Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) hold great therapeutic potential, but their activity is hindered by poor drug‐like properties that restrict cytosolic bioavailability. Here, this challenge is addressed through the synthesis and evaluation of a novel series of PEGMA‐co‐DEAEMA‐co‐BMA copolymers with pH‐responsive, membrane‐destabilizing activity to enhance intracellular delivery of the CDN, cGAMP. Copolymers are synthesized with PEGMA of two different molecular weights (300 and 950 Da) and over a range of PEG mass fraction and polymer molecular weight, and relationships between copolymer structure, self‐assembly, endosomal escape, and cGAMP activity are elucidated. A subset of polymers that self‐assembled into 50–800 nm nanoparticles is identified, which can be loaded with cGAMP via a simple mixing strategy, resulting in significantly enhanced immunostimulatory activity. Increased cGAMP activity is found to be highly correlated with the capacity of carriers to enhance intracellular CDN uptake and to promote endosomal destabilization, findings that establish efficient cytosolic delivery as a criterion for CDN carriers. Additionally, it is demonstrated that a lead CDN carrier formulation can enhance STING activation in vivo in a model of intratumoral immunotherapy. Collectively, these investigations demonstrate the utility of PEGMA‐co‐DEAEMA‐co‐BMA copolymers as carriers for CDNs and potentially other cytosolically‐acting drug cargo.

No abstract

Endosomal escape is a rate limiting step for many promising therapeutics, including cyclic dinucleotide (CDN) agonists of the stimulator of interferon genes (STING) pathway. In article number 2001056, John T. Wilson and co‐workers synthesize and screen a novel library of polymers with pH‐responsive, membrane‐destabilizing activity and evaluate their capacity to enhance cytosolic delivery of CDNs, resulting in identification of new drug carrier technologies for potent STING activation.

The delivery of biomacromolecular drugs to cytosolic targets has been a long-standing engineering challenge due to the presence of multiple biological barriers including cellular and endosomal membranes. Although many promising carriers designed to facilitate endosomal escape have been developed, the clinical translation of these carriers is often limited by complex production processes that are not amenable to scaled-up manufacturing. In this study, we employed flash nanoprecipitation (FNP) for the rapid, scalable, and reproducible assembly of nanocarriers composed of the pH-responsive, endosomolytic diblock copolymer poly[(ethylene glycol)x-block-[((2-diethylamino) ethyl methacrylate)0.6-co-(butyl methacrylate)0.4]y (PEG-b-DEAEMA-co-BMA). We found that varying the second block molecular weight, while holding the first block molecular weight constant, significantly influenced nanoparticle self-assembly and hence nanocarrier properties and function – including drug encapsulation, endosomolytic capacity, cytotoxicity, and in vitro activity of a cytosolically-active drug cargo, a cyclic dinucleotide (CDN) stimulator of interferon genes (STING) agonist. We found that while increasing second block molecular weight enhanced the capacity of nanocarriers to induce endosomal destabilization, larger second block molecular weights also lead to increased cytotoxicity, increased particle size and heterogeneity, increased the encapsulation efficiency of small (<0.5 kDa) hydrophilic drugs, decreased the encapsulation efficiency of large (10 kDa) hydrophilic biomacromolecules, and decreased long-term particle stability. Collectively, these results demonstrate the utility of FNP for the rapid and scalable production of uniform PEG-b-DEAEMA-co-BMA nanocarriers and implicate an optimal hydrophilic mass fraction for balancing desirable nanoparticle properties with cytosolic cargo delivery efficiency.

The delivery of biomacromolecular drugs to cytosolic targets has been a long-standing engineering challenge due to the presence of multiple biological barriers including cellular and endosomal membranes. Although many promising carriers designed to facilitate endosomal escape have been developed, the clinical translation of these carriers is often limited by complex production processes that are not amenable to scaled-up manufacturing. In this study, we employed flash nanoprecipitation (FNP) for the rapid, scalable, and reproducible assembly of nanocarriers composed of the pH-responsive, endosomolytic diblock copolymer poly[(ethylene glycol)x-block-[((2-diethylamino) ethyl methacrylate)0.6-co-(butyl methacrylate)0.4]y (PEG-b-DEAEMA-co-BMA). We found that varying the second block molecular weight, while holding the first block molecular weight constant, significantly influenced nanoparticle self-assembly and hence nanocarrier properties and function – including drug encapsulation, endosomolytic capacity, cytotoxicity, and in vitro activity of a cytosolically-active drug cargo, a cyclic dinucleotide (CDN) stimulator of interferon genes (STING) agonist. We found that while increasing second block molecular weight enhanced the capacity of nanocarriers to induce endosomal destabilization, larger second block molecular weights also lead to increased cytotoxicity, increased particle size and heterogeneity, increased the encapsulation efficiency of small (<0.5 kDa) hydrophilic drugs, decreased the encapsulation efficiency of large (10 kDa) hydrophilic biomacromolecules, and decreased long-term particle stability. Collectively, these results demonstrate the utility of FNP for the rapid and scalable production of uniform PEG-b-DEAEMA-co-BMA nanocarriers and implicate an optimal hydrophilic mass fraction for balancing desirable nanoparticle properties with cytosolic cargo delivery efficiency.