INTRODUCTION Photocrosslinkable hydrogels are being investigated for a number of biomedical applications, since they can be delivered in a minimally invasive manner (e.g. by injection) and then crosslinked in situ. In this work, photoreactive hydroxyethylcellulose (HEC) derivatives were synthesized by functionalization with cinnamate moieties, which are known to form dimers when UV exposed . The aim was to evaluate whether aqueous solutions of these HEC derivatives could photo-crosslink upon UV exposure. The resulting hydrogels might be extremely advantageous compared to non-crosslinked fillers currently used (e.g. hyaluronic acid) because of their higher stability in the physiological environment, as well as because of the absence of any additional chemicals (e.g. photoinitiator), which would be detrimental for biocompatibility. EXPERIMENTAL METHODS Cinnamated HEC (cHEC) was synthesized in toluene/water/ethanol mixtures of cinnamoyl chloride (CC) and HEC (Sigma-Aldrich), adapting a protocol described in the literature . Several parameters were varied in order to assess the extent and kinetic of chemical functionalization (Table 1). Fourier Transform Raman spectroscopy (FT-Raman), UV/Visible spectrophotometry and solubility tests in water (1% w/v) were used to evaluate the efficacy of the functionalizations, using unmodified HEC as a control. The cytotoxicity of the novel cellulose derivatives was assessed by evaluating the viability and adhesion of human HeLa cells when cultured either in direct or indirect contact with the derivatives, also comparing the results with those obtained for methacrylated HEC (mHEC). The latter, which requires the use of a photoinitiator to be crosslinked, was synthesized for the sake of comparison, according to a well established protocol for similar polysaccharides . The synthesis of hydrogels from aqueous solutions of cHEC (2% and 4% w/v) was then attempted by means of exposure to UV light (UV lamp with peak of emission at 365 nm), and compared with the synthesis of hydrogels made of mHEC, with the same polymer content. RESULTS AND DISCUSSION As confirmed by FT-Raman spectra, the synthesis of cHEC was performed successfully when using triethylamine (TEA) as a catalyst (Table 1). In addition to the peaks characteristic of cinnamate moieties, the spectra highlighted the presence of the ester linkage at 1700 cm-1, which is representative of the chemical bond between cellulose and cynnamate groups. In some cases, a signal due to cinnamic acid dimerization was detectable. The UV/Visible spectra of cHEC displayed a strong UV absorption at about 290-300 nm. As expected, grafting of CC to HEC reduced or inhibited the water solubility of the cellulose derivative; however, one formulation, among those prepared, was found to be water soluble and thus of interest for the synthesis of hydrogels (table 1 bold row). The cytotoxicity tests carried out on this formulation and on mHEC (both of which freeze dried, from a 2% w/v solution) revealed that both derivatives are not cytotoxic, as found for unmodified HEC. Whereas hydrogels based on mHEC could be formed by exposure of solutions to UV light for 5 min (using Irgacure 1173 as a photoiniator), solutions with the same percentages of cHEC, in the absence of any photoinitiator, did not crosslink upon UV exposure, for times of exposure up to 90 minutes. Since the reactivity of cinnamate groups was detected in UV/Visible spectra as well as in some FT-Raman spectra (i.e. cinnamic acid dimers), the lack of crosslinking in water solution could be ascribed to the use of either low cellulose concentrations or unsuitable UV wavelengths. Table 1. Functionalization of HEC by means of cinnamate CONCLUSION The results showed that photoreactive and cytocompatible cellulose derivatives can be synthesized by either cinnamate or methacrylate functionalization. Water-soluble cinnamated hydroxyethylcellulose (cHEC) shows potential for the synthesis of hydrogels upon UV exposure, starting from a photoinitiator-free aqueous solution. Although photocrosslinking was not achieved using the working parameters adopted in this work, the synthesis of cHEC hydrogels appears feasible, if properly tuning the cellulose concentration in solution and the emission band of the UV lamp.
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