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Supplementary Information

Receptor Mediated Transcytosis in Biological Barrier：The Influence

of Receptor Character and their Ligand Density on the

Transmembrane Pathway of Active-targeting Nanocarriers

Xiaoning Songa, Rui Lia, Hailiang Denga, Ye Lib, Yanan Cuia, Hua Zhanga, Wenbing Daia,

Bing Hea, Ying Zhengb, Xueqing Wanga*, and Qiang Zhanga,c

a. Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems,

School of Pharmaceutical Sciences, Peking University, Beijing 100191, China;

b. State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese

Medical Sciences, University of Macau, Macao, China;

c. The State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing

100191, China

*Address correspondence to this author at School of Pharmaceutical Sciences, Peking

University, Beijing 100191, China; Tel: +86-10-82805935; Fax: +86-10-82805935; E-mail:

wangxq@bjmu.edu.cn

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1. Materials and Methods

1.1. Materials

HOOC-PEG4000-b-PCL2500 (Mw 6500) and mPEG3000-PCL2500 (Mw 5500) were supplied

by Advanced Polymer Materials Inc. (Montreal, Canada). N-hydroxysuccinimide (NHS) and

N, N-dicyclohexycarbodiimide (DCC) was purchased from J&K Chemical Ltd. (Shanghai,

China). FcBP (Arg-Phe-Pen-Thr-Gly-His-Phe-Gly-Sar-NMeLeu-Tyr-Pro-Cys, Mw 1510.76),

7pep (His-Ala-Ile-Tyr-Pro-Arg-His, Mw 893.02) and c(RGDfK)

(c[Arg-Gly-Asp-(D-Phe)-Lys], Mw 603.70) were offered by ChinaPeptides Inc. (Shanghai,

China). Hoechst 33342 was purchased from Molecular Probes Inc. (Eugene, OR, USA). Lyso

Tracker and endoplasmic reticulum (ER) Tracker were offered by Invitrogen (Carlsbad, CA,

USA). Golgi complex Tracker and Methyl-β-cyclodextrin (MβCD) were purchased from J&K

Chemical Ltd. (Shanghai, China). Chlorpromazine (CPZ), genistein, Triton X-100,

5-(N-Ethyl-N-isopropyl)-amiloride (EIPA) and filipin were obtained from Sigma Aldrich (St.

Louis, MO, USA). Cytochalasin D was purchased from Gene Operation (Ann Arbor, MI,

USA); Dynasore and nystatin were purchased from Selleck (Shanghai, China). Rabbit

polyclonal to Transferrin Receptor, Rat monoclonal [5H10-27 (MFR5)] to Integrin alpha 5,

Rabbit polyclonal to Rab11, ALEXA 647 docky anti-rabbit IgG(H+L) and ALEXA 647 goat

anti-rat IgG(H+L) were purchased from Abcam (Cambridge, MA). CellLight® Early

Endosomes-RFP *BacMam2.0* and CellLight® Late Endosomes-RFP*BacMam2.0* were

offered by Molecular Probes Inc. (Eugene, OR, USA). Wheat germ agglutinin, Texas Red®-X

conjugate and Transferrin from human serum, Alexa Fluor® 633 conjugate were purchased

from Thermo Fisher Scientific Ltd. Invitrogen (Carlsbad, CA). Coumarin-6 (C6, Mw 350.43)

was purchased from Molecular Probes Inc. (Eugene, Oregon, USA). All other chemicals were

of analytical or HPLC grade. Transwells (12 wells, 3 μm pore size, polycarbonate membranes)

were provided by Corning Inc. Costar (Cambridge, MA, USA). 8 chambered coverglass

system was obtained from In Vitro Scientific (Hangzhou, China).

The Caco-2 cells line was obtained from China Center for Type Culture Collection

(Wuhan, China). Dulbecco’s modified Eagle’s medium (DMEM; pH 7.4) and

penicillin−streptomycin were obtained from Macgene Biotechnology Ltd. (Beijing, China).

