# Automatic Detoxification Medicine Delivery by Thermo-Sensitive Poly(ethylene glycol)-Based Nanogels
## 2 Materials and Methods
### Drug Loading and Release  
Three detoxification medicines, sinomenine (SIN), diltiazem (DIZ) and chlorpromazine (CPZ), were loaded into the PS@P(MEO\({}_{2}\)MA-\(co\)-MEO\({}_{5}\)MA)-2 nanogels by complexation method. The following steps take SIN as an example. Firstly, 5 mL of the PS@P(MEO\({}_{2}\)MA-\(co\)-MEO\({}_{5}\)MA)-2 nanogel dispersion was stirred in an ice-water bath for 30 min to allow the nanogels to fully swell. Subsequently, 5 mL of 2 mg mL\({}^{-1}\) fresh SIN solution was added to the nanogel dispersion drop by drop. After stirring for 1 day, the dispersion was centrifuged at 10,000 rpm min\({}^{-1}\) for 15 min. The upper solution was collected and the precipitate was redispersed in 5 mL of buffer solution (pH = 7.4). The sample was further purified by repeated centrifugation/washing steps at least three times. All the upper clear solution from each centrifugation process was combined. The concentration of free SIN in the upper clear solution was determined by UV-vis spectrometry at 263 nm, based on the linear calibration curve (R\({}^{2}\) > 0.99) which was measured using the SIN solutions with known concentrations under the same conditions. The amount of SIN loaded into the PS@P(MEO\({}_{2}\)MA-\(co\)-MEO\({}_{5}\)MA)-2 nanogels was calculated from the decrease in SIN concentration. The encapsulation efficiency of the PS@P(MEO\({}_{2}\)MA-\(co\)-MEO\({}_{5}\)MA)-2 nanogels was calculated as follows:  
\[\text{SIN encapsulation efficiency }(\%)=\frac{\text{W}(\text{feeding SIN})-\text{W}( \text{free SIN})}{\text{W}(\text{feeding SIN})}\times 100\%\]  
The in vitro release test of the SIN-loaded PS@P(MEO\({}_{2}\)MA-\(co\)-MEO\({}_{5}\)MA)-2 nanogels was evaluated by the dialysis method. First, the purified SIN-loaded PS@P(MEO\({}_{2}\)MA-\(co\)-MEO\({}_{5}\)MA)-2 solid was redispersed in deionized water (5 mL). Then, 1 mL of the SIN-loaded PS@P(MEO\({}_{2}\)MA-\(co\)-MEO\({}_{5}\)MA)-2 dispersion was transferred into a Spectra/Por molecularporous membrane tube (molecular weight cutoff of 12,000-14,000) for dialysis. Subsequently, the dialysis bag was immersed into 50 mL of phosphate buffer solution (0.005 M) at both 22 \({}^{\circ}\)C and 38 \({}^{\circ}\)C. At defined time intervals, the SIN released outside of the dialysis bag was sampled and assayed at 263 nm using UV-vis absorption. The total percentage of SIN released outside the dialysis bag was expressed as a cumulative release. Blank release experiments, which contained a free SIN solution with the same amount of SIN loaded into the PS@P(MEO\({}_{2}\)MA-\(co\)-MEO\({}_{5}\)MA) nanogels, were carried out under the same conditions at 38 \({}^{\circ}\)C.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Sample** & **MEO\({}_{2}\)MA** & **MEO\({}_{5}\)MA** & **AAPH** & **PEGDMA** \\  & **(mmoL)** & **(mmoL)** & **(mmoL)** & **(mmoL)** \\ \hline PS@P(MEO\({}_{2}\)MA-\(co\)-MEO\({}_{5}\)MA)-1 & 0.25 & 0.75 & 0.02 & 0.75 \(\times\) 10\({}^{-2}\) \\ PS@P(MEO\({}_{2}\)MA-\(co\)-MEO\({}_{5}\)MA)-2 & 0.50 & 1.50 & 0.02 & 1.51 \(\times\) 10\({}^{-2}\) \\ PS@P(MEO\({}_{2}\)MA-\(co\)-MEO\({}_{5}\)MA)-3 & 0.75 & 2.25 & 0.02 & 2.06 \(\times\) 10\({}^{-2}\) \\ \hline \hline \end{tabular}
\end{table}
Table 1: Recipes used to prepare PS@P(MEO\({}_{2}\)MA-\(co\)-MEO\({}_{5}\)MA) nanogels with the same \(n\)(MEO\({}_{2}\)MA)/\(n\)(MEO\({}_{5}\)MA) feeding molar ratio of 1:3 but different initial feeding amounts of MEO\({}_{2}\)MA and MEO\({}_{5}\)MA.  
The same loading and release methods were applied for DIZ and CPZ, except the tested maximum adsorption wavelengths were 236 and 306 nm for DIZ and CPZ, respectively.