Supplemental Information

Zwitterion-Functionalized Dendrimer-Entrapped Gold

Nanoparticles for Serum-Enhanced Gene Delivery to Inhibit
Cancer Cell Metastasis
Zhijuan Xiong,1 Carla S. Alves,2 Jianhua Wang,3,4 Aijun Li,1 Jinyuan Liu,1 Mingwu Shen,1 João
Rodrigues,2 Helena Tomás,2 Xiangyang Shi*1,2

1
State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of
Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620,
People’s Republic of China
2
CQM-Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9000-
390 Funchal, Portugal
3
Cancer Institute, Fudan University Shanghai Cancer Center, Fudan University, Shanghai
200032, People's Republic of China
4
School of Medicine, Anhui University of Science & Technology, Huainan 232001, People's
Republic of China

*Corresponding author: E-mail address: xshi@dhu.edu.cn (X. Shi)

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Experimental Section

Materials. Ethylenediamine core amine-terminated generation 5 poly(amidoamine)

(PAMAM) dendrimers (G5.NH2) with a polydispersity index less than 1.08 were purchased from

Dendritech (Midland, MI). PEG monomethyl ether with the other end of carboxyl group (mPEG-

COOH, Mw = 2000) and PEG with one end of carboxyl group and the other end of amine group

(COOH-PEG-NH2, Mw = 2000) were from Shanghai Yanyi Biotechnology Corporation

(Shanghai, China). N-[3-(Dimethylamino)propyl] acrylamide (DMAPA, 98%) was from TCI

(Shanghai, China). 2,6-Bis(1,1-dimethylethyl)-4-methylphenol (BHT), β-propiolactone (98%),

(R)-morpholine-3-carboxylic acid hydrochloride (Mor-COOH, 98%), 1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) were

from J&K Scientific (Shanghai, China). Rabbit anti-human HIC1 polyclonal antibody, acetic

anhydride, triethylamine, and all the other chemicals and solvents were from Sigmal-Aldrich (St.

Louis, MO) and used as received. The HIC1 pDNA was provided by Prof. Jianhua Wang

(Shanghai Jiaotong University, China). The Luc assay kit was from Promega (Fitchburg, WI).

The bicinchoninic acid (BCA) protein quantitation kit was purchased from Biomiga (San Diego,

CA). The Label IT® Tracker™ Nucleic Acid Labeling kit containing Cy3™ reagent (MIR 7020)

was from Mirus Bio LLC (Madison, WI). LysoTracker DND-26 was from YEASEN (Shanghai,

China). Primary Amino Nitrogen (PANOPA) Assay Kit was provided by Megazyme (Bray,

Ireland). HeLa cells (a human cervical cancer cell line) was from Institute of Biochemistry and

Cell Biology (the Chinese Academy of Sciences, Shanghai, China). Dulbecco's modified eagle

medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS) were provided by

Hangzhou Jinuo Biomedical Technology (Hangzhou, China). Water used in all experiments was

purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) with a

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resistivity higher than 18 MΩ·cm. Regenerated cellulose dialysis membranes with a molecular

weight cut-off (MWCO) of 1000 or 8000-14000 were acquired from Fisher (Pittsburgh, PA).

Synthesis of G5.NH2-CBAA. CBAA was first synthesized according to the literature .

G5.NH2 dendrimers were then modified by CBAA according to the protocols reported in our

previous work . In brief, G5.NH2 dendrimers (50.00 mg, in 10 mL methanol) were reacted with

CBAA (75.00 mg) dissolved in an aqueous solution of NaCl (10 mL, 0.138 M) under stirring.

BHT (0.10 mg) was then added to inhibit the polymerization of CBAA. The reaction mixture was

stirred for 48 h at room temperature, followed by extensive dialysis against phosphate buffered

saline (PBS, 3 times, 4 L) and water (3 times, 4 L) through an 8000-14000 molecular weight cut-

off (MWCO) membrane for 3 days, and then lyophilized to get the product of G5.NH2-CBAA..

Characterization Techniques. 1H NMR measurements were carried out on a Bruker DRX

500 NMR spectrometer. Samples were dissolved in D 2O or DMSO before measurements.

UV−vis spectroscopy was carried out using a Lambda 25 UV−vis spectrophotometer

(PerkinElmer, Boston, MA). Zeta-potential and dynamic light scattering (DLS) measurements

were performed using a Malvern Zetasizer Nano ZS system (model ZEN3600, Worcestershire,

UK) equipped with a standard 633 nm laser. The concentration of Au for Au DENPs was

determined by Leeman Prodigy inductively coupled plasma-optical emission spectroscopy (ICP-

OES, Hudson, NH). The size and morphology of the formed Au core particles within dendrimers

were characterized by transmission electron microscopy (TEM, JEOL 2010F, Tokyo, Japan). The

number of the primary amines of different Au DENPs was determined using PANOPA Assay Kit

according to the manufacturer’s instruction. Before measurements, the Au DENPs were dissolved

in water (2 mg/mL).