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1.2. Cell Culture

Caco-2 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS,

Gibco, US), 100 units/mL penicillin, 100 mg/mL streptomycin, 1% non-essential amino-acid

and 1% sodium pyruvate, changed every 2 days. Subconfluent Caco-2 cells were plated at

1×105 cells/cm2 on 35mm glass-bottom chambers and used 24 h later. Polarized Caco-2 cells

were plated at 2×105 cells/cm2 on 12 mm Transwell filters and used after 21 days culture. The

TEER of transwell was over 400 Ω.

1.3. Synthesis of the Functional Polymer

The synthesis route of functional polymer was showed in Figure S3. DCC was added in

the solution of HOOC-PEG-PCL and NHS in DMF (N,N-Dimethylformamide) at the molar

ratio of 1.5:1:1.5 to active the carboxyl. After stirred overnight in N2 at 0 °C, the solution was

dried and redissolved in DMF. The intermediate products were dropwise added into functional

peptide (20% excess) DMF solution. Moderate N-methylmorpholine was added to adjust the

pH to 8.0~9.0. The reaction was maintained in N2 for about 36 h at room temperature. The

product was purified by dialysis against 50% ethanol and water in a dialysis bag (MW 3500).

The final solution was lyophilized and stored at -20 °C. High performance liquid

chromatography (HPLC) system (Shimadzu, Japan) was used to monitor the reaction.

1.4. Preparation of the Micelles

PEG-PCL micelles were prepared using the thin-film hydration method [1]. Proper

amount of PEG-PCL polymer and modified PEG-PCL polymer were dissolved in acetonitrile

in a pear-shaped flask to form a dry film after vacuum rotary evaporation at 60 °C for 30

minutes. Then the film was hydrated in PBS at 60 °C by vortex for 3 minutes. After

sonificated for 3 minutes, the solution would be clear and became micelles. The molar ratio of

PEG-PCL polymer and functional PEG-PCL polymer was listed in Table S7. C6 was added at

0.1% w/w to the polymer for fluorescence imaging.

1.5. Characterization of the Micelles

The particle size and polydispersity were determined by dynamic light scattering (DLS)

using Malvern Zetasizer Nano ZS (Malvern, UK) at 25 °C (Table S6). The shape and surface

morphology of micelles were investigated by transmission electron microscope (JEOL,

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JEM-200CX, Japan).

1.6. Receptor Expression Studies

For confocal microscope studies, Caco-2 cells were cultured on coverslips for 4 days

prior to study. After washed by PBS, the cells were then fixed with 4% paraformaldehyde,

treated with TPBS (PBS containing 0.1% Triton) and blocked in 5% BSA. Monoclonal

antibody to specific receptor (including human FcRn receptor, transferrin receptor and αvβ3

receptor, Abcam, UK) were incubated with cells overnight at 4 °C (PBS used as negative).

After washed by PBS for 3 times (5min per time), cells were incubated with secondary

antibody cells for 2 h at 37 °C. Nuclear staining was performed by Hoechst 33258 for 20 min,

and the fluorescent images of cells were acquired using a Leica TCS SP8 confocal laser

scanning microscope (CLSM, Heidelberg, Germany).

For flow cytometry studies, cells were seeded in a 6-well plate at a density of 4×105 per

well 4 days prior to study. Cells were removed by a cells scraper and dispersed. Cells were

then fixed and treated as mentioned above. After incubated with secondary antibody, cells

were washed with PBS three times and then analyzed by a flow cytometer (FAC scan, Becton

Dickinson, San Jose, CA).

1.7. Co-localization analysis

Pearson’s correlation coefficients: Pearson’s correlations were determined using LAS X

(Leica Microsystems, Heidelberg, Germany).

Manders’ coefficients: The percentage of C6-loaded micelles in different endosomes to

that in a whole cell was quantified with the Manders’ coefficient, which is a common method

to measure co-localization between different cellular markers [2-4]. A few endosome-labelled

polarized Caco-2 cells incubated with different micelles were scanned consecutively from the

apical side to the basolateral along the z axis, 1μm per layer, about 20 layers per cell. Manders’

coefficients (M2i) of micelles to specific endosome in the region of interest (that is, one cell)

in different layers were determined using Image J software (NIH Image). The total green

intensities (Gi) of the region of interest in different layers were recorded as a weight for

combining the different layers as well. Series of Manders’ coefficients in different layers were

weighted by the total green intensities of each layer and summed to gain the final percentage

as below fomula.

