The mechanism of catalase loading into porous vaterite CaCO3 crystals by co-synthesis

Porous vaterite CaCO3 crystals are nowadays extensively used as high-capacity bio-friendly sacrificial templates for the fabrication of such protein-containing nano- and micro-particles as capsules and beads. The first step in the protein encapsulation is performed through loading of the protein molecules into the crystals. Co-synthesis is one of the most useful and simple methods proven to effectively load crystals with proteins; however, the loading mechanism is still unknown. To understand the mechanism, in this study, we focus on the loading of a model protein catalase into the crystals by means of adsorption into pre-formed crystals (ADS) and co-synthesis (COS). Analysis of the physico-chemical characteristics of the protein in solution and during the loading and simulation of the protein packing into the crystals are performed. COS provides more effective loading than ADS giving protein contents in the crystals of 20.3 and 3.5 w/w%, respectively. Extremely high loading for COS providing a local protein concentration of about 550 mg mL−1 is explained by intermolecular protein interactions, i.e. formation of protein aggregates induced by CaCl2 during the co-synthesis. This is supported by a lower equilibrium constant obtained for COS (5 × 105 M−1) than for ADS (23 × 105 M−1), indicating a higher affinity of single protein molecules rather than aggregates to the crystal surface. Fitting the adsorption isotherms by classical adsorption models has shown that the Langmuir and BET models describe the adsorption phenomenon better than the Freundlich model, proving the aggregation in solution followed by adsorption of the aggregates into the crystals. We believe that this study will be useful for protein encapsulation through CaCO3 crystals using the COS method

Inter-protein interactions govern protein loading into porous vaterite CaCO3 crystals

The fast development of protein therapeutics has resulted in a high demand for advanced delivery carriers that can effectively host therapeutic proteins, preserve their bioactivity and release them on demand. Accordingly, vaterite CaCO3 crystals have attracted special attention as sacrificial templates for protein encapsulation in micro- and nanoparticles (capsules and beads, respectively) under mild biofriendly conditions. This study aimed to better understand the mechanism of protein loading into crystals as a primary step for protein encapsulation. The loading of three therapeutic proteins (250 kDa catalase, 5.8 kDa insulin, and 6.5 kDa aprotinin) was investigated for crystals with different porosities. However, unexpectedly, the protein loading capacity was not consistent with the protein molecular weight. It solely depends on the inter-protein interactions in the bulk solution in the presence of crystals and that inside the crystals. The smallest protein aprotinin aggregates in the bulk (its aggregate size is about 100 nm), which prohibits its loading into the crystals. Insulin forms hexamers in the bulk, which can diffuse into the crystal pores but tend to aggregate inside the pores, suppressing protein diffusion inward. Catalase, the largest protein tested, does not form any aggregates in the bulk and diffuses freely into the crystals; however, its diffusion into small pores is sterically restricted. These findings are essential for the encapsulation of protein therapeutics by means of templating based on CaCO3 crystals and for the engineering of protein-containing microparticles having desired architectures

Combination of IAP antagonist and IFNγ activates novel caspase-10- and RIPK1-dependent cell death pathways

Peptido-mimetic inhibitor of apoptosis protein (IAP) antagonists (Smac mimetics (SMs)) can kill tumour cells by depleting endogenous IAPs and thereby inducing tumour necrosis factor (TNF) production. We found that interferon-γ (IFNγ) synergises with SMs to kill cancer cells independently of TNF − and other cell death receptor signalling pathways. Surprisingly, CRISPR/Cas9 HT29 cells doubly deficient for caspase-8 and the necroptotic pathway mediators RIPK3 or MLKL were still sensitive to IFNγ/SM- induced killing. Triple CRISPR/Cas9-knockout HT29 cells lacking caspase-10 in addition to caspase-8 and RIPK3 or MLKL were resistant to IFNγ/SM killing. Caspase-8 and RIPK1 deficiency was, however, sufficient to protect cells from IFNγ/SM-induced cell death, implying a role for RIPK1 in the activation of caspase-10. These data show that RIPK1 and caspase-10 mediate cell death in HT29 cells when caspase-8-mediated apoptosis and necroptosis are blocked and help to clarify how SMs operate as chemotherapeutic agents