mucin and anionic SiNP interaction analysis was performed on Biotin CAPture kit, series S

(Cat. log # 28920234). Six different anionic SiNP (three anionic varieties as shown in Fig. 1

and of two sizes of 50 and 100 nm) were tested for binding with mucin. Cationic aminated 50

nm polystyrene NPs (Sigma Aldrich; L0780) were used as positive control. The sensor chip,

prior to analysis was regenerated by passing PBS buffer solution (0.01 M phosphate buffer,

0.0027 M KCl and 0.137 M NaCl, pH 7.4) through flow cells over the chip surface for 24–48

h. Interaction analysis was performed as per the manufacturer’s manual. Briefly, the biotin

CAPture reagent, Catalog # 28920234, 50 µg/ml in 0.01 M HEPES pH 7.4, with 0.15 M

NaCl, 3 mM EDTA, and 0.005% surfactant P20 was injected through the two flow cells over

the chip surface at a reduced flow rate of 2 µl/min for 300 s. Subsequently, 60 µl injections of

biotinylated PJM at a flow rate of 10 µl/min for 180 s were flown over one of the flow cell,

leaving the other flow cells bank on biotin CAPture coated sensor chip surface. 100 µM of

control cationic polystyrene NPs of 50nm flown over the blank- and PJM-coated chip surface

at a flow rate of 10 µl/min for 180 s. PBS was injected at 10 µl/minute for 120 s through the

two flow cells with PM and NPs interacting surface to check the stability of binding. The

chip surface was regenerated using regeneration solution (3 parts of stock solution containing

8 M guanidine hydrochloride and 1 part of stock solution of 1 M NaOH) to denature the

mucin hydrogel by a short 2 min injection of guanidine–HCl and NaOH. The above

mentioned interaction analysis process was repeated for all the six anionic NPs.

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Figure S9: Relative binding of six different anionic (uncoated and PEG1000/2000ylated) SiNP

with PJM as measured by SPR.

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