R ESULTS AND DISCUSSION

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SEC and MALDI-TOF spectrometry were used at first to purify and assess the masses of the final freeze-dried chitosan fractions. As revealed by SEC chromatography (see Figure 4-2), only one peak was observed with an elution time corresponding to about 10 min. This result assesses the purity of the product.

Figure 4-2: SEC chromatogram of collected fractions of chitosan after enzymatic digestion

To assess the degree of acetylation (DA) of the resulting low molecular chitosan, proton nuclear magnetic resonance ¹H NMR measurement was performed.

As can be observed in Figure 4-3, different peaks corresponding to individual unit residues of chitosan are revealed. The peak at 2.2-2.4 ppm in the ¹HNMR spectrum is assigned to the three protons of N-acetyl glucosamine (GlcNAc), and the peak at 3.4-3.6 ppm to the H2 proton of glucosamine (GlcN) residues. The peaks at 3.9-4.3 ppm correspond to the cumulative signals of the H2, H3, H4, H5, and H6 protons of glucosamine (GlcN) and the peak at 5.0-5.2 ppm is

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assigned to the proton H1 of glucosamine (GlcN). Therefore, using equation 7, the degree of acetylation (DA) could be evaluated to 3.8 %.

Figure 4-3: ¹H NMR spectrum (500 MHz) of chitosan dissolved in D2O and 2% DCl.

Chitosan, though semi-crystallizable, was mixed with appropriate 2,5 dihydroxybenzoic acid (DHB) matrix in order to be analysed by MALDI-TOF spectrometry (see Figure 4-4). As could be seen from the spectrum, several peaks corresponding to different masses were obtained for chitosan, varying from 3049 to 5801 Da. Native chitosan was successfully digested into low molecular weight chitosan with an average mass of (4479 ±1376) Da. of 1.3 polydispersity (PDI).

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Figure 4-4: MALDI-TOF spectrum of chitosan

As it was denoted earlier in the part material and methods, separation of chitosan-grafted-ssDNA from non-reacted DNA was performed through SEC. The chromatogram obtained by SEC (see Figure 4-5) showed two distinct fractions: the first at shorter elution time corresponding to the copolymer. The second peak at much longer elution times corresponds to the unreacted nuclei acid strands [183]. It is important to mention that the second peak was always more intense than the first one, which is due to the fact that the scattering of the light by the grafted copolymer structures tends to reduce the intensity of the UV signal of the first fraction [183].

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Figure 4-5: SEC chromatogram upon separation of chitosan-g-ssDNA from non-reacted DNA.

Chitosan-g-ssDNA is an amphiphilic copolymer with the chemical structure shown in Scheme 4-1. Analysis by analytical ultracentrifugation (AUC) sedimentation-velocity (see Figure 4-6) reveals distinct sedimentation profiles of the starting materials of chitosan, ssDNA and the final product of chitosan-g-ssDNA, which supports the success of the solid phase synthesis route (see solid phase synthesis part 4.3.2).

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Figure 4-6: Apparent sedimentation coefficient distribution of chitosan-g-ssDNA, ssDNA and chitosan as assessed by analytical ultracentrifugation

After synthesis, chitosan-grafted-ssDNA copolymer could not be fully dissolved in any solvent, either presenting some level of aggregation, in polar solvents, or not being soluble at all, in apolar solvents. An acetonitrile-water solvent mixture at 70:30 v/v was the solvent in which the best solubility was achieved. In order to determine the mass of the chitosan-g-ssDNA copolymer, MALDI-TOF spectrometry analysis was performed. Nucleic acids, though crystallizable, are quite sensitive to the ionization process, requiring an appropriate matrix, such as 2,5 dihydroxybenzoic acid, and a low laser intensity in order to be analysed. As can be observed in Figure 4-7, the MALDI-TOF spectrum of chitosan-g-ssDNA revealed different peaks varying from 7907 to 8889 DA. An average mass of (8250 ± 491) Da was calculated. In comparison to the theoretical mass of chitosan-g-ssDNA (8243 Da), calculated from the mass of chitosan (4479

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Da) grafted to DNA (3764 DA), the experimental value of 8250 Da, is thus in agreement with the expected mass of chitosan-g-ssDNA of 8243 Da at a 1:1 grafting ratio. Thus, this result confirms that coupling of a nucleic acid strand to a chitosan segment occurs at 1:1 molar grafting ratio counting about 30 monomers (about 28 of D-glucosamine and 2 of N-acetyl-D-glucosamine). In average, the coupling of chitosan to the nucleic acid strand enables achieving a copolymer with a reaction efficiency of about 40 %.

Figure 4-7: MALDI-TOF spectrum of chitosan-g-ssDNA copolymer

To evidence however that there are no copolymer of higher molecular weight than the hybrid resulting from the coupling of one nucleic acid graft to one chitosan backbone we performed gel permeation chromatography (GPC) (see Figure 4-8, Figure 4-9) Calibration was achieved using

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thirteen various molecular weights of polystyrene analytical standards (see Figure 4-8). The calibration plot (logarithm molecular weight versus retention volume) was generated by replicate analysis (n=3) at all molecular weight levels and the linear relationship was evaluated using the least square method (log Mw = -0.47 VR + 9.27, r² =0.99). The retention volume of 11.96 and 11.42 mL were obtained with chitosan andchitosan-g-ssDNA respectively. (see Figure 4-8 and Figure 4-9). According to the calibration, the molecular weight of chitosan and chitosan-g-ssDNA are 4511 and 8092 Dalton respectively. These values are in good agreement with values assessed by MALDI-TOF spectrometry.

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Figure 4-8: a) GPC calibration achieved with analytical polystyrene standards. b) Chromatogram for chitosan andchitosan-g-ssDNA (only LALS response).

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Figure 4-9: a) Chromatogram for chitosan. b) Chromatogram for chitosan-g-ssDNA. For both cases the LALS, RALS and RI responses are provided.

After synthesis, chitosan-g-ssDNA copolymer could not to be fully dissolved in any solvent, either presenting some degree of aggregation, in more polar solvents, or not being soluble at all, in less polar solvents. Solvent mixture of acetonitrile (CH3CN) with distilled water (H2O) was the

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best, in which the solubility was achieved. Acetonitrile solvent is known to be a polar aprotic solvent of large dielectric constant and high polarity degree, which, dissolves charged species and a wide range of nonpolar compounds. Moreover, chitosan is not soluble in any organic solvents but in acidic aqueous solution at pH below 6.5 [184]. For oligonucleotides, both water and acetonitrile are good solvents. The solubility of chitosan-g-ssDNA diblock copolymer was achieved in two steps, first chitosan-g-ssDNA was dissolved rigorously in acetonitrile with sonication and vortex until the solution became opaque. Later distilled Milli Q water was added to the opaque solution. Chitosan is completely soluble and the solution transparent. The acetonitrile/water solvent ratio was of 70:30 (v/v) respectively.