Unleashing the Power of Proenzyme Delivery for Targeted Therapeutic Applications Using Biodegradable Lipid Nanoparticles

Proenzymes, functioning as inactive precursor forms of enzymes, hold significant promise for regulating essential biological processes. Their inherent property of latency, remaining inert until they arrive at the intended site of action, positions them as particularly promising candidates for the development of targeted therapeutics. Despite this potential, the therapeutic potential of proenzymes is challenged by designing proenzymes with excellent selectivity for disease cells. This limitation is further exacerbated by the inability of proenzymes to spontaneously cross the cell membrane, a biological barrier that impedes the cellular internalization of exogenous macromolecules. Therefore, efficacious intracellular delivery is paramount to unlocking the full therapeutic potency of proenzymes. In this Account, we first elucidate our recent advancements made in designing biodegradable lipid nanoparticles (LNPs) for the cell-specific delivery of biomacromolecules, including proteins and nucleic acids. Using a strategy of parallel synthesis, we have constructed an extensive library of ionizable lipids, each integrated with different biodegradable moieties. This combinatorial approach has led to the identification of LNPs that are particularly efficacious for the delivery of biomacromolecules specifically to tumor cells. This innovation capitalizes on the unique intracellular environment of cancer cells to control the degradation of LNPs, thereby ensuring the targeted release of therapeutics within tumor cells. Additionally, we discuss the structure-activity relationship governing the delivery efficacy of these LNPs and their applicability in regulating tumor cell signaling, specifically through the delivery of bacterial effector proteins. In the second segment, we aim to provide an overview of our recent contributions to the field of proenzyme design, where we have chemically tailored proteins to render them responsive to the unique milieu of tumor cells. Specifically, we elaborate on the chemical principles employed to modify proteins and DNAzymes, thereby priming them for activation in the presence of NAD-(P)-H:quinone oxidoreductase 1 (NQO1), an enzyme that is prevalently upregulated within tumor cells. We summarize the methodologies for intracellular delivery of these proenzymes using biodegradable LNPs, both in vitro and in vivo. The concomitant intracellular delivery and activation of proenzymes are examined in the context of enhanced therapeutic outcomes and targeted CRISPR/Cas9 genome editing. In conclusion, we offer a perspective on the chemical principles that could be leveraged to optimize LNPs for tissue-specific delivery of proenzymes. We also explore chemical strategies for the irreversible modulation of proenzyme activity within living cells and in vivo. Through this discussion, we provide insights into potential avenues for overcoming existing limitations and enhancing the delivery of proenzymes using LNPs, particularly for developing tumor-targeted therapies and genome editing applications.