# In Situ Transformation of Electrospun Nanofibers into Nanofiber-Reinforced Hydrogels
## 2 Materials and Methods
### Electrospinning of Nanofiber Scaffolds  
Different gelatin-containing samples were produced by uniaxial and coaxial electrospinning at 25 \({}^{\circ}\)C and 45% relative humidity. The electrospinning conditions were selected according to the stability of the electrospinning process and are summarized in Table 1. The uniaxial samples were a PDA-PEI-crosslinked gelatin sample (GPP) and an un-crosslinked gelatin sample (G) as a reference. The coaxial samples both contained zein in the core and PDA-PEI-crosslinked gelatin in the shell, either without (zeinPEO-GPP) or with T in the core (zeinPEO-GPP) (Figure 1).  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Sample**} & \multirow{2}{*}{**Core**} & \multirow{2}{*}{**Shell**} & **Flow Rate** & **Flow Rate** & **Injector** & **Collector** & **Distance**, \\  & & & **Core**, \#L h\({}^{-1}\) & **Shell**, \#L h\({}^{-1}\) & **Voltage**, \#V & **Voltage**, \#V & **cm** \\ \hline G & Gelatin & - & 250 & - & 8 & \(-1\) & 12 \\ \hline GPP & Gelatin, PEI, PDA & - & 250 & - & 9 & 0 & 12 \\ \hline zeinPEO-GPP & Zein, PEO & Gelatin, PEI, PDA & 400 & 400 & 9 & \(-7.5\) & 21.5 \\ \hline zeinPEOT-GPP & Zein, PEO, T & Gelatin, PEI, PDA & 400 & 400 & 9 & \(-6.5\) & 21.5 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Sample description and electrospinning.  
Figure 1: Different types of uniaxial and coaxial samples that were electrospun in the scope of this study (top four images) and concept of the in situ transformation of the nanofiber scaffold into a nanofiber-reinforced hydrogel (bottom image). Key: G, sample containing gelatin; GPP, sample containing gelatin crosslinked by polydopamine and polyethyleneimine; zein PEO-GPP, sample containing zein and polyethylene oxide in the core and gelatin crosslinked by polydopamine and polyethyleneimine in the shell; zeinPEOT-GPP, sample containing zein, polyethylene oxide and tetracycline hydrochloride in the core and gelatin crosslinked by polydopamine and polyethyleneimine in the shell.  
For uniaxial electrospinningning of GPP, 40% (\(w/v\)) gelatin was dissolved in 60% (\(v/v\)) aqueous AA in a sonication bath at 80 \({}^{\circ}\)C for 1 h and then continuously stirred for 1 h at 80 \({}^{\circ}\)C. After the solution cooled down to RT, 5% (\(w/w\) of gelatin) PEI and 2% (\(w/w\) of gelatin) dopamine HCl were added and allowed to mix overnight under shaking at 150 rpm at 25 \({}^{\circ}\)C. The control sample G contained pristine gelatin without PEI, dopamine HCl and T. For coaxial electrospinningning (samples zeinPEO-GPP and zeinPEOT-GPP), 20% (\(w/v\)) of zein and 5% (\(w/w\) of zein) PEO were used as a core solution using 80% (\(v/v\)) aqueous ethanol as a solvent. Zein was added 2 h prior to electrospinningning and dissolved at 60 \({}^{\circ}\)C. The composition of GPP from the uniaxial set-up was used as a shell solution. For drug-loaded fibers, 5% (\(w/w\) of zein) T was added to the core solution and mixed for 10 min.  
After electrospinningning, all samples except for G were crosslinked with ammonium carbonate vapors in a sealed desiccator with 5 g of ammonium carbonate for 48 h. All prepared fiber mats were stored at 0% relative humidity and RT and were sterilized with UV light at 254 nm for 2 h on each side prior to further analysis.