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%100)(

%2

ii

iii

G

GMP

The percentage (P%) could be used to represent the portion of C6-loaded micelles in

specific endosomes compared to total C6 intensity in a whole cell.

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2. Tables

Table S1 Summary of the receptor character and the influence of receptor character and theirligand density on the transmembrane pathway of active-targeting micells.

Micelles Receptor Receptor charactersLigand density

Low Medium High

F-M FcRn 1. Mediate maternal Ig G transportacross polarized epithelial barriers.2. Be transported to the basolateralmembrane or be recycled to the apicalmembrane with Ig G.

Recyclingpathway,CREpathwayand ERpathway

Recyclingpathway,CREpathwayand ERpathway

CREpathwayandDegradation pathway

T-M TfR 1. Transport iron-Tf complex fromsites of absorption.2. A classical recycling pathwayreceptor.

Recyclingpathway,Golgipathway

Recyclingpathwayand Golgipathway

Degradation pathway

R-M αvβ3 1. Mediate interactions between cellsand their extracellular environments.2. Enhance cell binding, the transportpathway in polarized epithelial cellsis not clear.

CREpathwayand Golgipathway

Degradation pathway,CREpathwayand Golgipathway

CREpathwayand Golgipathway

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Table S2 Inhibitors and their concentrations used in the endocytosis pathway study.Inhibitor Concentration FunctionDynasore 80 μM Blocks formation of pinched off vesicles,inhibitlipid

raft/caveolae and clathrin mediated endocytosis [5, 6]MβCD 10 mM Deplete cholesterol, inhibit lipid raft/caveolae

mediated endocytosis [7]Filipin 0.5 μg/mL Deplete cholesterol, inhibit lipid raft/caveolae

mediated endocytosis [8]Genistein 100 μM Inhibitor of protein tyrosine kinase (PTK) [9]Chloropromazine 30 μM blocks the reassembling of subunit AP2 on

membrane [10]Nystatin 30 μM Inhibitor lipid raft/caveolin-mediated endocytosis [8]Hypertonicsucrose

0.4 M Dissociate the clathrin lattice, inhibit clathrinmediated endocytosis [7]

Cytochalasin D 25 nM Stimulates actin depolymerization, inhibit potentactin polymerization [11]

EIPA 40 μM Block Na+/H+ ion channel, inhibit macrocytosis[12,13]

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Table S3 Transport rate constant coefficients of FcBP decorated PEG-PCL micelles.B-M 5%-F-M 10%-F-M 15%-F-M

K01 1.06192 1.75328 1.47743 1.8056K12 0.018393 0.01752 0.011541 0.029659K23 0.030475 0.016242 0.010512 0.047296K1 5.79E-04 1.34E-04 1.00E-06 1.00E-06K2 1.04E-11 7.68E-11 2.79E-08 5.64E-08K3 0.005421 0.014532 0.010513 0.008839

Table S4 Transport rate constant coefficients of 7pep decorated PEG-PCL micelles.B-M 5%-T-M 15%-T-M 20%-T-M

K01 1.06192 2.40215 4.35344 3.41701K12 0.018393 0.01566 0.019144 0.023432K23 0.030475 0.011775 0.019429 0.029298K1 5.79E-04 4.40E-04 8.58E-04 7.21E-05K2 1.04E-11 2.09E-09 7.35E-10 2.72E-09K3 0.005421 0.015405 0.02436 0.013758

Table S5 Transport rate constant coefficients of c(RGDfK) decorated PEG-PCL micelles.B-M 5%-R-M 15%-R-M 20%-R-M

