CONSPECTUS The use of protein to precisely manipulate cell signaling is an effective approach for controlling cell fate and developing precision medicine. More recently, programmable nucleases, such as CRISPR/Cas9, have shown extremely high potency for editing genetic flow of mammalian cells, and for treating genetic disorders. The therapeutic potential of proteins with an intracellular target, however, is mostly challenged by their low cell impermeability. Therefore, a developing delivery system to transport protein to the site of action in a spatiotemporal controlled manner is of great importance to expand the therapeutic index of the protein. In this Account, we first summarize our most recent advances in designing combinatorial lipid nanoparticles with diverse chemical structures for intracellular protein delivery. By designing parallel Michael addition or ring-opening reaction of aliphatic amines, we have generated a combinatorial library of cationic lipids, and identified several leading nanoparticle formulations for intracellular protein delivery both in vitro and in vivo. Moreover, we optimized the chemical structure of lipids to control lipid degradation and protein release inside cells for CRISPR/Cas9 genome-editing protein delivery. In the second part of this Account, we survey our recent endeavor in developing a chemical approach to modify protein, in particular, coupled with the nanoparticle delivery platform, to improve protein delivery for targeted diseases treatment and genome editing. Chemical modification of protein is a useful tool to modulate protein function and to improve the therapeutic index of protein drugs. Herein, we mostly summarize our recent advances on designing chemical approaches to modify protein with following unique findings: (1) chemically modified protein shows selective turn-on activity based on the specific intracellular microenvironment, with which we were able to protein-based targeted cancer therapy; (2) the conjugation of hyaluronic acid (HA) to protein allows cancer cell surface receptor-targeted delivery of protein; (3) the introduction of nonpeptidic boronic acid into protein enabled cell nucleus targeted delivery; this is the first report that a nonpeptidic signal can direct protein to subcellular compartment; and (4) the fusion of protein with negatively supercharged green fluorescent protein (GFP) facilitates the self-assembly of protein with lipid nanoparticle for genome-editing protein delivery. At the end of the Account, we give a perspective of expanding the chemistry that could be integrated to design biocompatible lipid nanocarriers for protein delivery and genome editing in vitro and in vivo, as well as the chemical approaches that we can harness to modulate protein activity in live cells for targeted diseases treatment.

The selective and temporal control of protein activity in living cells provides a powerful tool to manipulate cellular function and to develop pro-protein therapeutics (PPT) for targeted therapy. In this work, we reported a facile but general chemical approach to design PPT by modulating protein activity in response to endogenous enzyme of disease cells, and its potential for targeted cancer therapy. We demonstrated that the chemical modification of a protein with quinone propionic acid (QPN), a ligand that could be reduced by tumor-cell-specific NAD­(P)H dehydrogenase [quinone] 1 (NQO1), was reversible in the presence of NQO1. Importantly, the QPN-modified cytochrome c (Cyt c-QPN) and ribonuclease A (RNase A-QPN) showed NQO1-regulated protein activity in a highly selective manner. Furthermore, the intracellular delivery of RNase A-QPN using a novel type of lipid-based nanoparticles, and subsequent protein activation by cellular NQO1, selectively inhibit cancer cell growth in vitro and effectively suppress tumor growth in vivo. We believe that our approach increases the number of potentially useful chemical tools for reversibly controlling the structure and function of protein using a disease-cell-specific enzyme, opening opportunities in the study of dynamic biological processes and developing precise protein therapeutics.

Messenger RNA (mRNA) is an emerging class of biotherapeutics for vaccine development and genome editing. Efficacious delivery and control of mRNA functionality selectively to disease cells remains the major challenge in developing mRNA therapeutics. Herein, reactive oxygen species (ROS)degradable lipid nanoparticles containing a thioketal (TK) moiety to deliver mRNA into cells are reported, selectively releasing mRNA in tumor cells for enhanced gene expression. By screening a library of parallelly synthesized ROS-degradable lipids, it has been identified that BAmP-TK-12 delivers mRNA one-fold more potent in tumor cells than in non-cancerous cells. Furthermore, the delivery of mRNA encoding DUF5, a bacterial-derived RAS protease using BAmP-TK-12 enables generic depletion of mutant RAS of tumor cells, showing a significantly improved antitumor effect than small molecule-based RAS inhibitor. It has been believed that the strategy of tumor cell-selective mRNA delivery using ROS-degradable lipid nanoparticles can be expanded to the broad range of bacterial effectors for rewiring cancer cell signaling and developing advanced biotherapeutics.

The precision and therapeutic potential of CRISPR/Cas9 genome editing are greatly challenged by the less control over Cas9-mediated DNA cleavage. Herein, we introduce a conditional and cell-selective genome editing system controlled by disease-associated enzymes, termed enzyme-inducible CRISPR (eiCRISPR). eiCRISPR comprises Cas9 protein, a self-blocked inactive single-guide RNA (bsgRNA), and a chemically caged deoxyribozyme (DNAzyme) that activates bsgRNA and eiCRISPR in a controllable manner. We design chemical modifications of DNAzyme to suppress its ability to cleave the blocking region of bsgRNA, while the decaging of DNAzyme triggered by the tumor cell-overexpressed enzyme, for instance, NAD(P)H:quinone oxidoreductase (NQO1), restores the activity of bsgRNA and switches on eiCRISPR selectively for genome editing in cancer cells. Moreover, using a biodegradable lipid nanoparticle to deliver eiCRISPR in a tumor-bearing xenograft, we show that the in vivo activation of eiCRISPR enables the editing of human papillomavirus 18 E6 for potential cancer therapy. The strategy of postsynthetic and site-specific modification of DNAzyme is compatible with endogenous chemistries for regulating eiCRISPR for cell-selective genome editing and targeted gene therapy.

The delivery of protein into mammalian cells enables the dissection and manipulation of biological processes;h owever,t his potency is challenged by the lacko fa n efficient protein delivery tool and am eans to monitor its intracellular trafficking. Herein, we report that the hierarchical self-assembly of tetraphenylethylene (TPE)-featured metalorganic cages (MOCs) and b-cyclodextrin-conjugated polyethylenimine can generate fluorescent supramolecular nanoparticles (FSNPs) to deliver protein into neural cells,acell line that is hardtotransfect using conventional strategy.Further,the aggregation-induced emission (AIE) of TPE enabled the fluorescent monitoring of cytosolic protein release.Itisfound that FSNPs can deliver and release protein into cytosol for subcellular targeting as fast as 18 hpost-delivery.Moreover,the delivery of molecular chaperone DJ-1 using FSNPs activates MAPK/ERK signaling of neural cells to protect cells from oxidative stress.

We report herein a modular approach to design the Michael addition between glutathione and coumarin derivatives for fluorescence imaging of the reversible and dynamic change of oxidative stress in living cells and rat brain.

Bioorthogonal catalysis provides a powerful tool to perform non-natural chemical reactions in living systems to dissect complex intracellular processes. Its potency to precisely regulate cellular function, however, is limited by the lack of bioorthogonal catalysts with cell selectivity. Herein, we report that palladium nanoparticles deposited on metal−

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