Intracellular CO Release from Composite of Ferritin and Ruthenium Carbonyl Complexes

Abstract

Protein cages have been utilized as templates in the development of biomaterials. Here we report protein engineering of the ferritin (Fr) cage for encapsulating carbon monoxide releasing molecules (CORMs) and release of CO gas which serves as a cell signaling molecule. The protein cages enable us to increase the half-life for CO release, providing a release rate that is 18-fold slower than the rate of a typical CORM, Ru(CO)3Cl(glycinate) (CORM-3). Moreover, the uptake ratio of the composite is about 4-fold greater than that of CORM-3. We found that these effects enhance the activation of nuclear factor κB 10-fold higher than CORM-3. The protein cage of Fr thus provides the basis for new CORMs that can be used for in vitro cell research.

A Photoactive Carbon‐Monoxide‐Releasing Protein Cage for Dose‐Regulated Delivery in Living Cells

“…Them utant has two cysteine residues at positions 48 and 52 (C48, R52C), which are located close to each other on the accumulation center ( Figure 1b). [41] The residues are expected to coordinate to aMnCO moiety with S, S-bidentate ligation as reported for ap hotoactive Mn CORM. [53] Ac omposite of apo-R52C-rHLFrw ith photoactive MnCO moieties (MnCO·apo-R52 C-rHLFr 1)w as prepared by adding aM eOH solution of Mn(CO) 5 Br (50 equiv) to ab uffer solution of apo-R52C-rHLFr( 50 mm Tr is/HCl (pH 8.0), 0.15 m NaCl).…”

“…[28,56] Ap hosphate-buffered saline (PBS) solution (10 mm,pH7.4) containing deoxy-Mb (24 mm)with dithionite (10 mm)u nder Ar atmosphere was used for the assay as previously reported. [41] TheP BS solution containing deoxyMb was irradiated with visible light at 456 nm (15 mW cm À2 ) for defined periods after addition of 1 (final concentration of [Mn] = 6 mm,in10mm PBS buffer of pH 7.4). Theabsorption spectrum of the deoxy-Mb solution clearly changes to that of carbonmonoxy-Mb (MbCO) with light irradiation for 1-20 minutes ( Figure S3).…”

“…Theuptake ratio of 1 and CORM-1 into the cells are found as 0.35 %( 1.76 10 À10 mmol/cell) and 0.33 %( 1.64 10 À10 mmol/ cell), respectively,a fter incubation for 6h.N oc ytotoxic effects of 1 and CORM-1 on HEK293/kB-Fluc cells after 12 h incubation were observed according to the results of a3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay ( Figure S7). [41] Theeffects of CO released from 1 on NF-kBinHEK293/ kB-Fluc cells were evaluated using aluciferase reporter assay system. [41] Several NF-kB-based assay systems have been applied to evaluate cellular CO functions because it is believed that the nuclear factor is essential for processes against inflammation and apoptosis and for cell proliferation by CO. [25,51,52,57] TheH EK293/kB-Fluc cells were pre-incubated for 1h with 1 or CORM-1 (final concentration of [Mn] = 10 mm)b efore addition of tumor necrosis factora (TNF-a).…”

“…[41] Theeffects of CO released from 1 on NF-kBinHEK293/ kB-Fluc cells were evaluated using aluciferase reporter assay system. [41] Several NF-kB-based assay systems have been applied to evaluate cellular CO functions because it is believed that the nuclear factor is essential for processes against inflammation and apoptosis and for cell proliferation by CO. [25,51,52,57] TheH EK293/kB-Fluc cells were pre-incubated for 1h with 1 or CORM-1 (final concentration of [Mn] = 10 mm)b efore addition of tumor necrosis factora (TNF-a). Then, the cells were incubated for 12 ha t3 78 8C in 5% CO 2 .T he cells were irradiated by 456 nm light at six setup time points (0.5 hbefore and 0.5 h, 1h,3h, 6h,and 9h after the addition of TNF-a; Figure 4a and b).…”

Preparation of a Cross-Linked Porous Protein Crystal Containing Ru Carbonyl Complexes as a CO-Releasing Extracellular Scaffold

“…The results suggest that the activation is caused by (1) effective delivery of CO from Ru·CL-HEWL with cell surface adhesion and (2) slower release of CO from the interior space constructed in solvent channels of CL-HEWL as reported previously. 28,70 According to the literature, dozen micromolar levels of CORM induce activation of NF-κB in the presence of LPS, but this effect is accompanied by a decrease in Conversion of Mb to MbCO was monitored with a spectrophotometer. After calculation of the equivalents of CO/Ru, t 1/2 was determined by fitting to first-order kinetics.…”