K01 1.06192 1.99613 2.75394 2.25044K12 0.018393 0.017953 0.020216 0.030336K23 0.030475 0.008891 0.011636 0.017154K1 5.79E-04 2.61E-04 1.73E-09 1.25E-08K2 1.04E-11 3.49E-08 2.69E-08 1.29E-07K3 0.005421 0.013977 0.012262 0.022133

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Table S6 Particle size distribution of micelles with different ligand density by dynamic lightscattering (DLS)

FcBPcontent(%)

Size(nm) PDI7pepcontent(%)

Size(nm) PDI

c(RGDfK)

content(%)

Size(nm) PDI

0 30.67±2.76 0.131±0.06 0 31.85±0.83 0.189±0.03 0 31.85±0.83 0.189±0.03

1 31.76±1.74 0.194±0.06 2.5 30.35±0.13 0.191±0.02 2.5 35.36±0.62 0.184±0.03

2 32.05±1.65 0.180±0.01 5 36.22±0.64 0.195±0.01 5 34.78±0.72 0.211±0.01

5 32.32±1.72 0.180±0.03 10 36.66±0.33 0.198±0.01 10 35.59±0.27 0.196±0.06

10 33.54±1.25 0.200±0.03 15 33.59±0.29 0.188±0.01 15 36.42±0.71 0.194±0.03

15 35.20±1.23 0.210±0.01 20 40.45±0.85 0.236±0.01 20 40.79±0.51 0.199±0.05

Table S7 Concentration of PEG-PCL and modified PEG-PCL in micelles with different liganddensity.ligand density (%) 0 2 2.5 5 10 15 20PEG-PCL (μmol) 1000 980 975 950 900 850 800modified PEG-PCL (μmol) 0 20 25 50 100 150 200

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3. Figures

Figure S1 Endosomal itinerary of micelles in polarized cells. Micelles apicallyendocytosed by polarized Caco-2 cells has three fate: 1. Transported into lysosome(degradation pathway); 2. Recycled to apical membrane (recycling pathway); 3. Released tothe basolateral compartment (transcytosis pathway). 3a represent the pathway travelingthrough Golgi complex to the basolateral membrane. 3b represent the pathway travelingthrough CRE to the basolateral membrane.

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Figure S2 Transport model of degradation pathway in Caco-2 cells. The constantcoefficients, K01, K12 and K23 denote transfer rates for sequential movement of micellesthrough the compartments, while K1, K2 and K3 correspond to micelles escaping from earlyendosome, late endosome and lysosome. EE: early endosome. LE: late endosome. Ly:lysosome.

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Figure S3 Synthesis of peptide decorated PEG-PCL. (A)The activation ofHOOC-PEG-PCL. (B, C, D) The synthesis route of FcBP-PEG-PCL, 7pep-PEG-PCL andc(RGDfK)-PEG-PCL.

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Figure S4 Monitoring of the peptide decorated PEG-PCL synthesis. (A, B, C) The FcBP,7pep and c(RGDfK) concentration during the reaction at different time point were tested byHPLC.

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Figure S5 Transmission electron microscopy (TEM) image of micelles. (A) Blank micelles.(B) A representative image for functional micelles (10%-F-M).

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Figure S6 Characterization of Caco-2 cells for receptor expression. Caco-2 cells werefixed and incubated with primary antibody of FcRn, TfR and αvβ3 receptors separately, anddyed with secondary antibodies. Cells incubated with PBS instead of primary antibody wereused as negative control. (A) Confocal image of FcRn expression. The red channel representsthe staining of FcRn. The blue channel was Hoechst 33258, representing the nucleus of cells.(B) Confocal image of TfR expression. The red channel represents the staining of TfR. Theblue channel was Hoechst 33258, representing the nucleus of cells. (C) Confocal image ofαvβ3 receptors expression. The red channel represents the staining of αvβ3 receptors. Theblue channel was Hoechst 33258, representing the nucleus of cells.

