Ultra centrifugal filters

For EE, 22 NPs were ultra-filtrated by Milipore Ultra centrifugal filters (NMWL = 10 kDa). The filtrate was used for determination. For DLC, 22 NPs were treated with vacuum freeze-drying, and weighed. The EE (%) and DLC (%) for 22 NPs were detected via high performance liquid chromatography (HPLC, Agilent Technologies 1260 Infinity, Germany) by analyzing COP-22. For HPLC detection, methanol/H₂O (90/10, v/v) was used as the mobile phase at the flow rate of 1 mL/min. The standard curves were determined using 20 μL of 20, 15, 10, and 5 μM COP-22 in methanol/H₂O (1/1, v/v) solution (Figure 13). (COP-22, 3.812 min, 310.4 nm, S (mAU∗S) = 5.414 + 21.8824 C (μM)).
EE (%) and DLC (%) were calculated as follows.

EE (%) = (Total amount of the bound drug in the NPs)/(Total feed amount of the drug) × 100%

= (Total feed amount of the drug—the amount of free drug)/(Total feed amount of the drug) × 100%

DLC (%)

= (Total amount of the bound drug in the NPs)/(Total weight of the NPs) × 100%

= (Total feed amount of the drug—the amount of free drug)/(Total weight of the NPs) × 100%.

pET-28a was used as the vector for His-tagged protein expression with E. coli BL21(DE3) as the host strain. Expression of the protein was induced with 1 mM isopropyl-d-thiogalactopyranoside (IPTG) at 28°C for 8 h. According to the manufacturer’s instructions, His-tagged proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) columns (GE Healthcare). Ultra centrifugal filters (Millipore) were used to desalt the purified protein in phosphate-buffered saline (PBS) buffer. The quality and amount of isolated proteins were evaluated by using SDS-PAGE and a Micro BCA protein assay kit (Cwbiotech), respectively. The purified protein was then kept at 80°C for storage.

Plasma protein binding (PPB) of NQ (0.1 and 1.0 μg/mL) was determined in pooled plasma collected from female or male mice, rat or human, using the ultrafiltration method. Briefly, stock solution of NQ was diluted with blank plasma to achieve the test concentrations. Incubations were performed in a shaking water bath at 37 °C for 1 h to allow equilibration. Plasma samples were loaded into the Ultra centrifugal filters (Millipore, USA) with 10 kDa molecular weight cutoff, and the filtrate was centrifuged at 6000 rpm for 20 min at 37 °C. Phosphate buffered saline (PBS) was used to test non-specific binding (NSB). The NSB was calculated according to the equation: $$\text{NSB}=\left({\text{C}}_{\text{BD}}-{\text{C}}_{\text{BF}}\right)/{\text{C}}_{\text{BD}},$$ where C_BD was the total drug concentration in PBS before centrifugation and C_BF was the drug concentration in the PBS filtrate after centrifugation. The PPB was calculated based on the equation: $$\text{PPB}\%=100\times \left(1-{\text{C}}_{\text{SF}}/\left[\left(1-\text{NSB}\right)\times {\text{C}}_{\text{SD}}\right]\right),$$ where C_SF was the NQ concentration in the plasma ultrafiltrate and C_SD was the nominal plasma concentration. All drug concentrations were determined by LC–MS/MS.

Loading of the PCP containing proteins (PCP or PCP-X) with peptidyl-CoA substrates (T7P(d/l-Hpg7)-CoA, T7P(d-Hpg7)-CoA, T7P(l-Hpg7)-CoA) was catalysed by an engineered phosphopantetheinyl transferase from B. subtilis (Sfp R4-4)36 (link). PCP proteins (60 μM) were incubated with a 3-fold molar excess of peptidyl-CoA and 6 μM Sfp in PCP-loading buffer (50 mM Hepes pH 7.0, 50 mM NaCl, 10 mM MgCl₂) for 1 h at 30 °C. Following the loading reaction, the excess of free peptidyl-CoA was removed from the loading reaction by a concentration dilution (4 × 1:5 dilution) procedure using 50 mM Hepes pH 7.0, 50 mM NaCl low salt buffer (0.5 mL Ultracentrifugal filters, 10,000 MWCO, Merck Millipore). The generated peptidyl-PCP constructs were used immediately after the PCP-loading reaction as substrates for the P450 activity assays.

Purified from the E. coli-based expression system, recombinant cathD is prone to aggregate into insoluble inclusion bodies. Therefore, we applied a mammalian expression system to produce recombinant proteins from transient or stably transfected HEK293T cells. Cells were collected and disrupted in 5 mM imdazole, 500 mM NaCl, and 20 mM Tris-HCl (pH 7.9) by sonication. His-tagged cofilin, cathD and its mutated proteins were subsequently purified under native conditions by standard affinity chromatography procedures using a purification resin (Roche Applied Science). Eluted proteins were desalted and concentrated twice by sequential centrifugation using 100-kDa and 50-kDa ultra centrifugal filters (Merck). The molecular weights of purified proteins were checked by silver staining following SDS-PAGE separation for quality control. The protein stability and the secondary structure of mutated cathD proteins were evaluated by a circular dichroism spectroscopy. Purified cathD was mainly in the 48-kDa immature form and stored at −80 °C in 150 mM NaCl, pH 7.4, 0.05% sodium azide and 25% glycerol.

All the chemicals were purchased from commercial sources and were either analytical grade or molecular grade. The growth media components were obtained from BD Difco (Franklin Lakes, NJ, USA) and Himedia (Mumbai, India). The amino acids, glutathione (GSH) (reduced), β‐nicotinamide adenine dinucleotide phosphate sodium salt hydrate (NADP), glucose 6‐phosphate (G6P), mevalonolactone and 6‐phosphogluconic dehydrogenase from yeast were obtained from Merck (Darmstadt, Germany). Zymolase‐20T was obtained from MP biomedicals (USA). Ultra centrifugal filters were obtained from Merck (Burlington, MA, USA). Oligonucleotides were obtained from Integrated DNA Technologies (IDT) and Merck (Bangalore, India). Vent DNA polymerase and restriction enzymes were obtained from New England Biolabs (Ipswich, MA, USA). Plasmid miniprep and gel/PCR clean‐up kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The NADPH kit was obtained from Promega (Madison, WI, USA), nickel‐nitrilotriacetic acid agarose (Ni‐NTA), and polypropylene columns were obtained from Qiagen (Hilden, Germany).