Supporting Information

Tailored Synthesis of Octopus-type Janus Nanoparticles for Synergistic

Actively-Targeted and Chemo-Photothermal Therapy
Lingyu Zhang+, Yinyin Chen+, Zilu Li, Lu Li,* Philippe Saint-Cricq, Chunxia Li,* Jun Lin,
Chungang Wang,* Zhongmin Su, and Jeffrey I. Zink

anie_201510409_sm_miscellaneous_information.pdf
EXPERIMENTAL SECTION
Materials. Tetraethyl orthosilicate (TEOS, ≥98%), 3-aminopropyltrimethoxysilane
(APTMS), fluorescein isothiocyanate (FITC), hydrogen tetrachloroaurate (HAuCl4·H2O),
doxorubicin hydrochloride (DOX), polyacrylic acid (PAA, Mw ≈ 1800), silver nitrate
(AgNO3), L-ascorbic acid (AA) and methoxy-poly(ethylene glycol)-thiol (PEG) were
purchased from Sigma (USA). N-hydroxysuccinimide (NHS), lactobionic acid (LA), and
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC) was obtained from Thermo
Fisher Scientific. Isopropyl alcohol (IPA), trisodium citrate and aqueous ammonia solution
were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd and used without further
purification. Deionized (DI) water was used in all experiments.
Characterization. Transmission electron microscopy (TEM) was performed on a
JEOL-2100F transmission electron microscope under 200 kV accelerating voltage. Fourier
transform infrared (FTIR) spectra were obtained on a Magna 560 FTIR spectrometer (Nicolet,
USA). Fluorescence microscopy was operated on Olympus DP73. Ultraviolet-visible (UV-Vis)
spectra were recorded by a U-3010 spectrophotometer (Hitachi, Japan). N2 sorption analysis
was performed by an intelligent gravimetric analyzer Autosorb-iQ (Quantachrome).
Inductively coupled plasma atomicemission spectroscopy (ICP-AES) was measured with
Leeman ICP-AES Prodigy. Confocal laser scanning microscopy (CLSM) was operated on
Olympus Fluoview FV1000. Thermal imaging was taken by a GX-A300, infrared camera
(Shanghai Guixin Corporation). A continuous-wave (CW) diode laser (LSR808H) with
wavelength of 808 nm was used for the laser irradiation experiment (power density of 2 W
cm-2).
Scalable synthesis of Au-PAA Janus NPs. Monodispersed Au NPs about 50 nm were
prepared by reduction of tetrachloroauric acid by sodium citrate.[1] 100 mL of the
as-synthesized AuNPs were washed with water, which could remove the unreacted specie.
Then, the AuNPs were re-dispersed into 25 mL of DI water. After that, PAA aqueous solution
(100 µL, 0.2 g mL-1) and NH3·H2O (125 µL, 2 mol L-1) were added into 1 L flask,
ultrasonically dispersed for 10 min. Under magnetic stirring, 100 mL of IPA was dropped into
the flask, and then another 625 mL of IPA was added to obtain the Au-PAA Janus NPs for
further experiment (water : IPA = 1 : 29).
To synthesize the Au-PAA Janus NP with different thicknesses of PAA shell (20, 37 or 70
nm), 0.2 g mL-1 PAA aqueous solution (60, 80 or 100 µL) were mixed with 2 mol L-1
NH3·H2O (75, 100 or 125 µL), respectively. After sonication for 10 min, 100 mL of IPA was