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Figure S7 (A) Colocalization of micelles and early endosomes. Confocal images oftransfected Caco-2 cells incubated with blank micelles and functional micelles. The greenchannel was C6 trapped in micelles. The red channel was RFP, representing early endosomes.(B) Colocalization of micelles and late endosomes. Confocal images of transfected Caco-2cells incubated with blank micelles and functional micelles. The green channel was C6trapped in micelles. The red channel was RFP, representing late endosomes. (C)Colocalization of micelles and lysosomes. Confocal images of Caco-2 cells incubated withblank micelles and functional micelles. The green channel was C6 trapped in micelles. Thered channel was lysotracker, representing lysosomes.

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Figure S8 the Global Pearson colocalization of micelles and endosomes in degradationpathway in 20min. The Global Pearson colocalization correlations was determined usingLAS X (Leica Microsystems, Heidelberg, Germany) in confocal images in Figure S7. EE:early endosome. LE: late endosome. Ly: lysosome.

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Figure S9 Confocal image of ASE staining by WGA. Polarized Caco-2 cells were incubatedapically with 5 mg/ml AF594-WGA in PBS (10 min at 37℃ ). (A) Cartoon displaying ofrelevant endosomal compartments in polarized Caco-2 cells, showing the distribution ofendosomes. (B) The z view of the ASE stained polarized Caco-2 monolayer. (c) The xy viewof ASE. The punctate distribution indicated that the ASE was dyed properly. (D) The series ofimage in different focal distance. The image was captured every 2μm in z-axis. The greychannel was reflection of laser at 633nm, representing transwell membrane. The blue channelwas Hoechst 33258, representing the nucleus of cells. The red channel was AF594-WGA,representing ASE.

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Figure S10 Colocalization of micelles and ASE in polarized Caco-2 monolayer. Confocalimage of Caco-2 cell dyed with WGA and incubated with micelles for 10mim, 20min and30min. The green channel was C6 trapped in micelles. The red channel was AF594-WGA,representing the ASE.

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Figure S11 Confocal image of ARE staining by antibodies. polarized Caco-2 monolayerwere fixed with 4% paraformaldehyde and incubated with anti-Rab11 antibody at 4 ℃

overnight and Alexa-647 anti-rabbit antibody at 37℃ for 2h in sequence. (A) Cartoondisplaying of relevant endosomal compartments in polarized Caco-2 cells, showing thedistribution of endosomes. (B) The z view of ARE stained polarized Caco-2 monolayer. (c)The xy view of ARE. The punctate distribution indicated that the ARE was dyed properly. (D)The series of image in different focal distance. The image was captured every 2μm in z-axis.The grey channel was reflection of laser at 561 nm, representing transwell membrane. Theblue channel was Hoechst 33258, representing the nucleus of cells. The red channel wasAlexa-647 anti-rabbit antibody, representing ARE.

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Figure S12 Colocalization of micelles and ARE in polarized Caco-2 monolayer. Confocalimage of Caco-2 cell dyed by Alexa-647 anti-rabbit antibody incubated with micelles for10mim, 20min and 30min. The green channel was C6 trapped in micelles. The red channelwas Alexa-647 anti-rabbit antibody, representing the ARE.

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Figure S13 Confocal image of CRE staining by antibodies. polarized Caco-2 cells wereincubated basolaterally with 10 mg/ml AF633-Tf in PBS (60 min at 37℃ ). (A) Cartoondisplaying of relevant endosomal compartments in polarized Caco-2 cells, showing thedistribution of endosomes. (B) The z view of CRE stained polarized Caco-2 monolayer. (c)The xy view of CRE. The punctate distribution indicated that the CRE was dyed properly. (D)The series of image in different focal distance. The image was captured every 2μm in z-axis.The grey channel was reflection of laser at 561 nm, representing transwell membrane. Theblue channel was Hoechst 33258, representing the nucleus of cells. The red channel wasAF633-Tf, representing CRE.

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Figure S14 Colocalization of micelles and CRE in polarized Caco-2 monolayer. Confocalimage of Caco-2 cell dyed by AF633-Tf incubated with micelles for 10mim, 20min and 30min. The green channel was C6 trapped in micelles. The red channel was AF633-Tf,representing the CRE.

