Amphiphilic Polyelectrolyte Graft Copolymers Enhance the Activity of Cyclic Dinucleotide STING Agonists

Abstract

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.

Immunostimulatory Polymers as Adjuvants, Immunotherapies, and Delivery Systems

“…Such advanced applications are advantageous for the coadministration of STING agonists with other PRRs to generate synergistic activation by a single construct. 6 , 111 Similar strategies for the (co)delivery of STING agonists have been employed by others, 112 − 116 and more advanced formulations are expected to emerge as the STING receptor is better understood.…”

“…Using the PEG-Q11R9-CDN complexes, they could achieve selective delivery of the STING agonists to dendritic cells and subsequent activation in mice using a sublingual route of administration. Such advanced applications are advantageous for the coadministration of STING agonists with other PRRs to generate synergistic activation by a single construct. , Similar strategies for the (co)­delivery of STING agonists have been employed by others, − and more advanced formulations are expected to emerge as the STING receptor is better understood.…”

Flash nanoprecipitation for the production of endosomolytic polymersomes for cytosolic drug delivery

“…Following endosomal acidification, the DEAEMA groups became protonated resulting in solubilization of the second block, disassembly of the nanoparticle, and exposure of the membrane-lytic DEAEMA-co-BMA domains, resulting in interactions with and the disruption of the endosomal membrane due to electrostatic attraction, resulting in disruption of the endosomal membrane. 12,31 A library of polymers was synthesized to examine the effect of second block molecular weight (MW) on the properties of the resultant nanoparticles while first block MW was held constant at 2 kDa, as it has been reported that polymer hydrophilic mass fraction affects nanoparticle self-assembly as well as endosomolytic capacity. Note: For the remainder of the paper, a PEG2k-EB'x'k copolymer will refer to a copolymer with a [DEAEMA-co-BMA] (E-co-B) block molecular weight of 'x' kDa.…”

“…[5][6][7][8][9][10] Previously, our laboratory has described several classes of STING-activating nanoparticles (STANs). [11][12][13] STANs are 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), and exploit the aforementioned mechanism to induce disruption of the endosomal membrane for the cytosolic delivery of cyclic dinucleotides (CDNs), which are agonists of the stimulator of interferon genes (STING) pathway. 11,12 The STING pathway is triggered when the enzyme cyclic-GMP-AMP synthase (cGAS) detects DNA in the cytoplasm, a common indicator of viral infection.…”

“…11,23 In our previous studies, we utilized a previously-described direct hydration approach for STAN production, 24 which enabled the self-assembly of polymeric vesicles, or polymersomes, with an aqueous core for cGAMP loading and a pH-responsive shell to induce endosomal membrane destabilization. 11,12 This approach involved the dropwise addition of solubilized cGAMP to a highly concentrated solution of swollen polymer in ethanol (EtOH). Although this direct hydration method allowed for high encapsulation efficiency (EE) of cGAMP, the process is conducted on the scale of a few hundred microliters and accordingly cannot be implemented into a practical scaled-up manufacturing process.…”