Protein Resistance Assay. We used protein resistance assay to investigate the antifouling

property of the {(Au0)25-G5-CBAA-PEG-Mor} and {(Au0)25-G5-CBAA-mPEG} DENPs

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according to the literature . UV−vis spectroscopy was employed to check the absorbance of

bovine serum albumin (BSA, 1 mg/mL, in PBS) at 278 nm before and after interaction with the

Au DENPs at different concentrations (0.125, 0.25 and 0.5 mg/mL, respectively) at 37 oC for 4 h.

After interaction with the Au DENPs, the mixtures were centrifuged and the respective

supernatant was measured. The reduced absorbance at 278 nm was calculated to quantify the

protein resistance ability of the Au DENPs.

Cytotoxicity Assay. HeLa cells were continuously cultured and passaged in DMEM

supplemented with 10% FBS, 100 U/mL penicillin, 100 U/mL streptomycin at 37 oC in a 5% CO2

incubator. The viability of HeLa cells after treatment with vector or vector/pDNA polyplexes

with a dendrimer concentration ranging from 0 to 3000 nM (1 μg of siRNA for each sample) was

tested by Cell Counting Kit-8 (CCK-8) according to literature protocols .

Expression of pDNA Encoding Enhanced Green Fluorescent Protein (EGFP) and

Luciferase (Luc). Expression of the EGFP Gene: The transfection of EGFP gene was conducted

to compare the gene transfection efficiency of the vector/pDNA polyplexes in serum-free and

serum-containing medium. The cells were firstly seeded into each well of a 24-well plate at a

density of 5  104 cells per well and cultured overnight. Then the medium in each well was

replaced with 400 μL fresh FBS-free medium or 10% FBS-containing medium, and then each

well of cells was treated with a polyplex (100 μL) containing 1.0 μg EGFP pDNA under the N/P

ratios of 1, 2, 4, 8, and 16, respectively. After 3 h incubation, the cells were cultured with fresh

complete medium for 24 h. Then HeLa cells transfected by the above polyplexes were observed

by Nikon Ti-S invert fluorescence microscope (Tokyo, Japan) to validate the EGFP expression.

Expression of the Luc Gene: The cells were firstly seeded into each well of a 24-well plate at

a density of 5  104 cells per well and cultured overnight. Then the medium in each well was

replaced with 400 μL fresh FBS-free medium or 10% FBS-containing medium, and then each
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well of cells was treated with a polyplex (100 μL) containing 1.0 μg Luc pDNA under the N/P

ratios of 1, 2, 4, 8, and 16, respectively. After 3 h incubation, the cells were cultured with fresh

complete medium for 24 h. Then the Luc gene transfection efficiency in HeLa cells was

measured according to the literature protocols .

Cellular Uptake of Au DENPs/pDNA Polyplexes. Flow cytometry (BD FACS Calibur,

Franklin lake, NJ) was used to detect the cellular uptake of Cy3-labeled pDNA encoding EGFP

in HeLa cell using Au DENPs as a vector. The Cy3-labeled EGFP plasmid was prepared using

the protocolof the Label IT® TrackerTM intracellular nucleic acid localization kit. Different vectors

were mixed with the Cy3-labeled EGFP plasmid to form the polyplexes. Under the same cell

culture and transfection condition as described above for the expression of EGFP gene at an N/P

ratio of 4, HeLa cells were washed with PBS for three times, trypsinated, centrifuged and

collected after 3 h incubation with polyplexes. The Cy3 fluorescence of 1  104 cells was

measured in the FL2-H channel and each measurement was repeated three times.

Impact of Endocytic Pathway Inhibitors on the Cellular Uptake of the Polyplexes. In

order to determine the endocytic pathway of the vector/pEGFP DNA polyplexes, several

endocytic pathway inhibitors including chlorpromazine, dynasore and nystatin were used.

Chlopromazine is a cationic amphipathic drug that blocks the clathrin-dependent uptake; nystatin

is a lipid raft blocker that specifically interferes with the caveolin-dependent endocytosis

pathway; dynasore is a noncompetitive chemical inhibitor of dynamin activity and inhibits

dynamin-mediated endocytic pathway including both caveolin-dependent and clathrin-mediated

pathway. HeLa cells were seeded into a 24-well plate at the density of 5  104 cells per well and

cultured overnight. Then, each well of cells were treated with chlopromazine (10M), nystatin

(20 M), or dynasore (60 M), respectively to inhibit the corresponding endocytic pathway

before pEGFP DNA transfection. After 2 h preincubation, the medium of each well was
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substituted by polyplex-containing medium at an N/P of 4 (1 g of Cy3-labeled pEGFP DNA

was used). After 3 h, the cells were rinsed with PBS, trypsinated, centrifuged, collected and

measured by flow cytometry (BD FACS Calibur, Franklin lake, NJ). The Cy3 fluorescence was

measured for 1  104 cells in the FL2-H channel and each measurement was in triplicate.

