Synthesis PolySpermine

Reduciable Polyspermine (amide)

Synthesis and characterization of RPS

RPS was synthesized by Michael addition reaction between spermine and N,N'-Cystaminebisacrylamide (Polysciences Inc, Dry Ice, 5 g, $143.00) CBA. In this polymerization reaction, CBA (M n : 260.4) and spermine (Mn : 202.35) were separately dissolved in a brown flask with freshly prepared 80% aqueous methanol (3 ml) in two different stoichiometric ratios 1:2 and 1:4 of CBA to spermine. The spermine solution was added into CBA solution in a slow controlled fashion in an inert environment. The reaction was performed at 55 o C for 15 days with stirring and additional 6 days at room temperature till highly viscous solution was obtained. During the entire course of reaction, the reactants and product were strictly protected from light and air which otherwise significantly affected the final product.

After completion of reaction, the final volume was adjusted to 20 ml with cold acidified (pH w3) water and the product was purified by ultrafiltration at 4 o C (MWCO: 1000) (Pall Life Sciences, USA). After purification, product was lyophilized immediately and stored at -20 o C. The confirmation of synthesis and the composition of the prepared RPS were determined by 1 H nuclear magnetic resonance ( 1 H NMR) (AdvanceÔ 600, Bruker, Germany). The molecular weight of RPS copolymer was measured using gel permeation chromatography (Amersham Biosciences, USA)

Polyspermine Imidazole-4,5-imine

Imine Structure

The synthesis of PSI was as simple as a single condensation reaction (Scheme 1): adding an approximately equivalent molar amount of imidazole-4,5-dialdehyde to spermine (both dissolved in dimethyl formamide (DMF)) in the presence of a catalytic amount of p-toluenesulfonic acid lead to the
formation of PSI. The polymerization was allowed to proceed at 80 8C for 24 h, with stirring, followed by filtration, evaporation, and dialysis in ultrapure water through a cellulose membrane (10 kDa molecular weight cut off). The final product was lyophilized to give a yellowish powder, prior to storage at -80 oC.

the average molecular weight (MW) of PSI was determined to be 21.9 kDa (with polydispersity of 1.44) by eluting a solution of the polymer (2 mg mL/1 in water) through an SEC column (PL aquagel-OH), using a series of polyethylene glycol (PEG) standards and 25 kDa polyethylenimine (PEI) for calibration.

Synthesis and characterization of polyspermine imidazole-4,5-imine (PSI)
Spermine (170 mg, 0.84 mmol) was dissolved in DMF (5 mL), to which p-Toluenesulfonic acid
monohydrate (1 mg, 0.005 mmol) and a small amount of freshly activated molecular sieves of type 4A
were added at room temperature under a high-purity nitrogen atmosphere. 1H-Imidazole-4,5-
dicarboxaldehyde 1 (100 mg, 0.8 mmol) dissolved in DMF (5 mL) was added dropwise to the mixture
slowly over 4 hours, and heated quickly to 80°C. After stirring for another 24 hours, the resulting solution
was filtrated in vacuum and evaporated to remove the solvent. The viscous residue was dissolved again in

Synthesis of polyspermine carbamate (PSP-Carb) and polyspermine amide (PSP-Amide)

Spermine (420 mg, 2.08 mmol) was dissolved in methanol (30 ml) at −78 ◦ C under a dry nitrogen atmosphere via Schlenk technique. Ethyl trifluoracetate (0.46 ml, 4.16 mmol) was then added dropwise and the resulting solution was magnetically stirred at 0 ◦ C for 1 h (Geall and Blagbrough, 2000). Protected spermine, N 1 ,N 14 -bis (trifluoracetyl) spermine, was isolated from this solution by evaporating the solvent and unreacted trifluoracetate. Then the
product was recrystallized in ethyl acetate with over 98% yield. Ethylenebis-(chloroformate) (144.0 mg, 0.77 mmol) or succinyl chloride(119.0 mg, 0.77 mmol) dissolved in chloroform (12 ml) was added dropwise to N 1 ,N 14 -bis (trifluoroacetyl) spermine (303.4 mg, 0.77 mmol) dissolved in chloroform (15 ml) and triethylamine (5 ml) at 0 ◦ C under a dry nitrogen atmosphere. The resulting solution was then continuously stirred at room temperature for 12 h (Xu et al., 2008). After the solvents evaporated, the reaction products were treated with a mixture of 30% ammonium water (1.4 ml) and methanol (8 ml) at 60 ◦ C in a sealed vessel for 8 h to remove the trifluoroacetate protecting groups (Garrett et al., 2000). The de-protected polyspermine biscarbamate (PSP-Carb) and bisamide (PSP-Amide) linkages were obtained after evaporating their respective liquid residues and dialyzed using a membrane with molecular weight cut-off (MWCO) of 3500 (Fig. 1). water and dialyzed through a cellulose membrane of molecular weight cutoff of 10000 (Thermo
Scientific) for 24 hours to remove the small fragments. The dialysate was stored at -80°C for 4 hours and
then freeze-dried (Christ Alpha 1-2 LD Freeze dryer, Germany) to obtain PSI (20 mg, 7.4 % yield)

The molecular weight (M w ) of PSI at the concentration of 2 mg/mL in water
measured by SEC relative to PEG standards was 21.9 KDa with polydispersity (PDI) 1.44.

Synthesis of polyspermine imine (PSP-Imine)

Spermine (490 mg, 2.43 mmol) was dissolved in 18 ml anhydrous  ethanol over molecular sieve (4 A) and then continuously stirred while 40% glyoxal solution  (Sigma 128465-1KG $54.50)(0.28 ml, 2.43 mmol) was added slowly at 0 ◦ C under nitrogen. Afterwards, the mixture was brought to room temperature and stirred overnight. The filtrate was then evaporated in vacuo, and the resulting polymer was transferred to dialysis membranes (MWCO = 3500). It was dialyzed against doubly distilled water and subsequently lyophilized.

Stability

 Mw of PSP-Carb was nearly unchanged when it was incubated in an aqueous buffer of pH 7.4 for 85 days. When incubated at pH 5.8 and 5.0, M w of PSP-Carb dropped to 74.1% and 69.8%, respectively, of its original value in 85 days, suggesting a pH-responsive degradability of the carbamate linkage. PSP-Amide showed higher degradability under the same conditions in that M w of which dropped to 66.3%, 59.6%, and 58.3% of the original M w values over 85 days incubation at pH 7.4, 5.8 and 5.0, respectively (Fig. 2(b)). PSP-Imine degraded remarkably faster than the other two polymers. By incubating at 37 ◦ C and pH 7.4, 5.8 and 5.0 for only 11 h, the Mw of PSP-Imine dropped to 57%, 48% and 42% of its original value, respectively. The rate of degradation of PSP-Carb, PSP-Amide and PSP-Imine accelerated with the dropping of pH.