X-ray Structure of the Carboplatin-Loaded Apo-Ferritin Nanocage

Abstract

The second-generation Pt anticancer agent carboplatin (CBDCA) was encapsulated within the apo horse spleen ferritin (AFt) nanocage, and the X-ray structure of the drug-loaded protein was refined at 1.49 Å resolution. Two Pt binding sites, different from the one observed in the cisplatin-encapsulated AFt, were identified in Ft subunits by inspection of anomalous electron density maps at two wavelengths and difference Fourier electron density maps, which provide the necessary sensitivity to discriminate between Pt from CBDCA and Cd ions that are present in the crystallization conditions. Pt centers coordinate to the NE2 atom of His49 and to the NE2 atom of His132, both on the inner surface of the Ft nanocage.

Encapsulation of the Dinuclear Trithiolato‐Bridged Arene Ruthenium Complex Diruthenium‐1 in an Apoferritin Nanocage: Structure and Cytotoxicity

“…DiRu‐1‐encapsulated AFt was crystallized by the hanging‐drop vapor diffusion method at 298 K using 5 mg mL −1 protein adduct. Drops were prepared by mixing 1 μL of a protein solution with an equal amount of the precipitant solution (0.6–0.8 m (NH 4 ) 2 SO 4 , 0.1 m Tris⋅HCl pH 7.7, 50 m m CdSO 4 ), as described previously …”

“…Drops were prepared by mixing 1 mLo faprotein solution with an equal amount of the precipitant solution (0.6-0.8 m (NH 4 ) 2 SO 4 , 0.1 m Tris·HCl pH 7.7, 50 mm CdSO 4 ), as described previously. [6,14] X-ray diffraction data on these crystals were first collected at the CNR Institute of Biostructure and Bioimages, Naples, Italy,a nd then recollected at higher resolution at ESRF synchrotron in Grenoble, France. Data frames were indexed and scaled using HKL2000 [52] or AutoProc.…”

“…Apoferritin (AFt) has been widely used as ap rotein cage to entrapm etal-based drugs, as it can be reversibly dissociated and reassembledu pon pH variation.A dducts formedu pon encapsulation of drugs within AFt cages have the advantage of enhanced selectivity for tumor cells, [6] because H-and L-chains as well as H/Lheteropolymers can be recognized by surface receptors that are overexpressed by various tumor cell lines. [7,8] Recently,c isplatin, [9][10][11][12][13] carboplatin, [11][12][13][14] ab imetallic cytotoxic gold-platinum compound (4PF 6 ), [15] and cytotoxic gold(III) compounds, namely Auoxo3 ([Au 2 (bipy Me ) 2 (m-O) 2 ][PF 6 ] 2 ,w here bipy Me = 6-methyl-2,2'-bipyridine), [6] Auoxo4 ([Au 2 (bipy nP ) 2 (m-O) 2 ] [PF 6 ] 2 ,w here bipy nP = 6-neopentyl-2,2'-bipyridine) and Au 2 phen ([Au 2 (phen 2 Me ) 2 (m-O) 2 ][PF 6 ] 2 ,w here phen 2 Me = 2,9-dimethyl-1,10phenanthroline), [16] have been successfully encapsulatedw ithin the horse spleen AFt nanocage. X-ray diffraction analysis showst hat cisplatin and carboplatin bind the AFt chain close to the imidazole rings of His132, and His132a nd His49, respectively.…”

“…[13,14] On the other hand, gold compounds degrade upon encapsulation within the cage, and gold(I)i ons bind the side chains of Cys126, Cys48, His49, His132, and His147, with ap referencef or Cys126. [6][7][8][9][10][11][12][13][14][15][16] Incorporation of Auoxo3 inside the cage also leads to the formationo fg old nanoparticles. [6] These protein adducts are cytotoxic towardv arioush uman malignant cells (HeLa, MCF-7, and HepG2) and, at least in some cases, moderately selective.…”

“…[6] However, the inclusiono ft he compounds within the cage decreases their activity. [6][7][8][9][10][11][12][13][14][15][16] The effects of encapsulatingt he cytotoxic dinucleart rithiolatobridged arene ruthenium complex [(h 6 -p-MeC 6 H 4 iPr) 2 Ru 2 (m 2 -S-p-C 6 H 4 tBu) 3 ]Cl (DiRu-1)w ithin the apoferritin (AFt) nanocage were investigated. The DiRu-1-AFt nanocarrier was characterized by UV/Vis spectroscopy,ICP-MS, CD and X-ray crystallography.I nc ontrastt op reviouslyr eported Au-and Pt-basedd rugloaded AFt carriers, we found no evidenceo fd irect interactions between DiRu-1 and AFt.…”