Alpha-alumina nanoparticles induce efficient autophagy-dependent cross- presentation ... ·...

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2011.153

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 1

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Alpha-alumina nanoparticles induce efficient autophagy-dependent cross-

presentation and potent antitumour response

Haiyan Li, Yuhuan Li, Jun Jiao, Hong-Ming Hu

Supplementary methods.

Bioconjugates and immunocomplex (IC). Oxide nanoparticles including α-Al2O3

nanoparticles (60 nm and 200 nm, Nanoamor) (2 mg/mL), anatase TiO2 nanoparticles (25 nm

and 100 nm, Sigma-Aldrich) (2 mg/mL), α-Fe2O3 nanoparticles (25 nm, Nanoamor) (2 mg/mL)

were functionalized with 4-aminophenol (Sigma) (50 µg/mL) in deionized water at 90 °C for 2

hours. The amine functionalized oxide nanoparticles were washed by centrifuging at 9000 × g

for 1 hour and re-suspended in deionized water. Following the suggested protocol (Solulink),

the amine group-functionalized oxide nanoparticles were further modified using 4-

formylbenzoate (4FB). Soluble OVA (Sigma) was modified using 4-hydrazinonicotinate

acetone hydrazone (SANH). After mixing the suspension of SFB modified oxide NPs (10

mg/ml) and the same volume of modified soluble OVA solution (2 mg/ml) for 12 hrs, the metal

oxide-OVA conjugates were purified by centrifuging at 9000 × g for one hour and re-

suspended in PBS. Both the OVA protein loaded by oxide nanoparticles (released by

extracting with 0.2 M citric acid, pH 3.0) and the OVA protein remaining in the supernatant was

determined using the bicinchoninic acid (BCA) assay. The amounts of OVA conjugated to 1

mg/mL α-Al2O3 nanoparticles (60 nm), α-Al2O3 nanoparticles (200 nm), anatase TiO2

nanoparticles (25 nm), anatase TiO2 nanoparticles (100 nm) and α-Fe2O3 nanoparticles (25

nm) were 0.089 mg/mL, 0.086 mg/mL, 0.263 mg/mL, 0.030 mg/mL and 0.245 mg/mL,

respectively. The endotoxin content of all kinds of nanoparticles we presented was determined

using a Limulus Amebocyte Lysate Kit (QCL-1000, BioWhittaker). The endotoxin content was

below the level that is required to activate DCs (less 0.05 EU/µg NPs).

© 2011 Macmillan Publishers Limited. All rights reserved.

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For the preparation of immunocomplexes of OVA/anti-OVA, equal amount of OVA (2 mg/ml)

and goat-anti-OVA (2 mg/m) were incubated at room temperate overnight before they were

used to loading DCs.

Fourier transform-infrared (FT-IR) analysis. FT-IR spectra were recorded on a Perkin-Elmer

2000 FT-IR spectrometer. The samples were prepared by applying pressure to the mixture of

potassium bromide powder and α-Al2O3 nanoparticles (or 4-amimophenol-modified α-Al2O3

nanoparticles, 4-amimophenol).

Uptake of α-Al2O3-OVA by DCs. The uptake of α-Al2O3-OVA by bone marrow-derived

immature DCs was monitored using a Zeiss Axiovert 40 CFL inverted microscope with an

excitation source at 488 nm. α-Al2O3-OVA conjugates were labelled using Alexa 488

(Invitrogen). Bright field and fluorescent images of live cells were acquired and then overlaid

using Photoshop imaging software.

Major histocompatibility complex class I expression. Immature DCs were derived from

bone marrow of C57BL6 mice after in vitro culture with complete media supplemented with 20

µg recombinant GM-CSF (Peprotech). DCs were incubated with OVA and α-Al2O3-OVA for 18

hrs. The washed cells were stained using biotin-conjugated 25D-1.16 mAb, which recognizes

the Kb-SIINFEKL complexes (OVA257-264) derived from OVA proteins1. The Kb-SIINFEKL

complexes were quantified by BD FACS AriaTM II flow cytometer after staining with PE-

conjugated strepavidin.

Transmission electron microscopy (TEM) and Scanning electron microscope (SEM). The

shape, internal structure and chemical composition of α-Al2O3 nanoparticles and α-Al2O3-OVA

conjugates were analyzed using an FEI Tecnai F20 TEM equipped with an energy dispersive

X-ray (EDX) spectrometer. For TEM analysis, α-Al2O3-OVA-pulsed DCs were washed and

fixed with 4% paraformaldehyde in 0.25 M PBS buffer (pH 7.4) for one hour at room

temperature. The fixed DCs were stained with 0.1 M sodium cacodylate buffer and then 2 %


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osmium tetroxide in 0.1 M sodium cacodylate buffer. After being dehydrated gradually in

ethanol solution, the stained DCs were embedded in epoxy resin at 60 °C overnight. The

cross-sections of the embedded DCs were imaged using a Philips/FEI CM120/Biotwin TEM.

The morphology and chemical composition of the α-Al2O3-autophagosome conjugates were

analyzed by an FEI Sirion field emission SEM equipped with an EDX spectrometer.

