The selective detection of specific cells of interest and their effective visualization is important but challenging, and fluorescent cell imaging with target-specific probes is commonly used to visualize cell morphology and components and to track cellular processes. Multiple displays of two or more targeting ligands on a polyvalent single template would make it possible to construct versatile multiplex fluorescent cell imaging probes that can visualize two or more target cells individually without the need for a set of individual probes. To achieve this goal, we used encapsulin, a new class of protein cage nanoparticles, as a template and implanted dual targeting capability by presenting two different affibody molecules on a single encapsulin protein cage nanoparticle post-translationally. Encapsulin was self-assembled from 60 identical subunits to form a hollow and symmetric spherical structure with a uniform size. We genetically inserted SpyTag peptides onto the encapsulin surface and prepared various SpyCatcher-fused proteins, such as fluorescent proteins and targeting affibody molecules. We successfully displayed fluorescent proteins and affibody molecules together on a single encapsulin in a mix-and-match manner post-translationally using bacterial superglue, the SpyTag/SpyCatcher ligation system, and demonstrated that these dual functional encapsulins can be used as target-specific fluorescent cell imaging probes. Dual targeting protein cage nanoparticles were further constructed by ligating two different affibody molecules onto the encapsulin surface with fluorescent dyes, and they effectively recognized and bound to two individual targeting cells independently, which could be visualized by selective colors on demand.

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The aggressive proliferation of tumor cells often requires increased glucose uptake and excessive anaerobic glycolysis, leading to the massive production and secretion of lactate to form a unique tumor microenvironment (TME). Therefore, regulating appropriate lactate levels in the TME would be a promising approach to control tumor cell proliferation and immune suppression. To effectively consume lactate in the TME, lactate oxidase (LOX) and catalase (CAT) were displayed onto Aquifex aeolicus lumazine synthase protein nanoparticles (AaLS) to form either AaLS/LOX or AaLS/LOX/CAT. These complexes successfully consumed lactate produced by CT26 murine colon carcinoma cells under both normoxic and hypoxic conditions. Specifically, AaLS/LOX generated a large amount of H2O2 with complete lactate consumption to induce drastic necrotic cell death regardless of culture condition. However, AaLS/LOX/CAT generated residual H2O2, leading to necrotic cell death only under hypoxic condition similar to the TME. While the local administration of AaLS/LOX to the tumor site resulted in mice death, that of AaLS/LOX/CAT significantly suppressed tumor growth without any severe side effects. AaLS/LOX/CAT effectively consumed lactate to produce adequate amounts of H2O2 which sufficiently suppress tumor growth and adequately modulate the TME, transforming environments that are favorable to tumor suppressive neutrophils but adverse to tumor-supportive tumor-associated macrophages. Collectively, these findings showed that the modular functionalization of protein nanoparticles with multiple metabolic enzymes may offer the opportunity to develop new enzyme complex-based therapeutic tools that can modulate the TME by controlling cancer metabolism. Graphical Abstract

Targeted cancer therapies have been extensively tested with the purpose to selectively suppress tumor growth and to avoid harming healthy tissue. However, failure to escape endosomes upon receptor‐mediated endocytosis is a major obstacle limiting the efficacy of targeted cancer therapeutics. Here, novel target‐switchable intracellular toxin delivery systems (TiTDS) are presented which use the catalytic and translocation domain of diphtheria toxin (dtA‐T) as an intracellular toxin delivery platform and affibody molecules targeting human epidermal growth factor receptor 2 or epidermal growth factor receptor (HER2Afb or EGFRAfb) as target‐specific ligands. The intracellular toxin delivery platform and the affibody molecules are genetically fused with SpyCatcher (SC) protein and SpyTag (ST) peptide, respectively, to generate dtA‐T‐SC and ST‐HER2Afb or ST‐EGFRAfb modules. These modules can be individually purified and post‐translationally ligated to produce dtA‐T/HER2Afb or dtA‐T/EGFRAf. dtA‐T/HER2Afb and dtA‐T/EGFRAfb can selectively bind to their corresponding target cancer cells, efficiently enter the cells through receptor‐mediated endocytosis, successfully escape endosomes, and release toxins into the cytosol. They exhibit high target‐specific cytotoxicity in vitro and can significantly reduce tumor masses in vivo. TiTDS is a promising targeted cancer therapy platform because of its high target specificity, effective intracellular delivery of active toxins with improved therapeutic efficacy, and target switchability.