Supporting Information

� Wiley-VCH 2010

69451 Weinheim, Germany

Polyacrylate Dendrimer Nanoparticles: A Self-Adjuvanting VaccineDelivery System**Mariusz Skwarczynski, Mehfuz Zaman, Carl N. Urbani, I-Chun Lin, Zhongfan Jia,Michael R. Batzloff, Michael F. Good, Michael J. Monteiro,* and Istvan Toth*

anie_201002221_sm_miscellaneous_information.pdf

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37

37

J14peptide

epitope

J14peptide

epitope

37

J14

pept

ide

epito

pe

J14

pept

ide

epito

pe

37

37

37

37

Scheme S1. Synthesis of dendrimer 1 and construct 2.

S1

a

b

Figure S1. Dynamic light scaterring analysis of nanoparticles. (a)Particle size and (b) zeta

potential of 2 (multiplied measurements).

S2

-30000

-20000

-10000

0

10000

20000

30000

40000

190 210 230 250

mea

n re

sidu

e el

iptic

ity (d

eg c

m2 /d

mol

)

wavelengh (nm)

Figure S2. CD spectra of peptides free J14 (blue) and 2 (pink) at 100 μM concentration in

water.

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1PBS

2 + C

FA

J14 +

CFA 2

J14 +

1 mix

0

2

4

6 IgG1

IgG2a

IgG2b

IgG3

J14-

spec

ific

IgG

isot

ype

titre

(log

10)

Figure S3: J14-specific serum IgG isotype titer (log10) at the final bleed (day 37) after

subcutaneous immunization with J14-D (2) and variants for each individual mouse.

S4

Supplementary Methods

Materials and Methods. All chemicals used in this study were of analytical grade or

equivalent unless otherwise stated. Boc-L-amino and Fmoc-L-amino acids and pMBHA resin

were purchased from Novabiochem (Läufelfingen, Switzerland), Renanal (Budapest,

Hungary) and Peptides International (Louisville, Kentucky). Peptide synthesis grade N,N′-

dimethylformamide (DMF), dichloromethane (DCM), trifluoroacetic acid (TFA), HBTU, and

di-t-butyldicarbonate were purchased from Auspep (Melbourne, VIC, Australia). HPLC

grade acetonitrile (ACN), isopropyl alcohol (IPA), and methanol (MeOH) were supplied by

Labscan (Dublin, Ireland) or Honeywell Burdick and Jackson (Morristown, NJ). Hydrogen

bromide in acetic acid and ninhydrin were obtained from Merck (Kilsyth, VIC, Australia).

Anhydrous hydrofluoric acid (HF) was supplied by BOC gases (Sydney, NSW, Australia).

Tert-butyl acrylate (tBA, 99 % Aldrich) was passed through a basic alumina column (activity

I) to remove inhibitor. CuBr (99.9%, Aldrich), CuBr2 (99.9 %, Aldrich), basic alumina

(Aldrich: Brockmann I, standard grade, ~ 150 mesh, 58 Å), sodium azide (99.9 %, Sigma),

triethylamine (TEA, 99 %, Fluka), tripropargyl amine (TPA, 98 %, Aldrich), tetrahydrofuran

(THF, 99.8 %, LAB-SCAN), DMSO (99 %, Aldrich), dimethylformamide (DMF, A.R.

Grade, Lab-Scan), methanol (MeOH, A.R. grade, Univar) were used as received.

Pentaerythritol tetrakis(2- bromopropionate) (4BrPr) was used to prepare 4-arm stars and was

synthesized according to a published procedure.[1] All other reagents were purchased from

Sigma-Aldrich (Castle Hill, NSW, Australia). A Kel-F HF apparatus (Peptide Institute,

Osaka, Japan) was used for HF cleavage. ESIMS were performed on a Perkin-Elmer-Sciex

API3000 using the Analyst 1.4 (Applied Biosystems/MDS Sciex, Toronto, Canada) software.

Samples (1–10 μL) were injected into ACN-H2O mobile phases containing 0.1% (v/v) acetic

acid and run in positive ion mode. 1H NMR spectra were recorded on Bruker Avance 500

MHz spectrometer (Bruker Biospin, Germany) at 298 K. Analytical RP-HPLC was

S5

performed using Shimadzu (Kyoto, Japan) instrumentation (Class Vp 6.12 software, SCL-

10AVp controller, SIL-10A autoinjector, LC-10AT pump, LC-10AD pump, Waters 486

tunable absorbance detector) using a 0–100% linear gradient of solvent B over 40 min with a

1 mL/min flow rate and detection at 214 nm. Solvent A consisted of 0.1% (v/v) aqueous TFA

and solvent B consisted of 90% ACN/H2O + 0.1% TFA. Separation was achieved on a Vydac

(Hesperia, CA) analytical C8 column. Preparative RP-HPLC was performed on a Waters

Delta 600 system in linear gradient mode using a 10 mL/minute flow rate, with detection at

230 nm. Separations were performed on a Vydac C8 preparative column. CD spectra were

measured on a JASCO (Tokyo, Japan) J-710 spectropolarimeter using quartz cuvette of 1 mm

path length at 23 °C.

