*3.2. In vitro Performance of NPs*

Although clear influences of the polymer crystallinity and physical state on NP formulation and enzymatic degradation were found, other effects might come into play in the more complex environment of a cell. The PCL[BRP-187] particles were hence tested in human PMNL for bioactivity. PMNL are the most abundant leukocytes in the blood and

are a major source for FLAP-dependent 5-LO product biosynthesis, thus they are suitable cells for evaluation of various anti-LT agents. Note that FLAP as helper-protein of 5-LO has no enzyme activity that can be experimentally assessed, but instead assists 5-LO in LT formation by facilitating the access towards the substrate for the 5-LO enzyme. At first, the PCL[BRP-187] NPs were compared to the free drug for their influence on the cell viability of PMNL (cytotoxicity). No cytotoxic effects of the particles were found within a 5 h incubation as shown in the SI (Figure S2). These results were in agreement with previous studies that demonstrated PCL NPs to be biocompatible [40,41]. Considering their good biocompatibility, all PCL[BRP-187] particles as well as the free drug were studied for their efficiency to inhibit the drug target FLAP in PMNL and, thus, to prevent 5-LO product formation [42]. Therefore, a drug concentration of 0.3 µM was chosen for free and encapsulated BRP-187, which were investigated at different preincubation times (15 min, 1 h, 2 h and 5 h, respectively). As shown in Figure 4A, 5-LO product formation was clearly suppressed after 15 min of incubation with the PCL[BRP-187] particles to variant degrees, but essentially the particles performed as efficiently as the free drug. Apart from this, there was no significant difference between the different PCL[BRP-187] polymers at longer incubation time points (i.e., 1–5 h; Figure 4B and SI, Figure S3). More specifically, the NPs prepared with ε75-δ25 prevented the 5-LO product formation most after 15 min of incubation (Figure 4A). This observation also correlated with the fastest apparent degradation of the ε75-δ25 copolymer (Figure 3B), which might be promoted by its melting point of 42 ◦C, which is around the temperature of cell incubation (i.e., 37 ◦C). Karavelidis et al. reported that other polyesters with melting points around 37 ◦C exhibited a faster drug release [8]. It can be inferred that the rapid degradation of ε75-δ25 led to an accelerated release of the BRP-187, thereby considerably preventing the 5-LO product formation at early time points (Figure 2C,D). NPs formed from PCL with higher εCL fraction and hence higher Tm, as well as a higher degree of crystallinity, were less effective. As shown in Figure 2D, the 5-LO product formation was almost linearly dependent on the polymer crystallinity if only the semicrystalline materials are considered. The better performance of the NPs with lower crystallinity could be explained by two effects based on two different release mechanisms. Firstly, less crystalline materials with a larger fraction of amorphous domains enable a faster diffusion of the drug through the polymer matrix without barriers formed by crystalline domains [11,43]. Secondly, if the drug release is promoted through polymer degradation, these amorphous domains would most likely be more accessible for enzymes catalyzing the polyester hydrolysis [11]. *Polymers* **2021**, *13*, 2557 10 of 13 reported that other polyesters with melting points around 37 °C exhibited a faster drug release [8]. It can be inferred that the rapid degradation of ε75-δ25 led to an accelerated release of the BRP-187, thereby considerably preventing the 5-LO product formation at early time points (Figure 2C,D). NPs formed from PCL with higher εCL fraction and hence higher Tm, as well as a higher degree of crystallinity, were less effective. As shown in Figure 2D, the 5-LO product formation was almost linearly dependent on the polymer crystallinity if only the semicrystalline materials are considered. The better performance of the NPs with lower crystallinity could be explained by two effects based on two different release mechanisms. Firstly, less crystalline materials with a larger fraction of amorphous domains enable a faster diffusion of the drug through the polymer matrix without barriers formed by crystalline domains [11,43]. Secondly, if the drug release is promoted through polymer degradation, these amorphous domains would most likely be more accessible for enzymes catalyzing the polyester hydrolysis [11]. Based on the apparent enzymatic degradation of the PCL NPs, a burst release of the drug is conceivable considering the immediate decrease in the count rate of at least 5 to 10% of all PCL NPs (Figure 3B). The slightly higher efficiency of the NPs to inhibit 5-LO product formation in PMNL within 15 min supports this idea (Figure 4A). For the polymers with a degree of crystallinity below 10% and T<sup>g</sup> < 37 °C, namely ε61-δ39, ε55-δ45 and ε00-δ100, inhibition of 5-LO product formation is less apparent after 15 min of incubation (Figure 2D, black-circled data points). This is presumably because these polymers are molten and more viscous at 37 °C. As a consequence, they could delay the release of the drug and therefore hamper the drug action in the cells. Furthermore, the coating effect of PVA might reduce the influence of the crystallinity of NPs or their intracellular drug release. It is reported that increasing amounts of residual surfactant decrease the cellular uptake of the NPs [44,45]. This could explain why the PCL copolymers with a higher fraction of δCL containing more residual PVA (Figure 2B) were less efficient to inhibit 5-LO product formation after 15 min of incubation (Figure 2C, Figure 4A) when compared to the PCL copolymers with a higher εCL fraction containing less residual PVA. However, no correlation was observed between suppression of 5-LO product formation and the PVA content in the formulation, showing that the trend cannot be generalized (SI, Figure S4).

