## 3 Results and Discussion  
### Synthesis and Characterization of Plx-g-Cmp  
The grafted copolymer (Plx-\(g\)-CMP) was prepared by the coupling reaction between the amino groups of functionalized poloxamer and carboxylic groups present on the pullulan chains. The reaction occurred with the formation of an amide bond (Scheme 1) in the presence of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ). It is well known that EEDQ is a specific coupling reagent for carboxyl groups widely used in the formation of peptide bonds [52]. The synthesis of the monoamine derivative of poloxamer was performed according to the method described by [PERSON] et al. (2003) [34] (Scheme 2a) and CMP was obtained by the carboxymethylation reaction of pullulan, using sodium chloroacetate as a carboxymethylating agent (Scheme 2b), according to [PERSON] et al. [53]. The \({}^{1}\)H NMR spectrum of CMP in D\({}_{2}\)O confirmed that the carboxymethyl reaction of the pullulan occurred, by the presence of the characteristic signal of methylene protons at 4.16 ppm (Figure 1). Since this signal overlaps with those specific to the protons in the glucoside units, the determination of the substitution degree (\(DS\)) was not possible by this method. Therefore, the \(DS\) of pullulan with carboxymethyl groups was determined by the titration method with 0.1 N HCl solution and found to be 0.87 (29/100 sugar units) [54] (data not shown). The \(M\)n value of CMP estimated by the gel permeation chromatography was found to be 1.94 \(\times\) 10\({}^{4}\) g/mol (\(M\)w/\(M\)n = 2.3), close to the \(M\)n of pullulan, which is an indication that the carboxymethylation reaction took place without the chain cleavage. The \({}^{1}\)H NMR spectrum of the grafted copolymer (Figure 1) shows the presence of the typical signal corresponding to methyl groups in the propylene oxide units of the poloxamer at 1.14 ppm. The other signals from methylene and methylene protons in PPO and PEO units of Plx appeared together with the specific protons of the anhydrolucosidic unit of CMP in the region between 3.0 and 4.6 ppm. Additionally, the (1\(\rightarrow\)6) and (1\(\rightarrow\)4)omeric protons at 5.67 and 4.95 ppm and the hydrogen atoms H2-H7 from the glucosidic unit of CMP are present. Based on this spectrum, the composition of the polymer was determined by reporting the integral value of \(CH_{3}\) protons at 1.14 ppm to that of \(H\)-1 protons of CMP observed at 4.95 and 5.67 ppm (Equation (8)). \[DS\ (\%)=\frac{I_{CH3}/3\times y}{I_{H(1,A)}+I_{H(1,6)}}\times 100 \tag{8}\]  
where \(y=56\) (considering the poloxamer composition (PEO)\({}_{x}\)(PPO)\({}_{y}\)(PEO)\({}_{x}\) (\(x=101\), \(y=56\)). The weight content of 83.88% Plx of the grafted polymer was obtained, equivalent to a \(DS\) equal with 10 mol% (mol of Plx/100 anhydrol cytosolic unit of CMP) and a carboxymethyl groups content of 0.59 mmol/g. The average number of graft chains/100 sugar units of Plx-\(g\)-CMP calculated from \({}^{1}\)H NMR spectrum was 2.9 and the \(M\)n value was \(57.7\times 10^{4}\) g/mol. The evidence for a successful grafting of Plx to carboxymethyl groups of CMP via amide linkages was also revealed in the \({}^{13}\)C NMR spectrum of Plx-\(g\)-CMP (Figure 2) where, in the region between 170 and 185 ppm, three distinct signals are present. Besides the signal at 180 ppm belonging to C8 in CMP component, the peak at 174 ppm could be attributed to C11 (carbonyl in amide group of Plx) and the small signals at 185 ppm corresponded to C=O in unprotonated carboxymethyl groups of CMP. The FT-IR spectra of native pullulan, CMP derivative and Plx-\(g\)-CMP copolymer are presented in Figure 3. The characteristic absorption bands of pullulan (Figure 3a) are present in the FT-IR spectrum of CMP (Figure 3b). Moreover, the appearance of new bands specific to newly introduced carboxymethyl groups confirms the successful achievement of the carboxymethylation reaction.