The synthesis of alginate-PEGAc was designed as a two-step procedure where the synthesis of alginate–thiol is performed first, followed by the conjugation of PEG-DA to the alginate backbone [26] (Figure 14.4). In brief, the synthesis of alginate–thiol [47,52] was achieved by activating the alginate carboxylic groups by the carbodiimide functional group within the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDAC) intermediate reagent. Next, EDAC was replaced with L-cystein through its amine end group to form an amide bond. The thiol concentration in the thiolated product was measured using Ellman’s assay. The resulting thiolated alginate was dissolved in tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution in order to prevent intramolecular disulfide formation [52]. Finally, a Michael-type addition, involving a nucleophilic reaction of the thiols on the thiolated alginate with the vinyl group on the acrylate functionalized poly(ethylene glycol), was performed. In order to lower the probability of multiple attachments of a single PEG-DA molecule to the backbone, a large molar excess of PEG-DA was used. The resultant product includes PEG chains, still carrying one acrylate end group, linked to the alginate backbone through the cystein spacer molecule. The molecular structure of alginate, alginate–thiol and alginate-PEGAc were verified using NMR experiments, where both methylene and vinyl protons of the PEG-acrylate chain were detected in the alginate-PEGAc product [26]. Lack of cytotoxicity was demonstrated using in vitro cell assay [26], where alginate-PEGAc samples prepared from two types of alginate were cultured with human foreskin fibroblasts (HFFs) cells for 24 hours and assayed for the live/dead cells using fluorescent calcien and ethidium homodimer labelling followed by fluorescent microscopy imaging. None of the alginate-PEGAc caused cytotoxic effects in HFFs cells.

Alginate PEGylation was also performed by Laurienzo et al. [48], who conjugated PEG molecules to alginate through its hydroxyl end groups in order to maintain the gelation ability of alginate, which consumes the carboxylic end groups. The synthetic approach described above reduces the number of carboxylic groups on the alginate backbone. Yet, the product retained its gelation ability and gel microparticles could be formed by adding 1% PEGAc solution into 1% CaCl₂ aqueous solution drop-wise [26].

14.5.2 Mucoadhesion Ability

The adhesion ability of alginate-PEGAc to mucosal surface was analysed by measuring the MDF needed to detach compressed polymer tablets from fresh small intestinal surface and compared to native alginate and thiolated alginate, a known covalently binding mucoadhesive polymer [26]. The MDF of two different alginate-PEGAc samples was significantly higher compared to both native alginate and thiolated alginate (Figure 14.5). However, no significant difference in adhesion properties was observed between the two native alginate samples. The adhesion of the thiolated alginate was higher than that of the native alginate in the case of alginate HF120 but not in the case alginate LF200 S. Moreover, the MDF values obtained from thiolated alginate HF120 were significantly higher compared to thiolated alginate LF200 S. This result was attributed to the higher thiol content of thiolated alginate HF120 (136.1 ± 6.6 μmol thiol/g polymer) compared to that of thiolated alginate LF200 S (56.7 ± 12 μmol thiol/g polymer), which is expected to increase the probability for disulfide interactions with mucin glycoproteins. A similar behaviour was also detected when comparing the two acrylated alginates. Alginate-PEGAc synthesized from HF120 demonstrated a significantly higher MDF compared to alginate-PEGAc prepared by modifying LF200 S. It can be assumed that the thiol content is strongly correlated with the PEGylation degree, since a larger thiol content increases the ability of PEG-DA to attach to the alginate, thus leading to a larger PEG content. An increase in PEG and acrylate content could be expected to increase the probability of chain entanglements, polymer noncovalent bonds such as hydrogen bonds and covalent bonds between the acrylate and mucin glycoproteins.

14.5.3 Thermal Properties of Alginate-PEGAc

Characterization of the thermal stability of polymers intended for drug delivery applications is essential due to the high temperatures used in the accelerated stability tests that are required in order to determine the sample’s shelf-life [53]. Furthermore, the thermal behaviour of polymers is sensitive to the presence of molecular interactions or chemical modification [54,55]. Thus, thermal studies can improve the understanding of the polymer structure and behaviour on the molecular level. Thermal gravimetric analysis (TGA) of alginate revealed three thermal steps (Figure 14.6), in agreement with a previous publication by Saores et al. [56], who attributed the first one to a dehydration process and the other two to decomposition. Weight loss analysis revealed dehydration at temperatures of up to 200 °C with around 20% weight loss. The first decomposition step involved additional weight loss of around 30%; in the second decomposition step about 30% of the initial weight was lost. The total remaining ash is about 20%. It is evident from Figure 14.6 that native alginate and alginate–thiol share a similar dehydration behaviour, suggesting that they have similar hydrophilicity.

It is worthwhile mentioning that while several studies have identified two distinct decomposition temperatures for alginate and its derivatives, and the reported values for the location of the first decomposition peaks are similar, the location of the second decomposition peaks varies. For example, decomposition temperatures of 200 °C and 600 °C were reported for alginate by Saores et al. [56], while values of 240 °C and 380 °C were measured by Shah et al. [57]. A two-step decomposition process occurring at 250 °C and 320 °C was reported by Laurienzo et al. for 2,3-dioctyl amine alginate [48], and peaks at 200 °C and 400 °C were observed for alginic acids [56]. The first decomposition step at around 250 °C was also detected by Caykara et al. for alginate samples [58]. Notably, alginate and alginate–thiol share a similar first decomposition temperature (256 °C for alginate and 245 °C for alginate–thiol); however, they differ in the position of the second decomposition step (367 °C and 604 °C for alginate and alginate–thiol, respectively). This result is in line with the behaviour of other alginate derivatives as stated above. The shift of the second decomposition temperature of alginate–thiol to a higher value could result from the formation of inter- and intramolecular disulfide bonds, which allude to higher polymer thermal stability. It is recognized that intramolecular interactions increase the thermal stability of polymers, thus leading to a higher decomposition temperature [54,55]. Moreover, thermal studies of amino acids have shown that the presence of sulfide end groups has led to disulfide bridges, which shifted the thermal peaks to a higher temperature [59].

