Prominin‐1‐Specific Binding Peptide‐Modified Apoferritin Nanoparticle Carrying Irinotecan as a Novel Radiosensitizer for Colorectal Cancer Stem‐Like Cells

Resistance of cancer stem cells to radiotherapy remains a major obstacle to successful cancer management. Prominin‐1 (PROM1) is a cancer stem cell marker. Nanoparticle (NP) chemotherapeutics preferentially accumulate in tumors and are able to target cancer and cancer stem‐like cells through cancer cell‐specific ligands, making them uniquely suited as radiosensitizers for chemoradiation therapy. Using a biocompatible apoferritin NP, a PROM1‐targeted NP carrying irinotecan (PROM1‐NP) is engineered. The synergistic effect of the NP and irradiation is evaluated in PROM1‐overexpressing HCT‐116 colorectal cancer cell lines in vitro and in vivo. PROM1‐NP has a size of 17.2 ± 0.2 nm and surface charge of ?13.5 ± 0.2 mV. It demonstrates higher intracellular uptake than nontargeted NP or irinotecan alone. Treatment with PROM1‐NPs decreases HCT‐116 cell proliferation in a dose‐ and time‐dependent manner. In vitro radiosensitization reveals that PROM1‐NP is significantly more effective as a radiosensitizer than nontargeted NP or irinotecan. HCT‐116 tumor xenograft growth is markedly slower following treatment with PROM1‐NP plus irradiation, suggesting that PROM1‐NP is more effective as a radiosensitizer than irinotecan and nontargeted NP in vivo. This study provides the first preclinical evidence of the effectiveness of PROM1‐targeted NP formulation of irinotecan as a radiosensitizer.     

Sedimentation as a tool for crystallization from protein mixtures

Crystals from apoferritin which is an iron‐free form of protein ferritin were obtained from protein mixtures lysozyme/apoferritin using sedimentation under high gravity. Solution containing apoferritin at concentration as high as 5mg/ml in the presence of 25mg/ml lysozyme and overlaid on 5%(w/v) CdSO₄ in 0,2M/L NaAC, pH=5 still favors apoferritin crystal formation under normal gravity conditions, but at apoferritin concentrations <0,5mg/ml (∼1,14µM/L) in 25mg/ml (∼1,71mM/L) lysozyme only the sedimentation in a centrifuge appears to be useful for separating the apoferritin molecules from the mixture followed by apoferritin crystallization in the same system. The very high molecule number ratio (∼1:10³) of two proteins is used to stress on the observed effect. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Layered crystals of apo‐ and holoferritin grown by alternating crystallization

We report on the use of alternating crystallization for deposition of layers of different (though closely related) proteins in a single crystal. Investigations were carried out with the unique protein couple consisting of two forms of ferritin, apoferritin and holoferritin from horse spleen, which, despite being of quite different molecular masses, still possess identical organic shells. Crystals of both proteins were used as substrates for subsequent contiguous growth of the partner protein in perfect alignment. We observed continuous growth of combined (onion‐like) single crystals; artificial structures of biological macromolecules can be designed in this way. The homoepitaxial layered growth shows in an unambiguous way that protein crystallization depends only on the surface protein conformation and amino‐acid composition, but not on the internal molecule structure. The limitations of protein crystal growth for designing layered structures of biological macromolecules were revealed by growing of heterogeneous protein crystals onto pre‐existing protein crystalline substrates. Tetragonal crystals of hen egg‐white lysozyme were grown onto cubic apoferritin crystals used as substrates. It was observed that the lysozyme crystals were not lattice‐matched to the ‘host’ apoferritin crystals; this led to mere aggregates of different crystals. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)