Attenuated Total Reflectance-Fourier Transform Spectroscopy (ATR-FTIR):

ATR-FTIR spectra were obtained using a horizontal, single bounce, diamond ATR accessory

on a Nicolet Nexus 870 FT-IR. Spectra were recorded between 4000 and 500 cm-1 for 32

scans at 4 cm−1 resolution with an OPD velocity of 0.6289 cm s−1. Solids were pressed

directly onto the diamond internal reflection element of the ATR without further sample

preparation.

Absolute Molecular Weight SEC:

Absolute molecular weights of polymers were determined using a Polymer Labs GPC50 Plus

equipped with dual angle laser light scattering detector, viscometer and differential refractive

index detector. HPLC grade tetrahydrofuran was used as eluent at flow rate 1 mL/min.

Separations were achieved using two PLGel Mixed C (7.8 x 300 mm) SEC columns

connected in series held at a constant temperature of 40 ○C. The triple detection system was

calibrated using a 2 mg/mL PSTY Standard (Mwt =110 K, dn/dc = 0.185 and IV = 0.4872

mL/g).

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Transmission electron microscopy:

The sample of the construct was added to glow discharged carbon coated 200 mesh grids for

3 min and then wicked off with filter paper. Pictures were taken from a JEM-1010

transmission electron microscope (JEOL Ltd., Japan) operated at 80 kV.

Particle size and zeta potential:

A Zetasizer Nano ZP instrument (Malvern Instruments, UK) with DTS software was used for

particle size and zeta potential measurements of 2. Sizes were analysed using a non-invasive

backscatter system and zeta potentials were measured using M3-PALS technique.

Measurements were taken at 25 °C with scattering angle of 173° using disposable capillary

cuvettes. The experiments were performed in multiplicate.

Immunogold Staining:

Carbon coated 200-mesh copper grids were prepared and used. The grids were first placed on

a drop of dendrimer (2) solution (suspended in distilled water) and was left for 1~5 minutes.

The grid was then placed into a drop of water or PBS buffer to wash off excess samples.

Sections on grids were then blocked for 15 minutes by using the PBS/Block solution

containing 10 x PBS, 0.2% fish skin gelatine, 0.2% BSA and 20mM glycine. Grids were then

incubated with the primary antibody (anti-J14 mouse IgG) for 30 minutes (diluted 1:200 in

PBS/Block solution) and washed with PBS/Block solution for 4 x 5 minutes. This is followed

by moving the grid to a drop of gold conjugated goat anti-mouse antibody (1:40 diluted with

PBS/Block solution) and washing the grids with PBS (4 x 5 minutes) and distilled water (4x2

minutes). The grids were then dried and observed under the TEM.

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Synthesis

Synthesis of 4-Arm Star P(tBA-Br)4 (4): [tBA]:[4BrPr(3)]:[CuBr]:[CuBr2]:[PMDETA] =

[250]:[1]:[4]:[0.4]:[4.4] freshly purified tBA (2.20 g, 1.72 x 10-2 mol), PMDETA (0.0525 g,

3.03 x 10-4 mol), pre-formed CuBr2 (0.0062 g, 2.76 x 10-5 mol), and pentaerythritol

tetrakis(2- bromopropionate) (4BrPr, 0.0465 g, 6.89 x 10-5 mol) were added to a 10 mL

Schlenk flask equipped with a magnetic stirrer and purged with N2 for 15 min. CuBr (0.0395

g, 2.76 x 10-4 mol) was added under positive N2 flow and purged with N2 for a further 10

min. The flask was placed in a temperature controlled oil bath at 35 ○C for 2 h. The reaction

was terminated by quenching in liquid nitrogen followed by exposure to air. The

polymerization mixture was diluted with THF, and the copper salts were removed by passage

through an activated basic alumina column. The solution was concentrated through

evaporation with air flow, and the polymer recovered by precipitation into methanol/water

(50:50 vol), filtered and dried for 48 h under high vacuum at 25 ○C. The polymer was

characterized by Absolute Molecular Weight SEC. (Mn = 19000, PDI = 1.09, P(tBA37-Br)4).

