Multi detection microplate reader

Hydrodynamic size and zeta potential of the nanoparticles were measured using the zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK). Each measurement was performed in triplicate at 25°C. The indirect encapsulation efficiency of siRNA-loaded NPs was determined using BLOCK-iT^®-loaded NPs and quantified at Ex555/Em595 using Multi-Detection Microplate Readers (Bio-Tek Instrument Inc., Winooski, VT, USA). The drug release studies of siRNA were performed using BLOCK-iT^®-encapsulated NPs at 37 °C. At each time point, samples were taken and calculated the released fraction of BLOCK-iT^® at Ex555/Em595 by using Multi-Detection Microplate Readers (Bio-Tek Instrument Inc., Winooski, VT, USA).

A cell counting kit-8 (CCK-8, Beyotime Institute of Biotechnology, China) was used to investigate the cytotoxicity of N₃–PEG₃–N₃ and Tetra-PEG-DBCO against 4T1 cells. Briefly, cells were seeded at a density of 1 × 10⁴ cells per well in a 96-well plate, and cultured overnight in RMPI 1640 (Hyclone, Thermo Scientific) supplemented with 10% fetal bovine serum (Hyclone, Thermo Scientific) and 1% penicillin & streptomycin (GIBICO, Invitrogen) at 37 °C in a humidified atmosphere containing 5% CO₂. Subsequently, 100 μl of fresh medium containing N₃–PEG₃–N₃ at different concentrations (100, 500, or 1000 μg/ml) or Tetra-PEG-DBCO at different concentrations (6000, 10000, or 60000 μg/ml) was added to replace the culture medium for 24 and 48 h, respectively. After incubation, the culture medium containing N₃–PEG₃–N₃ or Tetra-PEG-DBCO was removed, 100 μl of fresh medium and 10 μl of CCK-8 solution were added to each well. After incubation for 3 h, the OD value of cultures was recorded at 450 nm with the help of a Multi-Detection Microplate reader (BioTek, USA). The well with medium and CCK-8 solution but without cells, N₃–PEG₃–N₃, and Tetra-PEG-DBCO was used as a blank group. The well with medium, cells, and CCK-8 solution but without N₃–PEG₃–N₃ and Tetra-PEG-DBCO was employed as a control group. The experiment was replicated for four parallel samples.

Total ribonucleic acid (RNA) was extracted and purified from freshly isolated rat renal cortical tissues using a total RNA extraction kit (Amresco, Columbus, OH, USA). Briefly, rat renal cortical tissue was homogenized in extraction reagent as per the manufacturer’s protocol. The purity and integrity of total RNA were determined by a multidetection microplate reader (Biotek, Winooski, VT, USA). Subsequently, first-strand complementary deoxyribonucleic acid (cDNA) was synthesized using the iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). mRNA levels were determined quantitatively using the Sensi-FAST SYBR Lo-Rox Kit (Bioline, London, UK). Primer sets were used as previously published [13 (link)] and were purchased from Integrated DNA Technologies (Coralville, IA, USA). Gene expressions were normalized to the β-actin mRNA level and expressed as relative fold changes (RFC). qPCR amplification was performed in duplicate for each cDNA.