# Dual Stimuli-Responsive Polysaccharide Hydrogels Manufactured by Radiation Technique
## 2 Materials and Methods  
The modified polysaccharides used in this research were carboxymethylcellulose (CMC) of Mw = 250 kDa and a degree of substitution 1.2; hydroxypropylcellulose (HPC), Mw = 100 kDa, and a degree of substitution 1.4; the macromer of poly(ethylene glycol) diacrylate (PEGDA) was of Mn = 700 g/mol; all three components were purchased from Sigma-Aldrich (Germany); 1 M hydrochloric acid, 1 M perchloric acid, and 1 M sodium hydroxide were obtained from POCH (Poland). All compounds were used as received. The hydrogels of HPC and/or CMC were manufactured by radiation method. In order to obtain highly concentrated aqueous solutions of polysaccharides, the weighed amount of the polymer/s was mixed with water, or with PEGDA aqueous solution of a predetermined concentration and left for dissolution and homogenization for two days, with occasional mixing. Alternatively, viscous solutions, which were polysaccharide/s of lower concentrations (2.5 or 5% wt/vol) after mixing with water or PEGDA aqueous solution, were conditioned overnight. If the low pH of the polymeric solution was required, it was prepared by the dissolution of polysaccharide/s in an aqueous solution of perchloric acid of a predetermined concentration to end up with pH 2 of the resulting mixture. The samples for irradiation were heat-sealed in polyethylene bags, impermeable to air, and assured a uniform specimen thickness of ca. 1 mm, which was essential for swelling experiments. The specimens were exposed to an electron beam (EB) from the ELU-6 linear accelerator of 6 MeV energy. The actual absorbed dose was monitored by B3 WINdose film type dosimeter (GEX Corporation, USA). Applied doses were in the range of 5-100 kGy, and the dose rate was 5 kGy-min\({}^{-1}\)\(\pm\) 10%. As the polymers irradiated in aqueous solution undergo simultaneous crosslinking and degradation, the polymers comprising the resulting network do not constitute 100% of the polymers used for the preparation of the initial mixture that was subjected for irradiation. A part of the polymer is soluble (sol) and subsequently removed upon extraction. Therefore, the classical method of sol-gel analysis [3] was employed to assess (a) the gel fraction (GF (%)--the crosslinked part, Equation (1)) defined as a dry mass of the sample part that is insoluble upon autoclaving (\(m_{\mathrm{g}}\)) divided per mass of polymer/s (and PEGDA if used) in the initial mixture (\(m_{\mathrm{p}}\)); and (b) the equilibrium degree of swelling (EDS (g\({}_{\mathrm{j}}\)/g\({}_{\mathrm{dry\;gel}}\)), Equation (2)), defined as a ratio of the mass of absorbed water (\(m_{\mathrm{w}}\)) over the dried mass of the insoluble part (the aforementioned gel mass, \(m_{\mathrm{g}}\)). \[\mathrm{GF}=m_{\mathrm{g}}/m_{\mathrm{p}}\,\times 100\% \tag{1}\]  
\[\mathrm{EDS}=m_{\mathrm{w}}/m_{\mathrm{g}} \tag{2}\]  
The extraction of sol was accomplished by immersing the sample in water (which was regularly exchanged) for a prolonged amount of time--typically up to 7 days, until the hydrogel attained its EDS--and subsequent autoclaving (121 \({}^{\circ}\)C, 2 atm, 15 min). The HPC-CMC hydrogels were investigated in terms of thermo- and pH-responsive swelling. Initially, the gel was swollen for one week in water to remove all soluble partsnon-crosslinked and degraded chains (sol)--and attained EDS, a routine which was applied for all experiments. Then, its response to the temperature was examined--the gel was placed in a water bath at a particular temperature. It was subsequently weighed after 10 min of conditioning and transferred to water with a 5 degree higher temperature, etc. After reaching the highest temperature of 55 \({}^{\circ}\)C, the gel was placed in the room-temperature water and conditioned there with occasional weighing, up to 7 days, to record the reswelling. Similarly, the pH response experiment involved the media of various pH, adjusted with HCl and NaOH, to which the gel was consecutively transferred and conditioned 10 min prior to weighing.