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dropped into the flask, and then another (335, 480 or 625 mL) IPA was added with magnetic
stirring to get the products.
Synthesis of octopus-type Au-PAA/mSiO2 Janus NPs. The pH value of 90 mL
as-prepared Au-PAA Janus NPs solution was adjusted to ~8 with NH3·H2O solution (2 M).
With continuous stirring, TEOS (200 µL) and APTMS (20 µL) were dropwise added into the
solution for 24 h at room temperature (RT). Then, the obtained Au-PAA/mSiO2 Janus NPs
were centrifuged and washed with DI water repeatedly to dispose the redundant precursors,
and then re-dispersed in 10 mL DI water for next step. To synthesize octopus-type
Au-PAA/mSiO2 Janus NPs (OJNPs), 1 mL of above Au-PAA/mSiO2 Janus NPs solution was
dispersed into 70 mL water, then added 42 µL of 30 mM HAuCl4 solution and 5 µL of 1 M
HCl in a 100 ml flask with moderate stirring at RT. Quickly, 50 µL of 3 mM AgNO3 and 25
µL of 100 mM AA were simultaneously added. The OJNPs were obtained after stirring for 30
s, while its color rapidly turned from light red to greenish-black.
Under dark conditions, 4 mg of FITC was mixed with 44 µL of APTMS in 0.75 mL ethanol
for 2 days. The prepared FITC-APTMS stock solution was kept at 4 °C. For the preparing of
the FITC-labeled OJNPs, 5 µL of FITC/APTMS solution was added followed by the addition
of TEOS.
Dual surfaces modification. After washing, 1 mL of PEG (MW ≈ 5000, 10 mg mL-1 in
H2O) was added and sonicated for 30 s, then left to react over night. The octopus-type
PEG-Au-PAA/mSiO2 Janus NPs (PEG-OJNP) were centrifuged and washed twice to remove
excess PEG, then re-dispersed in water. With sonication, 0.5 mL of NHS (1 mg mL-1), EDC
(1 mg mL-1, 1 mL) and 1.5 mL of LA (2 mg mL-1) were mixed at the same time for 30 min at
0 °C. To form the covalent bonding between the carboxyl of LA and amine groups on the
surface of PAA/mSiO2, the PEG-OJNP were dropped into the solution for 4 h at RT to get
octopus-type PEG-Au-PAA/mSiO2-LA Janus NPs (PEG-OJNP-LA).
Photothermal effection of PEG-OJNP-LA. The PEG-OJNP-LA dispersions of different
concentrations (1 mL, 0.1 or 0.2 mg mL-1) in centrifuge tubes were irradiated by 808 nm NIR
laser (5 min, 2W cm-2). The temperature was measured every 20 s. DI water was also
irradiated with the NIR laser light as comparison group. The photothermal conversion
efficiency, η, was calculated according to the previous reported method.[2]
The photothermal conversion efficiency, η, was calculated by the following equations：
hS (Tmax − Tsurr ) − Qdis
η= (1)
I (1 − 10 − A808 )

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 In this equation, I expresses the incident laser power (2 Wcm-2), A808 means the absorbance
at 808 nm of the PEG-OJNP-LA, the heat-transfer coefficient is h, the surface area of the
container is S, the equilibrium temperature is Tmax, the ambient temperature of the
surroundings is TSurr, and the heat associated with the light absorbance by the solvent is Qdis.
The value of hS is derived according to Equation (2):
mDCD
τs = (2)
hS
In this equation, the τs means the sample system time constant, mD and CD express the mass
and heat capacity (4.2 J g-1), when DI water was used as the solvent. To measure the Qdis, pure
water in a test tube was utilized.
Drug loading and pH-NIR dual-stimuli responsive controlled release. The DOX aqueous
solution (10 mg mL-1, 40 µL) was mixed with PEG-OJNP-LA solution (1 mg mL-1, 1 mL) for
24 h to get the DOX loaded PEG-OJNP-LA. The loading efficiency (LE %) of DOX can be
calculated by Equation (3):
Abs (original DOX) - Abs (residual DOX)
LE % = × 100 % (3)
Abs (original DOX)

To investigate the DOX release at different pH values and under NIR irradiation, the
DOX-loaded PEG-OJNP-LA (1 mL, 1 mg mL-1) in pH 7.4 and 5.1 PBS buffer were putted in
a tube with or without NIR irradiation at RT, respectively. The 808 nm NIR laser (2 W cm-2)
irradiated the samples for 5 min, and then centrifuged to quantify the amount of the released
DOX by UV-Vis spectroscopy.
Cell culture. The human cervical carcinoma cancer Hela cells and human liver cancer
HepG2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented
with 10 % (v/v) fetal bovine serum (Gibco) at 37 °C in a humidified atmosphere containing
5 % CO2.
Celluar uptake. Firstly, HepG2 and Hela cells (1×105) were cultured onto glass cover slips
in a 24-well plate for 24 h. PBS (control), FITC labeled PEG-OJNP and FITC labeled
PEG-OJNP-LA at 25 µg mL-1 were added to the HepG2 cells. Meanwhile, FITC labeled
PEG-OJNP-LA with the same concentration were added to the Hela cells. Then, the cells
were washed by PBS to remove the excess particles and dead cells after 3 h incubation. To
stain the nuclei, 300 µL Hoechst 33342 (10 µg mL-1) was added to the cells for 15 min. Then,
extra dye molecules were washed and the cells were sealed with a microscope glass slide.
After that, CLSM was used to observe the cellular uptake.

