Infinite m1000

Caspase activity was measured by the luminescent Caspase-Glo^® 3/7 Assay (Promega, Madison, Wisconsin, United States). This homogeneous, luminescent assay provided a luminogenic caspase-3/7 substrate, which contained the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity, luciferase activity, and cell lysis. The protocol was performed according to the manufacturer’s guidelines. In short, equal amounts of Caspase-Glo^® 3/7 reagent and PBS were added to the cells upon removal of the medium. Cells and buffer were mixed for 30 s using a plate shaker at 400 rpm (Infinite M1000, Tecan) and incubated for 60 min at RT. Luminescence was measured with a microplate reader (Infinite M1000, Tecan) and normalized for statistical analysis.

Purified preparations of Luc2, CBR, and CBR2 were diluted to 50 µg mL⁻¹ in TBS pH 7.5 + 0.01% BSA and then 10-fold serial dilutions were prepared in TBS. Substrates were diluted into assay buffer (150 mM HEPES (pH 7.5), 1 mM CDTA, 16 mM MgSO₄, and 1% NP-9) (0.5 mM D-LH2, 0.02 mM NH₂-NpLH2, or 0.04 mM OH-NpLH2) containing 10 mM ATP. In triplicate, 50 µL of substrate dilution and 50 µL of enzyme dilution were combined and immediately measured on a Tecan Infinite® M1000 plate reader set to spectral scanning mode with 2 nm intervals (500‒850 nm). The following enzyme dilutions were used for D-LH2: 0.05 µg mL⁻¹ Luc2 or 0.5 µg mL⁻¹ CBR/CBR2. The following dilutions were used for NH₂-NpLH2 and OH-NpLH2: 50 µg mL⁻¹ for Luc2 or 5 µg mL⁻¹ CBR/CBR2.
Spectral measurements on live cells were carried out as follows: cells were transfected with Luc2 and CBR2opt as described in the “Transfections” section below. Substrates were pre-heated to 37 °C and then 30 μL of 100 mM D-LH2 and 30 μL of 4.5 mM NH₂-NpLH2 were each added to three wells of Luc2 and CBR2opt-transfected cells. The plate was manually shaken and immediately transferred to a Tecan Infinite® M1000 plate reader heated to 37 °C. The plate was incubated for 5 min and then measured in spectral scanning mode with 3 nm intervals (500‒850 nm).

Confocal images and fluorescence recovery after photobleaching (FRAP) experiments were conducted with a Zeiss LSM 700 confocal laser scanning microscope (Zeiss) equipped with a 63x oil immersion objective. Confocal images of isolated patterns were used to quantify the surface concentration of transferred BMP-2^CF thanks to a calibration curve obtained by UV-visible spectrometry using a microplate reader (Infinite M1000, Tecan) on PEM films homogeneously loaded with BMP-240 (link). Fluorescence recovery after photobleaching (FRAP) experiments were conducted to evaluate the possible diffusion of BMP-2^CF within (PLL/HA) films. To this end, a 15 μm diameter circular region of interest (ROI) was bleached using the 488 nm laser diode and the recovery after photobleaching was followed over time. The fluorescence intensity of the ROI was normalized to that of a control region. The release of BMP-2 from the patterns was evaluated by measuring the amount of BMP-2^CF in the medium every 10 min over 4 h at 37 °C with a UV–visible spectrometer (Infinite M1000, Tecan).
AFM images were obtained in PBS in peak force tapping mode using an AFM BioCatalyst instrument (Bruker). Pyramidal silicon nitride cantilevers (MSNL probes, Bruker) with a spring constant of 0.07 N m⁻¹ were used. The analysis of the topography of 5 patterns per condition was performed using Nanoscope analysis (Bruker).

The binding efficiency of PPNs to a fluorescently labelled negative control miRNA (NC-Alexa647, 1.6 µg miRNA/10⁹ particles/mL) was determined by measuring free, unbound miRNA in an aqueous solution with a TECAN Infinite M1000 microplate reader [25 (link)]. The fluorescence intensities of serially diluted NC-Alex647 working stocks were measured with a TECAN Infinite M1000 microplate reader prior to PPN conjugation. After the conjugation, PPN-miRNAs were centrifugated at 21,000× g for 5 min to pellet the PPN-miRNA complex. The fluorescence intensity of unbound miRNA was measured in the supernatant at EX/EM: 650/665–671 nm. In the release study, the PPN-miRNA complex was washed after conjugation and resuspended in nuclease-free water or sodium citrate buffer (pH 7.2, to stimulate physiological, cytosolic pH) with or without foetal bovine serum (FBS). At pre-determined time points (0, 5, 10, 30, 60, 90, and 120 min), samples were centrifugated at 21,000× g for 5 min. The fluorescence signal from collected supernatants were measured by a TECAN microplate reader.