1H NMR (CDCl3) δ: 1.11 (m, 12H, (=CH(CH3))4), 1.45 (b, methyl protons of t-BA repeat

units), 1.82, 2.23 (b, methylene and methine protons of polymer backbone), 2.47 (m, 4H,

(=CH(CH3))4), 3.94-4.22, (m, 12H, C(CH2-O-)4, (-CH-Br)4); IR (ATR) υ(max) 2985 (br), 1719

1450 1373 1245 1150 902 843(sharp, strong).

Synthesis of 4-Arm Star with azide end-groups P(tBA37-N3)4 (5): NaN3 (0.133 g, 2.1 x 10-3

mol) was added to a stirred solution of P(tBA37-Br)4 (1.00 g, 5.26x 10-5 mol) in 5 mL DMF.

The reaction mixture was stirred for 24 h at 50 oC in a temperature controlled oil bath. The

polymer recovered by precipitation into methanol/water (50:50 vol), filtered and then dried

under high vacuum at 25 oC. 1H NMR (CDCl3) δ: 1.11 (m, 12H, (=CH(CH3))4), 1.45 (b,

methyl protons of t-BA repeat units), 1.82, 2.23 (b, methylene and methine protons of

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polymer backbone), 2.47 (m, 4H, , (=CH(CH3))4), 3.63-3.75, (m, 4H, (-CH-N3)4), 3.94-4.22,

(m, 8H, C(CH2-O-)4); IR (ATR) υ(max) 2985 (br), 2113 (broad, weak) 1719 1450 1373 1245

1150 902 843 (sharp, strong).

Synthesis of 4-Arm Star with propagyl end-groups P(tBA37-(≡)2)4 (1): P(tBA37-N3)4 (0.500 g,

2.63 x 10-5 mol), PMDETA (0.183 g, 1.06 x 10-3 mol), TPA (0.275g, 2.1 x 10-3 mol) , CuBr

(0.150 g, 1.05 x 10-3 mol) and 5 mL of DMF was added to a 10 mL Schlenk flask equipped

with a magnetic stirrer. The reaction mixture was stirred for 4 h at 80 ○C in a temperature

controlled oil bath. The polymer was recovered by precipitation into an acidified

MeOH/water (50:50 vol) mixture and then filtered. The polymer was re-dissolved in DMF (6

mL) and re-precipitated into an acidified MeOH/water (50:50 vol) mixture, recovered by

filtration and washed exhaustively with water. The dendrimer (1) was dried under high

vacuum at 25 ○C. 1H NMR (CDCl3) δ: 1.11 (m, 12H, (=CH(CH3))4), 1.45 (b, methyl protons

of t-BA repeat units), 1.82, 2.23 (b, methylene and methine protons of polymer backbone),

2.47 (m, 4H, , (=CH(CH3))4), 3.48, (s, 16H, (-N(-CH2-C≡CH)2)4), 3.88, (s, 8H, (methylene

protons close to the 1,2,3-triazole ring), 3.94-4.22, (m, 8H, C(CH2-O-)4), 5.24, (m, 1H,

methine protons of PtBA close to the 1,2,3-triazole ring) 7.72, (m, 4H, methine protons of

1,2,3-triazole ring); IR (ATR) υ(max) 2985 (br), 1719 1450 1373 1245 1150 902 843 (sharp,

strong).

S9

1H NMR spectra of A) 4-Arm Star PtBA-Br (4), B) 4-Arm Star PtBA-N3 (5), C) 4-Arm Star

PtBA-Alkyne (1), (500MHz, CDCl3).

ATR-IR spectra of A) 4-Arm Star PtBA-Br (5), B) 4-Arm Star PtBA-N3 (5), C) 4-Arm Star

PtBA-Alkyne (1).

S10

Synthesis of J14 azide (6): Azide peptide epitope (6, N3CH2CO-

KQAEDKVKASREAKKQVEKALEQLEDKVK-NH2) was synthesized by HBTU/DIPEA

method on a p-methylbenzhydrylamine (p-MBHA) resin using the standard Boc-SPPS[2] or

on Rink Amide MBHA resin using the Fmoc-SPPS.[3] In both cases before the final cleavage

and after coupling of last J-14 amino acid, azido acetic acid[4] was coupled to the peptide

under standard conditions but with prolonged reaction time (overnight). HPLC: tR =19.5 min

(purity over 98 %); m/z ESI 1719.6 [M+2H]2+, (calc. 1719.4), 1146.6 [M+3H]3+ (calc.

