Microbalance

To determine egg survival, we counted the number of larvae hatching from the twenty (P. brassicae) or eight (P. rapae) eggs deposited on a plant. After 4 days, presence/absence of HR-like necrosis was scored as previously described by Griese et al. (2017 (link)). After 5 days, survival of eggs was noted by counting the number of hatched caterpillars. To assess larval performance and the impact of the plant’s response to previous egg deposition on larval performance, we divided the neonate larvae hatching from egg-deposited plants into two groups. Half of them were placed back onto the previously egg-infested plant (labeled ‘egg and feeding’, EF) (on the adaxial side of the leaf where they hatched), and the other half was transferred to an egg-free plant (labeled ‘feeding’, F) plant of the same species and placed onto the adaxial side of the leaf as well. Three and seven days after hatching, caterpillar weight was measured on a microbalance (accuracy = 1 µg; Sartorius AG, Göttingen, Germany). We weighed each caterpillar individually, and afterwards the caterpillars were transferred back to their original position, on EF or F plants. Every EF and F plant was considered one replicate.

To determine if the thermal properties are impacted by thiol addition, the glass transition temperature of both PEGDMA and PEGDMA-thiol hydrogels were analyzed using differential scanning calorimetry (DSC) (TA Instruments, New Castle, Delaware, USA). Between 8 and 12 mg of each sample was weighed using the Sartorious microbalance, heated at a rate of 20 °C/min from room temperature to 110 °C, cooled to –70 °C using the cooling compartment of a DSC machine and reheated to 200 °C at a rate of 5 °C/min. From the subsequent thermographs, it was possible to determine the glass transition temperature for each sample (n = 2). Volatiles were removed from the purging head with nitrogen at a rate of 30 mL/min. The calibration of the instrument was performed using indium as the standard.

Commercial CuO (CAS 1317-38-0) and ZnO (CAS 1314-13-2) NPs, with size <50 nm, were purchased from Sigma-Aldrich (Sigma-Aldrich, Milan, Italy). Standard Diesel Exhaust Particles (DEP) were from the National Institute of Standard and Technology (NIST) (SRM^®2975) (Sigma-Aldrich). Following preparation protocols already set up in our lab [19 (link),29 (link)], NPs and DEP were weighed in a micro-balance (Sartorius, Goettingen, Germany) in sterile condition, under a laminar flow hood, suspended in sterile ultrapure water, and sonicated in an ultrasonic bath (SONICA Soltec, Milano, Italy) for 10 min.
DEP (2 mg/mL) was sonicated with a probe-type sonicator until it reached energy 3 kJ/s (Bandelin Sonopuls, Berlin, Germany), in order to obtain a well-dispersed suspension of particles. Suspensions were stored at room temperature, while DEP was stored at −20 °C for a period no longer than 15 days. The mixtures were freshly prepared before the experiments using a subcytotoxic concentration of DEP (100 µg/mL) and increasing concentrations of CuO and ZnO NPs (10, 15, 20, 25 µg/mL). Mixtures were sonicated in the ultrasonic bath (SONICA Soltec) for 10 min. The mixtures are indicated as DEP + CuO and DEP + ZnO and were produced by mixing DEP at 100 µg/mL with metal oxide NPs to achieve the final concentrations of CuO and ZnO used for the single NPs.

Catheter segments loaded with NIC (2, 5 and 10% (w/w)) and non-loaded, with a length of 1 cm were weighted using a microbalance (Sartorius, Germany). NIC is a hydrophobic drug, and therefore is poorly soluble in aqueous solutions such as PBS^{33 (link)}. To perform an accurate quantification of released NIC, we supplemented PBS with Tween 80 in order to increase the solubility of NIC in PBS while increasing the saturation concentration of the drug^{34 (link)}. Then, each catheter segment was placed in 1 mL of phosphate buffered saline (PBS; ThermoFisher, USA) with 2% of Tween 80 (ThermoFisher, USA) and incubated at 37 °C with an agitation of 120 rpm (n = 3 per group). The buffer solution was exchanged for the fresh solution at every time point (1, 3, 4, 6 and 24 h, daily on days 2–10, and at 13, 16, 20 and 27 days). The aliquots were stored at − 20 °C for later use. The concentration of NIC released at every time point was calculated by measuring the absorbance at 340 nm of 300 µL of each aliquot in the flat bottom 96 well microtiter plates (Greiner Bio-One, USA) with a multi-well plate reader (Synergy H1, BioTek, USA). A calibration curve was plotted for NIC to estimate the concentration of drug released from the catheter segments. This calibration curve ranged from 1 to 50 µg/mL with R² equal to 0.9998.

Differential scanning calorimetry was conducted to determine whether there was a difference in thermal transitions following different polymerisation processes. Hydrogels to be tested were dried in a vacuum oven with 50 mBar of pressure at 80 °C overnight. Samples with a weight between 8 and 12 mg were weighed using a Sartorius microbalance, heated to 200 °C at a rate of 20 °C/min and cooled to −50 °C using the cooling compartment of a modulated differential scanning calorimetry (DSC) machine. Samples were then heated at a rate of 5 °C/min to 200 °C. Glass transition temperatures were calculated using the resulting thermographs (n = 2). The calibration of the instrument was carried out using indium any volatiles were purged using nitrogen gas at a rate of 30 mL/min.

The thermophysical behavior of specimens was studied with the Perkin-Elmer DSC8500 (PerkinElmer, Waltham, MA, USA) differential scanning calorimeter (DSC). The DSC cell was flushed with dry nitrogen at a flow rate of 20 mL per minute. An IntraCooler III mechanical refrigerator was used for cooling. The specimens were weighed on a Sartorius microbalance with an accuracy of ±0.01 mg. The heating rate was 20 °C per min.