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 Fluorescence microscopy. HepG2 cancer cells were incubated with PBS (as control
group), PEG-OJNP-LA (25 µg mL-1), NIR laser only and PEG-OJNP-LA (25 µg mL-1) upon
NIR light irradiation, respectively. After incubation for 24 h, NIR laser only and
PEG-OJNP-LA upon NIR laser irradiation groups were exposed to an 808 nm NIR laser (2 W
cm-2, 5 min). Then, calcein AM was used to stain the cells to verify the photothermal effect by
a fluorescence microscopy.
In vitro cytotoxicity study. To estimate the in vitro cell cytotoxicity, a standard
3-(4,5-dimethylthialzol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was utilized.
HepG2 cancer cells incubated with NIR laser, PEG-OJNP-LA, PEG-OJNP-LA with NIR laser
irradiation, free DOX, DOX loaded PEG-OJNP-LA, DOX loaded PEG-OJNP upon NIR laser
irradiation and DOX loaded PEG-OJNP-LA under NIR laser irradiation with different
concentrations (12.5, 25, 50 and 100 µg mL-1), respectively. The concentrations of free DOX
were the same level as the DOX concentrations in the DOX-loaded groups (4.5, 9, 18 and 36
µg mL-1), respectively. 20 µL of MTT solution (5 mg mL-1) was added after 24 h, and
incubated for another 4 h. Before the absorbance was measured, the medium was changed to
DMSO (150 µL). Cell viability was measured by Equation (4):
Abs (test cells)
Cell viability (%) = × 100 % (4)
Abs (control cells)
Biodistribution of PEG-OJNP-LA and PEG-OJNP in Mice. H-22 bearing Kunming
mice were intravenously injected with PEG-OJNP and PEG-OJNP-LA (20 mg kg-1),
respectively. At different time points (3, 12 h, 1 d, and 3 d), the mice (n = 4) were euthanized
and weighed. After treating all the major organs (spleen, kidney, heart, liver, lung and tumor)
with HNO3 and H2O2 (v/v = 1:2) at 70 °C, ICP-AES was used to calculate the contents of Au
in each tissue.
In vivo NIR imaging. The H-22 bearing Kunming mice treated with PBS (control),
PEG-OJNP (2.2 mg kg-1) and PEG-OJNP-LA (2.2 mg kg-1) for 24 h, then irradiated with the
808 nm NIR laser (5 min, 2 W cm-2). At different times, an infrared camera was utilized to get
the temperature of the tumor sites.
In vivo antitumor effect. Five groups (n = 4 each group) of H-22 bearing Kunming mice
were injected by tail vein with PBS, free DOX, DOX loaded PEG-OJNP-LA, DOX loaded
PEG-OJNP with NIR laser irradiation and DOX loaded PEG-OJNP-LA with NIR laser
irradiation. The DOX dose was equivalent to the total amount of 0.792 mg kg-1 (the
equivalent NPs dose was 2.2 mg kg-1) body weight. After 24 h post-injection, tumors in
Kunming mice (DOX loaded PEG-OJNP with NIR laser irradiation and DOX loaded
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PEG-OJNP-LA with NIR laser irradiation groups) were irradiated by the NIR light (2 W cm-2)
for 5 min. Every two days, the tumor volumes, survival rate and body weights of mice were
measured. The tumor volume was calculated according the equation: Volume = (Tumor
Length) × (Tumor Weight)2/2. The mice were randomly selected and euthanized to get the
organs and tumors after treatment for 11 days. After washing with DI water, they were fixed
with 4% (weight) paraformaldehyde solution. The formula inhibition (%) = (C - T)/C × 100 is
used to calculate the tumor growth inhibition rate, where T means the average tumor weight
of each treated groups and C expresses the average tumor weight of the control group.
Histology analysis. After 11 days, the tissues (liver, heart, spleen and lung) from the
sacrified mice were obtained from control, PEG-OJNP-LA and DOX loaded PEG-OJNP-LA
treated groups, which were fixed in 10% neutral buffered formalin. Then, these tissues were
embedded in paraffin, sectioned (4-µm thick), and stained with hematoxylin and eosin (H&E).
Finally, an optical microscope was used to detect the histological sections.

Figure S1. TEM images of Au-PAA Janus NPs with different thickness of PAA shell (A) 20
nm, (B) 37 nm (C) 70 nm.

Figure S2. Photograph of octopus-type Au-PAA/mSiO2 Janus NPs and octopus-type

PEG-Au-PAA/mSiO2 Janus NPs in water, PBS buffer and culture medium solution (DMEM),
respectively. The yellow squares indicate the location of the precipitation of OJNPs.
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Figure S3. FTIR spectra of (a) OJNPs, (b) PEG-OJNP, (c) LA, (d) PEG-OJNP-LA.
The spectra of OJNPs (blue line) contains a vibrational band at 1561 cm-1 that is assigned
to the stretching mode of the carboxyl C=O, indicating the presence of PAA, a strong
absorption band at 1104 cm-1 ascribed to the O-Si-O asymmetric stretching modes. The
successful modification of PEG on the surface of Au spikes is suggested by the presence of a
band at 2890 cm-1, which is attributed to the symmetric stretching modes of the -CH3 groups
(red line). After LA was anchored onto the PAA/mSiO2 domain, the amide bond was formed
and the carbonyl stretching of LA which should at 1701 cm-1 (green line) was disappeared,
and the band at 3438 cm-1 was appeared (yellow line), indicating enhanced intermolecular
hydrogen bonding after introducing LA into the scaffold.