1146.3), 860.3 [M+4H]4+ (calc. 860.2), 688.4 [M+5H]5+ (calc. 688.4), 573.9 [M+6H]6+ (calc.

573.8), 492.1 [M+7H]7+ (calc. 492.0), 430.8 [M+8H]8+ (calc. 430.6); MW 3436.87 g mol−1.

Synthesis of 2 and “micellization” of 2 from DMF into water: Dendrimer 1 (2.0 mg, 0.10

µM) and peptide 6 (4.0 mg, 0.90 µM) were dissolved in DMF (1 mL). Cu wires, treated with

concentrated sulphuric acid (5 min), subsequently washed with water, methanol and dried

under reduced pressure, were added to the solution. The reaction mixture was stirred

overnight at 50 °C in a temperature controlled oil bath. The wires were filtered off from

warm solution and washed with 1 mL DMF. Millipore endotoxin-free water (7 mL) was

added slowly (at 0.025 mL/min) to a solution. The nanoparticles formed through the self-

assembly process were exhaustively dialysed against Millipore water pH = 6.8 using

presoaked and rinsed dialysis bags (Pierce Snakeskin, MWCO 3K). The final concentration

of construct was determined as 0.9 mg/mL.

In vivo study

Subcutaneous immunization

All protocols were approved by the Queensland Institute of Medical Research Animal Ethics

Committee and were carried out according to Australian National Health and Medical

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Research guidelines. Female B10.Br (H-2K) mice (4–6 week-old, Animal Resource Centre,

Perth, Western Australia, Australia) were used for immunization. Mice (n) 5/group) were

injected subcutaneously at the tail base on day 0 with 50 μg of immunogens in a total volume

of 50 μL of sterile-filtered phosphate buffered saline (PBS). Immunogens consisted of PAA

dendrimer alone (1), J14 chemically attached to dendrimer (2), 2 + CFA (2 + CFA), PBS,

J14 + 1 physical mixture (J14 + 1 mix) , and J14 + CFA. Mice received two further boosts

(days 21, 28,) with 50 μg of immunogens in a total volume of 50 μL of PBS. Two positive

control received either 37.5 μg of J14 emulsified in a total volume of 50 μL of CFA/PBS (1:

1) or 50 μg of 2 emulsified in a total volume of 50 μL of CFA/PBS (1: 1). Two negative

controls were administered a 50 μL of either PBS or 23.8 μg of dendrimer (1) in total volume

of 50 μL PBS. All boosts (days 21, 28) were done in the same manner as primary except the

positive controls which were administered equivalent amounts of J14 or 2 in PBS.

Collection of serum

Serum was collected on days 20, 27 and 37 after primary immunization for measurements of

systemic antibodies after subcutaneous immunization. Blood was collected from mice via the

tail artery and allowed to clot for at least 30min at 37°C. Serum was collected after

centrifugation for 10min at 3500 rpm, heat inactivated for 10 min at 56°C, and stored at -

20°C.

Determination of antibody titers by ELISA

An ELISA (enzyme linked immunosorbent assay) was used to measure J14 and p145-specific

murine serum IgG, as described elsewhere.[5] Tested compounds were compared to 2 + CFA,

J14 + CFA as well as PBS. The titer was described as the lowest dilution that gave an

absorbance of > 3 standard deviation (SD) above the mean absorbance of control wells

(containing normal mouse serum immunized with PBS).

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Statistics. Statistical analysis of antibody titers between groups was performed using a one-

way ANOVA followed by the Tukey post hoc test. GraphPad Prism 4 software was used for

statistical analysis, with p < 0.05 taken as statistically significant.

Supplementary References

[1] K. Matyjaszewski, P. J. Miller, J. Pyun, G. Kickelbick, S. Diamanti, Macromolecules

1999, 32, 6526-6535.

[2] W. Zhong, M. Skwarczynski, P. Simerska, M. F. Good, I. Toth, Tetrahedron 2009,

65, 3459-3464.

[3] Y. Fujita, A. B. M. Abdel-Aal, N. Wimmer, M. R. Batzloff, M. F. Good, I. Toth,

Bioorg. Med. Chem. 2008, 16, 8907-8913.

[4] J. T. Lundquist, J. C. Pelletier, Org. Lett. 2001, 3, 781-783.

[5] M. R. Batzloff, J. Hartas, W. G. Zeng, D. C. Jackson, M. F. Good, J. Infect. Dis.

2006, 194, 325-330.