Editing Synthesis Chitosan Arginine (section)

=== Protocol 2<br>  ===

Poly-l-arginine hydrochloride (PLR) (molecular weight = 13,300 Da): Sigma<br> 

1,2-distearoyl-sn-glycero-3-phos-phoethanolamine--[methoxy(polyethylene glycol)-2000] (DSPE-PEG): Avanti Polar Lipids<br> 

The synthesis procedure of PLR–PEG was based on the pre-<br>viously described method (Huang et al., 2002). Briefly, PLR was<br>dissolved in 25 ml of 50 mM sodium tetraborate buffer (STBB, pH<br>8.5) per gram of PLR. The resulting solution was stirred vigor-<br>ously for approximately 30 min and subsequently filtered through<br>a 0.22 ␮m Durapore ® membrane (Sterile Millex GV, Sigma–Aldrich,<br>Buchs, Switzerland) into a sterile culture tube. The appropriate stoi-<br>chiometric amount of DSPE-PEG powder was then slowly added to<br>the solution while it was continuously stirred. After another 6 h<br>of vigorous stirring at room temperature, the solution was trans-<br>ferred to a dialysis tube (Spectr/Por dialysis tubing, M.W.C.O. of<br>6–8 kDa, Spectrum Laboratories, Inc., Rancho Dominguez, CA). The<br>synthesized product was dialyzed out for 24 h in 10 mM phosphate-<br>buffered saline (PBS, pH 7.0), followed by an additional 24 h of<br>dialysis in deionized water. The product was then freeze-dried for<br>48 h at −70 ◦ C with a pressure of 0.2 mbar.<br> 

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The synthesis schemes of CS–PLR and PEG–CS–PLR conjugates are<br>summarized in Suppl. Fig. 1. PLR was covalently linked to CS via<br>formation of amide bonds between the amine groups (–NH 2 ) of CS<br>and the carboxyl groups (–COOH) of PLR. The formation of amide<br>bonds between the activated carboxyl groups (–COOH) of PLR and<br>amine groups (–NH 2 ) of CS resulted in the conjugates of CS–PLR. The<br>conjugation of PLR to CS and the pegylation of CS–PLR conjugates<br>were confirmed by 1 H NMR (data not shown). The grafting of PLR and<br>PEG to CS was tested by FI-IR spectra. PEG–CS–PLR showed the<br>characteristic absorption band around 2900 cm − 1 and 1650 cm − 1 ,<br>resulting from the increased C–H bond and amide (C O) bond for-<br>mation by grafting of PEG to CS–PLR.<br>