Supporting Information

 Wiley-VCH 2010
69451 Weinheim, Germany

Polyacrylate Dendrimer Nanoparticles: A Self-Adjuvanting Vaccine Delivery 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

37

J14 peptide epitope

J14 peptide epitope 37 37

37

37 37

37

Scheme S1. Synthesis of dendrimer 1 and construct 2.

37

J14 peptide epitope

J14 peptide epitope

S1

Figure S1. Dynamic light scaterring analysis of nanoparticles. (a)Particle size and (b) zeta potential of 2 (multiplied measurements).

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40000 30000
mean residue elipticity (deg cm2/dmol)

20000 10000 0
190 210 230 250

-10000 -20000 -30000

wavelengh (nm)

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

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6 J14-specific IgG isotype titre (log 10)

IgG1 IgG2a IgG2b

IgG3

0
1 FA FA S 2 PB C C + + 2 + J1 4 1 m ix

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.

J1

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 (Lufelfingen, Switzerland), Renanal (Budapest, Hungary) and Peptides International (Louisville, Kentucky). Peptide synthesis grade N,Ndimethylformamide (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 ), 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 (110 L) 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, SCL10AVp controller, SIL-10A autoinjector, LC-10AT pump, LC-10AD pump, Waters 486 tunable absorbance detector) using a 0100% 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 cm1 resolution with an OPD velocity of 0.6289 cm s1. 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). S6

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).
1

H NMR (CDCl3) : 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) (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) : 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 S8

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) (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) : 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-CCH)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) (max) 2985 (br), 1719 1450 1373 1245 1150 902 843 (sharp, strong).

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H 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).

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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 mol1.

Synthesis of 2 and micellization of 2 from DMF into water: Dendrimer 1 (2.0 mg, 0.10 M) and peptide 6 (4.0 mg, 0.90 M) 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 selfassembly 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 S11

Research guidelines. Female B10.Br (H-2K) mice (46 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 g of immunogens in a total volume of 50 L 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 g of immunogens in a total volume of 50 L of PBS. Two positive control received either 37.5 g of J14 emulsified in a total volume of 50 L of CFA/PBS (1: 1) or 50 g of 2 emulsified in a total volume of 50 L of CFA/PBS (1: 1). Two negative controls were administered a 50 L of either PBS or 23.8 g of dendrimer (1) in total volume of 50 L 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 37C. Serum was collected after centrifugation for 10min at 3500 rpm, heat inactivated for 10 min at 56C, and stored at 20C. 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 oneway 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] [5] J. T. Lundquist, J. C. Pelletier, Org. Lett. 2001, 3, 781-783. M. R. Batzloff, J. Hartas, W. G. Zeng, D. C. Jackson, M. F. Good, J. Infect. Dis. 2006, 194, 325-330.

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