# Crosslinked Bifunctional Particles for the Removal of Bilirubin in Hyperbilirubin Cases
## 3 Results
### Bilirubin Removal Tests  
In order to assess the potential application of these materials as an alternative treatment for hyperbilirubinemia, in vitro tests were conducted to evaluate their capacity to remove bilirubin in two different media (PBS and human plasma). The kinetics of bilirubin removal onto non-wetted St-DVB particles with or without BSA were determined (Figure 5). The best bilirubin uptake results were found for the particles functionalized with GMA and PEGMA, with 0.27 and 0.52 mg BR/g particles for the cases without and with BSA, respectively. In the case of particles without BSA, particles without functionalization and functionalized with PEGMA removed about 0.15 mg BR/g particles. In the case of particles functionalized with GMA, the result was only 0.05 mg BR/g particles, improving a little by the incorporation of BSA, up to 0.22 mg BR/g particles. In the case of particles functionalized with PEGMA or non-functionalized and containing BSA, no bilirubin was retained in the polymers. In order to improve the results, the particles were pre-wetted in ethyl lactate and tested with and without BSA for bilirubin removal from PBS media. The progress of bilirubin  
Figure 4: Kinetics of BSA immobilization in St-DVB pre-wetted particles. Wp = 2.5 g; T = 25 \({}^{\circ}\)C; t = 72 h and Fr = 8 mL/min. Figure 5: Kinetics of BR removal by non-pre-wetted particles without (**a**) and with BSA (**b**). Experiments without BSA were carried out with 7 g of particles and the experiments with BSA were carried out with 12 g of particles. T = 23 \({}^{\circ}\)C and Fr = 120 mL/min. removal over time was recorded and is presented in Figure 6, while the final bilirubin uptake values are summarized in Table 3.  
According to these results, the particles with BSA showed a faster kinetic for removing bilirubin since, while particles without BSA needed 90 min or more to reach the equilibrium, the particles with BSA needed less than one hour to reach the end point. Besides, the final BR uptake was up to 43% higher when BSA was attached (Table 3). In all cases, the incorporation of PEGMA and GMA improved the capacity for BR removal, but this effect is higher in the case of particles without BSA. Thus, the BR removal is affected in the presence of BSA but also by the PEGMA and GMA functionalization, probably because they form branches that facilitate the link with the BR. Finally, the ability of the particles to remove bilirubin from plasma was investigated. The initial concentration of bilirubin was set at 7.5 mg/dL, which is commonly found in patients with hepatic failure. It should be noted that, unlike in PBS, human plasma contains not only free bilirubin, but also conjugated or direct bilirubin (dBR) and non-conjugated or indirect bilirubin (iBR), resulting in a higher total initial bilirubin concentration in human blood. St-DVB-GMA-PEGMA particles previously wetted in ethyl lactate were chosen due to their superior BR uptake from PBS. To confirm the importance of albumin immobilization in BR removal, experiments were also conducted with wetted St-MMA-GMA-PEGMA without BSA under the same conditions. According to the results in Figure 7a, particles that did not contain BSA were not effective in removing bilirubin from plasma. On the contrary, Figure 7b shows the results of two experiments with the same initial bilirubin concentration, with the particles removing an average of 8.5 mg of bilirubin per gram of particles in less than 30 min. The amount of indirect bilirubin removed in each experiment was 8.3 and 8.75 mg BR/g of particles, respectively. Thus, the presence of albumin provided selectivity, preventing the particles from St-MMA-GMA-PEGMA becoming saturated with other blood components or metabolites. Figure 6: Kinetics of BR removal in St-DVB pre-wetted particles without (**a**) and with BSA (**b**). Experiments without BSA were carried out with 2 g of particles and the experiments with BSA were carried out with 1.5 g of particles. T = 25 \({}^{\circ}\)C and Fr = 120 mL/min.