The thermogram of alginate-PEGAc is shown in Figure 14.6 along with the thermograms of the native alginate and PEG-DA. Due to the overlap between the decomposition peaks of alginate and PEG-DA full peak assignment is not straightforward, yet general trends can be realized. Firstly, the weight loss percentage of alginate-PEGAc and alginate at temperatures up to ∼400 °C are similar. Secondly, the total ash remaining for alginate-PEGAc is 10%, which is smaller than the value observed for both alginate and alginate–thiol from the TGA analysis [71], PEG-DA decomposes without any ashes left. Therefore, it can be concluded that the PEGAc chain attached to the alginate backbone will decompose in this temperature range, leaving ash from the alginate backbone alone. The sharp dehydration peak of about 15% weight loss was shifted to a higher temperature of around 170 °C, possibly due to hydrogen bond interactions between the water molecules and the hydrophilic PEG chains. Three additional small and broad peaks, located at 200, 249 and 378 °C are evident. The peaks at ∼250 and ∼370 °C are common to alginate and alginate-PEGAc, while conjugation of PEG has led to broadening of the alginate decomposition peaks and to the appearance of an additional small peak at ∼200 °C. Thus, this curve seems to reflect the thermal characteristics of both PEG-DA and alginate. Overall, modification of alginate by attachment of PEG chains has a significant impact on the thermal behaviour of the final product. This phenomenon is most likely due to the large molecular weight of the PEG and its chemical characteristics. Laurienzo et al. [48] synthesized alginate-PEG by a different methodology and also detected both broad peaks at a similar temperature range of 200–300 °C, which were attributed to the alginate backbone, and an additional peak at around 400 °C, which was attributed to the grafted PEG molecules. A TGA curve of thiolated chitosan cross-linked by PEG-DA molecules displayed a thermal peak attributed to the PEG residues at approximately 380 °C [60].

In accordance with the TGA results, differential scanning calorimetry (DSC) analysis of alginate and alginate–thiol revealed similar behaviour for the two polymers (Figure 14.7). Both curves exhibit one endothermic peak at around 90 °C (92 °C for alginate and 89 °C for alginate–thiol) followed by a second exothermic peak located around 255 °C (258 °C for alginate and 253 °C for alginate–thiol). Similar characteristic peaks at 103 °C and 260 °C were previously attributed to water dehydration and alginate exothermic decomposition, respectively [56]. These results are in line with the TGA analysis, where dehydration was observed at temperature of up to 200 °C and the first decomposition step was detected at around 250 °C for both samples, with the alginate decomposition temperature being slightly higher than that of alginate–thiol. It is noted that DSC analysis was limited to temperatures of up to 300 °C due to technical limitations; therefore, the second decomposition step could not be detected.

The DSC curve of alginate-PEGAc displays a decomposition peak at 256 °C. It is located in a similar temperature range to that of alginate–thiol and alginate. However, its intensity is reduced, presumably due to the large molecular weight of the grafted PEG that reduces the alginate weight percentage in the sample. A second exothermic peak appears at 62 °C, which can be attributed to the PEG melting temperature in accordance with previously reported values of 59 [61], 67 [62], 60 [58] and 55 °C [48], depending on the details of the experimental protocol (heating rate, type of gas used and gas flow rate) [56]. The comparison of the thermograms obtained for the native alginate, PEG-DA and their conjugation product, suggests that the characteristic decomposition behaviour of alginate in the DSC temperature range is maintained after PEGAc grafting. The peak broadening and the small shoulder in the endothermic peak of alginate-PEGAc sample may result from dehydration that occurs in the same temperature range.

In conclusion, a good correlation between the TGA and DSC results was observed for alginate, alginate–thiol and alginate-PEGAc. Moreover, the results show that the conjugation of PEG chains to the alginate backbone influences its thermal behaviour and increases its hydrophilicity. However, this modification does not decrease the thermal stability in a temperature range below about 400 °C.

14.5.4 Gelation of Acrylated Alginate

Alginate-PEGAc could be expected to undergo cross-linking (gelation) by two independent mechanisms: physical gelation though the alginate backbone and chemical gelation involving the acrylated end groups carried by the PEG side chains. The cross-linking schemes can potentially be used to cross-link the polymer chains in different ways, thus manipulating its characteristics and altering its properties.

Alginate’s G and M monomers are organized in a block-wise pattern of homopolymeric regions of M and G interspersed with regions of an alternating structure of MG blocks [63]. Many of the physical properties of alginate depend on the proportion and distribution of these segments and their relative sequencing [64]. It is believed that the G units are responsible for stiff chain characteristics while the M units form a flexible chain structure, due to the differences in monomer conformation which was found to be and chair conformation for the guluronate and mannuronate residues, respectively. Thus, the overall conformation of the chain backbone is assumed to be a combination of stiff G blocks connected by flexible M blocks. Small angle X-ray scattering (SAXS) from such chains was previously described in terms of the ‘broken rod linked with flexible chain’ model [40,65]

(14.1)

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