Intracellular Trafficking and Localization of the Polyplexes. Colocalization assay was

performed to explore the influence of zwitterion and/or Mor modification of the vector on the

lysosomal release of the polyplexes in different transfection conditions. The Cy3-labeled pEGFP

DNA was used for intracellular trafficking and localization of the polyplexes. In brief, cover slips

with a diameter of 14 mm were pretreated with 5% HCl, 30% HNO 3, and 75% alcohol and then

placed in 12-well tissue culture plate. 5×104 HeLa cells were seeded into each well of a 12-well

plate and cultured overnight. Then the medium in each well was replaced with 400 μL fresh FBS-

free medium or 10% FBS-containing medium, and then each well of cells was treated with a

polyplex (100 μL) containing 1.0 μg Cy3-labeled pEGFP DNA at an N/P ratio of 4 for 3 h

incubation. Then cells were cultured with fresh complete medium. After 2, 4, 8 or 12 h, the cells

in each well were co-incubated with different dyes, respectively. Incubation with Lyso Tracker

Green for 20 min was used to label lysosomes, and incubation with DAPI for 7 min was used to

stain the cell nuclei. Finally, the cells were washed with PBS for 3 times and samples were

scanned using a 63× oil immersion objective lens by using a ZEISS LSM-700 laser scanning

confocal microscope.

Inhibition of Cancer Cell Metastasis after HIC1 pDNA Transfection. Wound Healing

Assay: Tumor suppressor gene HIC1 was transfected into HeLa cells using the designed Au

DENP vectors. HeLa cells were seeded to 24-well plates at a density of 5  104 cells/well and

cultivated overnight. Then a scratch was made by using a 100-L tip in each well, and the cells

were three times rinsed with PBS to get rid of the floating cells. Then, 400 μL fresh FBS-free
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medium or 10% FBS-containing medium was added into each well, and each well was added

with 100 μL polyplex solution containing 3.0 μg HIC1 pDNA at an N/P ratio of 4. After 3 h

incubation, the medium of each well was exchanged with fresh medium and images of the wound

were taken by a Leica DM IL LED inverted phase contrast microscope after 0 h, 12 h and 24 h,

respectively. Quantitative analysis of the migration rate was carried out by ImageJ software

(https://imagej.nih.gov/ij/download.html).

HIC1 Protein Expression: Western blot assays were performed to compare the HIC1 protein

expression in HeLa cells after they were transfected with different polyplexes under the same

conditions. HeLa cells were seeded into 6-well plates at a density of 25  104 cells/well and

incubated overnight. The next day, 1 mL of fresh FBS-free or 10% FBS-containing medium was

added into each well of cells, and each well was added with 100 μL of polyplex solution

containing 5.0 μg HIC1 pDNA at an N/P ratio of 4. After 3 h, the medium was substituted with

fresh complete medium. Forty eight hours later, HIC1 protein was collected and analyzed

following the literature procedures .

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Table S1. Physicochemical parameters of the {(Au0)25-G5.NH2-CBAA-PEG-Mor}, {(Au0)25-
G5.NH2-CBAA-mPEG} vectors
Sample {(Au0)25-G5.NH2-CBAA-PEG-Mor} {(Au0)25-G5.NH2-CBAA-mPEG}
Calculated Mw 74843 70566
Mean number of 50 53
primary amines per
dendrimer

Figure S1. 1H NMR spectra of Mor-PEG-COOH (a) in DMSO, G5.NH2-CBAA (b), {(Au0)25 -

G5.NH2-CBAA-PEG-Mor} (c) and {(Au0)25 -G5.NH2-CBAA-mPEG-Mor} (d) in D2O.

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Figure S2. UV-vis spectra of {(Au0)25-G5.NH2-CBAA-PEG-Mor} and {(Au0)25-G5.NH2-CBAA-

mPEG} NPs. Inset shows the photographs of the respective aqueous suspension of the Au DENPs

(1 mg/mL).

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 ]
Figure S3. Surface potential (a and c) and hydrodynamic size (b and d) of Au DENPs. The

pDNA in (a) and (b) encodes EGFP gene and in (c) and (d) encodes HIC1 gene.

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 3
Before incubation
After incubation

NT T
Absorbance 2

 0.05
 0.04
 0.04  0.05
1  0.03
 0.02

0
0.125 0.25 0.5 0.125 0.25 0.5
Concentration (mg/mL)
Figure S4. Histogram of change of BSA/Au DENP mixture absorbance at 278 nm. BSA (1

mg/mL) was incubated with {(Au0)25-G5-CBAA-mPEG} (NT) or {(Au0)25-G5-CBAA-PEG-Mor}

(T) at different concentrations for 4 h, respectively, followed by centrifugation (8000 rpm, 5

min). The absorbance was measured before and after centrifugation at 278 nm. The change of

absorbance was calculated.

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Figure S5. The viability of HeLa cells after treatment with vector and vector/pDNA polyplexes

at different dendrimer concentrations. The cells without treatment were used as control. The

pDNA 1 encodes EGFP gene and pDNA 2 encodes HIC1 gene.

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