How many active caspases does it take to induce apoptosis? Do pro-survival and pro-apoptotic intracellular balances influence a caspase’s ability to induce cell death (i.e. potency)?
Cysteine aspartate proteases (caspases) act as the molecular scissors of apoptotic cell death, disintegrating diverse cellular components necessary for cell survival and growth. As nanomaterials continue to be developed and cancers continue to evolve death resistance mechanisms, intracellular introduction of caspases offers an opportunity to induce cell death via exploitation of native apoptotic machinery. Nevertheless, caspases are a particularly challenging therapeutic subset, requiring careful delivery vehicle design and compatibility for exogenous introduction into cancer cells with sufficient proteolytic activity and native structural characteristics for function. Our state-of-the-art polymeric delivery platform enables us to intracellularly deliver caspase-3, as well as other members of the caspase family, without any non-native modification to the proteases. Most importantly, our amphiphilic redox-responsive polymers temporarily silence caspase activity prior to intracellular release, decreasing off-target effects and increasing cytotoxic therapeutic control. Furthermore, caspases can be co-encapsulated with other enzymes or small molecules in the same delivery vehicle, facilitating easy combinatorial therapeutic assessment. Using our technology, we have learned different mechanistic insights that not only contribute to caspase therapeutic development and optimization, but also innovatively underscore the comprehensive knowledge of caspase function.
We have demonstrated that exogeneous introduction of less enzymatically efficient caspases can induce higher levels of apoptosis to the most catalytically efficient caspase, caspase-3. This finding underscores the fact that each caspase is uniquely regulated and is subject to inactivation by different factors, recognizing the central importance of tailoring a specific therapy to individual disease variants. Furthermore, the mechanism by which each caspase induced apoptosis was observed to be distinct and thus, can be exploited or hindered depending on the pro-apoptotic and pro-survival cancer context, underlying possible pathways for combinatorial therapeutic intervention. Beyond assessing different caspase cargos, we have demonstrated that <10 µM of cytosolically delivered caspase-3 is sufficient to induce cell death and that the amount of caspase-3 that reaches the cytosol can be tuned via delivery vehicle modification. Finally, nanogel-mediated caspase delivery can be targeted to certain populations and can be delivered in combination with other pro-apoptotic moieties, enzymatic or small molecule.
The balance of pro-apoptotic and pro-survival proteins define a cell’s fate. These processes are controlled through an inter-dependent, finely-tuned protein network that enables survival or leads to apoptotic cell death. The caspase family of proteases are central to this apoptotic network, with initiator and executioner caspases, and their interaction partners, regulating and executing apoptosis. We interrogated and modulated this network by exogenously introducing specific initiator or executioner caspase proteins using redox-responsive polymeric nanogels. Although caspase-3 might be expected to be the most effective due to the centrality of its role in apoptosis and its heightened catalytic efficiency relative to other family members, we observed that caspase-7 and caspase-9 are the most effective at inducing apoptotic cell death. By critically analyzing the introduced activity of the delivered caspase, the pattern of substrate cleavage as well as the ability to activate endogenous caspases, we conclude that the efficacy of each caspase correlated with the levels of pro-survival factors that both directly and indirectly impact the introduced caspase. These findings lay the groundwork for developing exogenous introduction of caspases as a therapeutic option that can be tuned to the apoptotic balance in a proliferating cell.
The ability for caspases to induce cell death hinges on cytosolic translocation of an active, unmodified protease. After caspase-nanomaterial endocytosis and subsequent endosomal escape, caspases would reach the cytosol, granting their access to a reducing environment and intracellular targets for proteolysis. As endosomal escape efficiencies are typically only ~1%, the susceptibility of caspase-nanomaterial escape becomes the key determinant in delivery system efficacy. Distinguishing cytosolically released caspases from those that are endosomally entrapped upon cellular uptake is particularly challenging and is often characterized using fluorophores that can improperly influence interpretation. To implement a reliable, but functionally silent, tagging technology for therapeutically relevant caspase-3 cargos, we developed a casp-3 variant tagged with the 11th strand of GFP that retains both enzymatic activity and structural characteristics of wild-type casp-3. Using this variant, we can appropriately evaluate and compare endosomal escape efficiencies of competing delivery systems of disparate compositions. In particular, we use SEE as a means to enable systematic investigation of the effect of polymer composition, polymer architecture (random vs. block), hydrophobicity, and surface functionality. Although polymer structure had little influence on endosomal escape, nanogel functionalization with cationic and pH-sensitive peptides significantly enhanced endosomal escape levels and further, significantly increased the amount of nanogel per endosome. These findings serve as a guide for developing an optimal caspase-3 delivery system, as this caspase-3 variant can be easily substituted for a therapeutic caspase-3 cargo in any system that results in cytosolic accumulation and cargo release.
In addition to determining the most potent caspase cargo and quantifying the amount of caspase that reaches the cytosol, we further investigated the possibility of using caspases as therapeutics by quantifying caspase encapsulation within individual nanomaterials, targeting their delivery and exploring the addition of pro-apoptotic molecules to caspase systems. These investigations are critical as great effort has been undertaken to evaluate how biologic encapsulation efficiencies correlate with efficacy as well as how therapeutic combinations can overcome pitfalls of one treatment. Characterizing nanoparticle loading and encapsulation efficiencies for biologic cargos is facile on the bulk scale yet, are average estimations for the solution. Instead, we persevered the assessment of encapsulation efficiencies on a single particle level, a distribution of one-to-three caspases per particle, which could be further explored to reveal the interplay between supramolecular nanogel formation and guest encapsulation with the downstream therapeutic results. Moreover, we demonstrated that antibodies targeting human epidermal growth factor 2 (HER2) can be added to the nanogel surface to target caspases to HER2+ populations, representing aggressive cancer genotypes.