**Figure 4.** Measurement of 5-LO product formation as an indicator for the inhibition of the drug target FLAP by BRP-187 [36]. PMNL preincubated with DMSO, BRP-187 (0.3 µM), empty PCL particles (labeled as w/o) or PCL particles with BRP-187 (labeled with BRP-187; 0.3 µM respective BRP-187) for 15 min (**A**) or 5 h (**B**) at 37 °C. Values are given as 5-LO products as a percentage of control (DMSO) (n = 3). Statistical analysis was performed via one-way ANOVA and Tukeys multi comparison test with logarithmic transformed data (\* p<0.05). **Figure 4.** Measurement of 5-LO product formation as an indicator for the inhibition of the drug target FLAP by BRP-187 [36]. PMNL preincubated with DMSO, BRP-187 (0.3 µM), empty PCL particles (labeled as w/o) or PCL particles with BRP-187 (labeled with BRP-187; 0.3 µM respective BRP-187) for 15 min (**A**) or 5 h (**B**) at 37 ◦C. Values are given as 5-LO products as a percentage of control (DMSO) (n = 3). Statistical analysis was performed via one-way ANOVA and Tukeys multi comparison test with logarithmic trans-formed data (\* *p* < 0.05).

Based on the apparent enzymatic degradation of the PCL NPs, a burst release of the drug is conceivable considering the immediate decrease in the count rate of at least 5 to 10% of all PCL NPs (Figure 3B). The slightly higher efficiency of the NPs to inhibit 5-LO product formation in PMNL within 15 min supports this idea (Figure 4A). For the polymers with a degree of crystallinity below 10% and T<sup>g</sup> < 37 ◦C, namely ε61-δ39, ε55-δ45 and ε00-δ100, inhibition of 5-LO product formation is less apparent after 15 min of incubation (Figure 2D, black-circled data points). This is presumably because these polymers are molten and more viscous at 37 ◦C. As a consequence, they could delay the release of the drug and therefore hamper the drug action in the cells.

Furthermore, the coating effect of PVA might reduce the influence of the crystallinity of NPs or their intracellular drug release. It is reported that increasing amounts of residual surfactant decrease the cellular uptake of the NPs [44,45]. This could explain why the PCL copolymers with a higher fraction of δCL containing more residual PVA (Figure 2B) were less efficient to inhibit 5-LO product formation after 15 min of incubation (Figure 2C, Figure 4A) when compared to the PCL copolymers with a higher εCL fraction containing less residual PVA. However, no correlation was observed between suppression of 5-LO product formation and the PVA content in the formulation, showing that the trend cannot be generalized (SI, Figure S4).