Tumour cell-derived autophagosomes. The preparation of isolated autophagosomes

followed the method reported previously2. Briefly, tumour cells were cultured in RPMI complete

media supplemented with 100 nM Bortizomib (proteasome inhibitor) and 20 mM ammonium

chloride for 24 hrs. Cells were collected and secreted autophagosomes (DRibbles) were

dislodged from cells by vigorous washing of cells with solution D (150 mM NaCl and 5 mM

EDTA). Autophagosomes (300-800 nm double membrane particles) were enriched by

differential centrifugation method to remove large cell debris and small vesicles (<200 nm

particles). The amount of protein in the isolated autophagosomes was determined by BCA

after lysis of autophagosomes with RIPA buffer. The isolated autophagosomes were

conjugated to SFB modified α-Al2O3 nanoparticles after modification with SANH as described

above for OVA protein. The amounts of protein in the autophagosome or the α-Al2O3-

autophagosome suspensions were kept at 1 mg/ml, while the concentration of α-Al2O3 was

around 1 mg/mL.

References:

1. Porgador, A., Yewdell, J.W., Deng, Y., Bennink, J.R. & Germain, R.N. Localization, quantitation, and in situ

detection of specific peptide-MHC class I complexes using a monoclonal antibody. Immunity 6, 715–726 (1997).

2. Li, Y. et al. Efficient cross-presentation depends on autophagy in tumour cells. Cancer Res 68, 6889–6895

(2008).

3. Savina, A. et al. The small GTPase Rac2 controls phagosomal alkalinization and antigen crosspresentation

selectively in CD8(+) dendritic cells. Immunity 30, 544–555 (2009).


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Supplementary figures.

Supplementary Figure 1. Infrared spectra of dried α-Al2O3 NPs (top), 4-amimophenol-

modified α-Al2O3 NPs (middle), and 4-amimophenol (bottom) reveal that α-Al2O3 NPs

were modified with 4-amimophenol. The narrow absorption peak around 3700 cm-1

(vibration of Al-OH), the broad absorption peak centred at 3400 cm-1 (hydrogen-bonded OH

stretching vibration), the narrow absorption peak around 1620 cm-1 (the scissoring vibration of

adsorbed water) and the narrow absorption peak around 1382 cm-1 (bending vibrations of OH)

significantly reduced or disappeared. This indicates that the surface -OH of α-Al2O3 NPs

reacted with the OH group of 4-aminophenol.


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Supplementary Figure 2. TEM characterization of TiO2-OVA and α-Fe2O3-OVA

conjugates. a-d, TEM images. (a) anataseTiO2 nanoparticle (100 nm) before and (b) after

conjugating with OVA protein. (c) α-Fe2O3 nanoparticle (25 nm) before and (d) after

conjugating with OVA protein.


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Supplementary Figure 3. Efficient cross-presentation of α-Al2O3-OVA, but not the

mixture of α-Al2O3 nanoparticles and OVA. The mixture was prepared by mixing equal

volumes of OVA (0.2 mg/mL) and α-Al2O3 nanoparticles (2 mg/mL) suspensions for 12 hrs.

DCs were pulsed in vitro with OVA, α-Al2O3-OVA or the mixture of α-Al2O3 nanoparticles and

OVA (0.1 µg/mL OVA loaded for each sample) for 6 hrs before they were used to stimulate

CFSE-labelled naïve OT-I CD8+ T cells. 60 hrs later, the percentage of divided OT-I CD8+ T

cells was measured by flow cytometry analysis. This experiment was repeated more than three

times with similar results.


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Supplementary Figure 4. Majority of internalized α-Al2O3-OVA did not go to lysosomes

after 6 hrs pulsing of DCs. Representative confocal images of the DCs loaded with FITC-

labelled α-Al2O3-OVA after staining with lysosome tracker (red). This experiment was repeated

twice and received with similar results.


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Supplementary Figure 5. EDX spectrum confirms that the nanoparticles phagocytized

by DCs were Al2O3. The cross-sections of the embedded DCs pulsed with α-Al2O3-OVA were

also imaged on an FEI Tecnai F20 TEM equipped with an EDX spectrometer. The internalized

particles in DCs were analyzed by EDX spectrometer. It shows that these particles are

composed of only Al and O.


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Supplementary Figure 6. Phagocytosis of α-Al2O3-OVA did not induce efficient

conversion of LC3. (a) LC3 II production of DCs after uptake of OVA or α-Al2O3-OVA, DCs

pulsed with ammonium chloride were used as positive control. (b) ER-Glogi blocker,

brefeldin A did not affect the stimulatory activity of peptide-pulsed DCs.


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Supplementary Figure 7. The working hypothesis and model that depicts the most likely

route and fate of antigens chaperoned by α-Al2O3 nanoparticles. Generally, soluble

antigen, such as OVA was internalized by macropinocytosis or receptor mediated endocytosis

and then degraded in an acidic environment after fusion of endosomes and lysosomes. In

contrast, α-Al2O3-OVA was delivered to autophagosomes after phagocytosis. By blocking the

direct fusion of lysosomes and the endosomes encapsulating α-Al2O3-OVA, the autophagic

vacuoles maintained akylination and reduced antigen degradation, resulting in an efficient

cross-presentation of α-Al2O3 nanoparticle carried OVA3.


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Supplementary Figure 8. Tumour cell-derived autophagosomes from 3LL tumour cells

contain higher levels of LC3-II. A equal amount of protein lysates (10 µg each) from either

whole tumour cells or isolated autophagosomes were separated by SDS-PAGE and subjected

to western blot analysis with anti-LC3 antibody.


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Supplementary Figure 9. EDX analysis reveals that α-Al2O3 nanoparticles were

conjugated to autophagosomes. EDX spectra of (a-d), normalized elemental proportions of

(e-h) correspond to Si substrate, SFB modified α-Al2O3 nanoparticles, isolated

autophagosomes and α-Al2O3-autophagosome conjugates, respectively.


Alpha-alumina nanoparticles induce efficient autophagy-dependent cross- presentation ... ·...

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