Figure S4. Nitrogen adsorption/desorption isotherms and pore size distribution curve (inset)
of PEG-OJNP-LA.
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Figure S5. UV-Vis spectra of (I) Au NPs, (II) Au-PAA Janus NPs, (III) Au-PAA/mSiO2
Janus NPs and (IV) OJNPs (V) PEG-OJNP-LA and corresponding digital pictures.
The optical properties were explored in Figure S5. Compared with Au NPs which presented
a localized surface plasmon resonance (LSPR) band at 536 nm, the LSPR bands of Au-PAA
Janus NPs and Au-PAA/mSiO2 Janus NPs gradually shifted to 550 nm and 556 nm
respectively, confirming their successful preparation. The red shift in the spectra might be
attributed to the higher dielectric constant of surrounding PAA and mSiO2. After further
growth of Au spikes on the Au NPs, the red solution was changed to greenish-black and a
strong plasmon band at 794 nm was observed in NIR region.

Figure S6. (A) Photothermal effect of the irradiation of the aqueous dispersion of
PEG-OJNP-LA with the NIR laser (808 nm, 2 W cm-2), in which the irradiation lasted for 5
min, and then the laser was turned off. (B) Time constant for heat transfer from the system is
determined to be τs = 363 s by applying the linear time data from the cooling period (after 300
s) versus negative natural logarithm of driving force temperature, which is obtained from the
cooling stage of panel a.
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Figure S7. Concentration-dependent temperature increases of PEG-OJNP-LA solution
exposed to NIR laser. Pure water was used as negative control.

Figure S8. TEM image of the PEG-OJNP-LA after irradiation.

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Figure S9. Fluorescence microscopy investigation of calcein AM stained HepG2 cells treated
with (I) PBS, (II) PEG-OJNP-LA (25 µg mL-1), (III) NIR laser only and (IV) PEG-OJNP-LA
upon NIR laser irradiation (25 µg mL-1). The laser is the 808 nm NIR laser at a power density
of 2 W cm-2.

Figure S10. UV/Vis spectra of DOX (a) before and (b) after interaction with the
PEG-OJNP-LA. Inset: photograph of PEG-OJNP-LA just mixing with DOX aqueous solution
(a) and loading DOX for 24 h after centrifugation (b). UV-Vis spectroscopy was used to
determine the amount of DOX loading.
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Figure S11. CLSM images of (A) HepG2 cells incubated with PBS, (B) HepG2 cells
incubated with FITC labeled PEG-OJNP, (C) Hela cells incubated with FITC labeled
PEG-OJNP-LA, (D) HepG2 cells incubated with PEG-OJNP-LA.
We labeled the PEG-OJNP-LA and PEG-OJNP using a green fluorescent dye FITC. As
shown in Figure S11, less green fluorescence was observed in cytoplasm in HepG2 cells (LA
receptor positive) after incubated with FITC labeled PEG-OJNP for 3 h, which might due to
the passive targeting. Importantly, an increase in green specks was confirmed by CLSM
imaging when the HepG2 cells incubated with FITC labeled PEG-OJNP-LA, indicating the
LA conjunction was more efficient. In contrast, there was lower uptake of FITC labeled
PEG-OJNP-LA in Hela cells (LA receptor negative), which proved that the functionalization
of active targeting ligands endowed these NPs with specific interaction with the receptor on
the cell membrane, which make them only gathered in the targeted cells.

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Figure S12. In vivo antitumor efficacy on H-22 tumor bearing mice after treating with PBS as
control, free DOX, DOX loaded PEG-OJNP-LA, DOX loaded PEG-OJNP under 808 nm NIR
laser irradiation and DOX loaded PEG-OJNP-LA under 808 nm NIR laser irradiation at
density of 2 W cm-2. (A) Mean tumor weights and (B) tumor inhibition rate of each group at
the last day of experiment. Two asterisks indicate statistically highly significant discrepancy
(**P <0.01).

Figure S13. Hematoxylin and eosin (H&E) stained images of major organ tissues from mice
after 11 days post injection of PBS (control) and DOX loaded PEG-OJNP-LA.

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