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Figure S15 Confocal image of BSE staining by antibodies. polarized Caco-2 cells wereincubated basolaterally with 10 mg/ml AF633-Tf in PBS (20 min at 37℃ ). (A) Cartoondisplaying of relevant endosomal compartments in polarized Caco-2 cells, showing thedistribution of endosomes. (B) The z view of BSE stained polarized Caco-2 monolayer. (c)The xy view of BSE. The punctate distribution indicated that the BSE was dyed properly. (D)The series of image in different focal distance. The image was captured every 2μm in z-axis.The grey channel was reflection of laser at 561 nm, representing transwell membrane. Theblue channel was Hoechst 33258, representing the nucleus of cells. The red channel wasAF633-Tf, representing BSE.

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Figure S16 Colocalization of micelles and BSE in polarized Caco-2 monolayer. Confocalimage of Caco-2 cell dyed by AF633-Tf incubated with micelles for 10mim, 20min and30min. The green channel was C6 trapped in micelles. The red channel was AF633-Tf,representing the BSE.

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Figure S17 Uptake percentage of C6-loaded micelles in specific endosomes to that in awhole cell calculated with Manders’ coefficients. (A, E, I, M) Cartoon displaying ofrelevant endosomal compartments in polarized Caco-2 cells, and the confocal image ofdifferent endosomes staining. (B, C, D) The percentage in ASE. (F, G, H) The percentage inARE. (J, K, L) The percentage in CRE. (N, O, P) The percentage in BSE. N=6. The error barwas SEM. *p<0.05, **p<0.01, ***p<0.001.

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Figure S18 Dynamic changes of the uptake percentage of C6-loaded micelles in ASE tothat in a whole cell compared with Pearson’s correlation coefficients. The blue linesrepresent the uptake percentage of C6-loaded micelles in ASE to that in a whole cell. The redlines represent the Pearson’s correlation coefficients of C6-loaded micelles and ASE. (A)Cartoon displaying of relevant endosomal compartments in polarized Caco-2 cells, and theconfocal image of ASE staining. (B-E) Tendency comparison in F-M group. (F-I) Tendencycomparison in T-M group. (J-M) Tendency comparison in R-M group. The error bar wasSEM.

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Figure S19 Dynamic changes of the uptake percentage of C6-loaded micelles in ARE tothat in a whole cell compared with Pearson’s correlation coefficients. The blue linesrepresent the uptake percentage of C6-loaded micelles in ASE to that in a whole cell. The redlines represent the Pearson’s correlation coefficients of C6-loaded micelles and ARE. (A)Cartoon displaying of relevant endosomal compartments in polarized Caco-2 cells, and theconfocal image of ARE staining. (B-E) Tendency comparison in F-M group. (F-I) Tendencycomparison in T-M group. (J-M) Tendency comparison in R-M group. The error bar wasSEM.

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Figure S20 Dynamic changes of the uptake percentage of C6-loaded micelles in CRE tothat in a whole cell compared with Pearson’s correlation coefficients. The blue linesrepresent the uptake percentage of C6-loaded micelles in CRE to that in a whole cell. The redlines represent the Pearson’s correlation coefficients of C6-loaded micelles and CRE. (A)Cartoon displaying of relevant endosomal compartments in polarized Caco-2 cells, and theconfocal image of CRE staining. (B-E) Tendency comparison in F-M group. (F-I) Tendencycomparison in T-M group. (J-M) Tendency comparison in R-M group. The error bar wasSEM.

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Figure S21 Dynamic changes of the uptake percentage of C6-loaded micelles in BSE tothat in a whole cell compared with Pearson’s correlation coefficients. The blue linesrepresent the uptake percentage of C6-loaded micelles in BSE to that in a whole cell. The redlines represent the Pearson’s correlation coefficients of C6-loaded micelles and BSE. (A)Cartoon displaying of relevant endosomal compartments in polarized Caco-2 cells, and theconfocal image of BSE staining. (B-E) Tendency comparison in F-M group. (F-I) Tendencycomparison in T-M group. (J-M) Tendency comparison in R-M group. The error bar wasSEM.

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