Synthesis and characterization of CpG-PP, CpG-PM, and CpG-P-ss-M

1. Synthesis

   1.1 Synthesis of PP

    a) Connect PAMAM with PEG-NHS
        50 mg PEG-NHS (10 μmol; 8 equiv ) was dissolved in 2 ml of methanol.
        Add 220 μL PAMAM (1.25 μmol; 1 equiv) and 3 μL TEA.
        The mixture was stirred for 24 hours in the protection of argon to obtain PAMAM-PEG (PP).

     b) Purification of PP

         PP were purified by dialysis (cutoff molecular weight, 7000 Da) and lyophilization.

    1.2 Synthesis of PM

a) Synthesis of MAN-PEG-NHS

MAN (2.7 mg; 1 equiv.) and NHS-PEG₅₀₀₀-NHS (50 mg; 1 equiv.) were dissolved in 4 ml of methanol, and 4 μl of TEA was added. The mixture was stirred for 24 hours in the protection of argon (Figure 1).

Figure 1. Synthetic rout of MAN-PEG-NHS.

b) Purification of MAN-PEG-NHS

Purified MAN-PEG-NHS was obtained by precipitating with diethyl ether, then washing in dichloromethane for three times, and lastly drying in vacuum pressure.

c) Connect PAMAM with MAN-PEG-NHS

50 mg MAN-PEG-NHS (10 μmol; 8 equiv ) was dissolved in 2 ml of methanol.

Add 220 μL PAMAM (1.25 μmol; 1 equiv) and 3 μL TEA.

The mixture was stirred for 24 hours in the protection of argon to obtain PAMAM-PEG-MAN (PM)

d) Purification of PM

PM were purified by dialysis (cutoff molecular weight, 7000 Da) and lyophilization.

    1.3 Synthesis of P-ss-M

a) Synthesis of MAN-PEG₅₀₀₀-CH₂CH₂-S-S-CH₂CH₂-COOH (MAN-PEG-ss-COOH)

NHS-PEG5000-OH (100 mg), 3,3′-dithiodipropionic acid (3.56 mg), and DMAP (4-dimethylaminopyridine) (24 mg) were stirred in three-necked flask, dichloromethane as solvent, with argon protection.

8.25 mg of DCC (dicyclohexylcarbodiimide) dissolved in dichloromethane was slowly dropped into the flask under ice bath condition, and the mixture was proceeded further with stirring for 24 hours at 25°C. 

MAN (5.4 mg) was dissolved in methanol and added in the above flask for another 24 hours.

The whole synthetic rout was showed in Figure.2

Figure 2. Synthetic rout of MAN-PEG-ss-COOH.

b) Purification of MAN-PEG-ss-COOH

Purified by dialysis (cutoff molecular weight, 1000 Da) three times in dimethyl sulfoxide and three times in ultrapure water to remove unreacted MAN and other reagents.

Dried by lyophilization.

c) Connect PAMAM with MAN-PEG-ss-COOH

50 mg MAN-PEG-ss-COOH (10 μmol; 8 equiv ) was dissolved in 2 ml of methanol.

Add 220 μL PAMAM (1.25 μmol; 1 equiv) and 3 μL TEA.

The mixture was stirred for 24 hours in the protection of argon to obtain PAMAM-PEG-ss-MAN (P-ss-M).

d) Purification of P-ss-M
      P-ss-M were purified by dialysis (cutoff molecular weight, 7000 Da) and lyophilization.

    1.4 Connect CpG with PP, PM and P-ss-M

150 μg CpG and 10 mg PP, PM and P-ss-M were dissolved in 2 ml ultrapure water, respectively.

9.1 μg EDC, 5.5 μg NHS and 0.5 μL TEA were then added in the mixture.

The mixture was stirred for 24 hours in the protection of argon to obtain CpG-PP, CpG-PM, and CpG-P-ss-M, respectively.

Purified by ultrafiltration (cutoff molecular weight, 10 kDa) for three times

2. Characterization

     2.1 Synthesis 

The success of synthesis of PEG fragments was validated by 1H NMR spectrum.

The success of synthesis of PP, PM and P-ss-M was validated by 1H NMR spectrum and MALDI-TOF-MS (Shimadzu, Japan).

The decoration of CpG on nanoparticles was validated by agarose gel electrophoresis.

CpG was quantified by measuring absorption at 270 nm (Micro drop, BIO-DL, China).

2.2 Physical properties 

The sizes and zeta potential of all the nanoparticles were characterized by DLS (Malvern ZetaSizer Nano ZS).

Their morphologies were captured by TEM (H-600, Hitachi, Japan).

3. Materials

PAMAM dendrimers (ethylenediamine core, G4, Mw= 14214.7 Da)

NHS-PEG5000-NHS, mPEG5000-NHS, and HO-PEG5000-NHS

CpG 1826 (5′-TCCATGACGTTCCTGACGTT-3′) with C-terminal modification

4-Aminophenyl α-D-mannopyranoside (MAN)

Triethylamine (TEA)

N-hydroxysuccinimide (NHS)

4-dimethylaminopyridine (DMAP)

N,N'-dicyclohexylcarbodiimide (DCC)

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC)

ultrapure water

methanol

dichloromethane

diethyl ether

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