(123e) Endosomolytic Polymersomes Enhance Intracellular Delivery of Nucleic Acid Therapeutics for Improved Anticancer Immune Responses

Introduction: Cancer immunotherapy has revolutionized the fields of oncology and drug delivery, with immune checkpoint blockade (ICB) demonstrating remarkable potential as an alternative to traditional treatment regimens such as surgery, chemotherapy, and radiation therapy. Although ICB has demonstrated disease control in certain cancers, it has also indicated limited therapeutic efficacy in poorly immunogenic cancers. Through the activation of specific cellular pattern recognition receptors (PRRs), the immunosuppressive tumor microenvironment (TME) of these cancers can be reprogrammed to a more immunogenic, ‘hot’ phenotype with larger populations of infiltrating T cells, directly correlating with improved responses to ICB. Indeed, activation of the retinoic acid-inducible gene I (RIG-I) pathway has been found to elicit a downstream signaling cascade resulting in the production of type I interferons which can induce this shift in the TME. Unfortunately, most RIG-I-activating therapeutics are not able to freely cross the cell membrane where they are required to activate this cytosolic PRR. To overcome this challenge, our lab has designed pH-responsive polymeric nanoparticles capable of encapsulating and delivering anticancer therapeutics to the tumor site. The goal of this work is to optimize the loading of 5’-triphosphorylated double-stranded RNA (3pRNA), a potent RIG-I agonist, within polymeric nanocarriers via a facile and scalable flash nanoprecipitation (FNP) process and assess therapeutic efficacy in a cancer model. To accomplish this, we analyzed the effect of copolymer properties (second block molecular weight, alkyl chain length) on nanocarrier physical properties (size, polydispersity, surface charge, morphology), RNA loading, cytotoxicity, endosomolytic activity, and RIG-I activation in vitro. We then validated the ability of the system to mitigate disease progression in a murine cancer model by systemically administering our ‘lead’ formulation and monitoring tumor volume, weight loss, and survival over the course of the experiment.

Materials and Methods: Reversible addition-fragmentation chain transfer (RAFT) polymerization was utilized to synthesize a library of [PEG]_2kDa-bl-[DMAEMA_50%-co-A_nMA_50%]_x copolymers with a PEG first block molecular weight (MW) of 2kDa, a DMAEMA pH-responsive component, and a second block MW of ‘x’kDa where ‘A_nMA’ refers to an alkyl methacrylate monomer of chain length ‘n’ ranging from n = 2-12 carbon atoms. By inducing turbulent mixing within a confined impingement jet (CIJ) mixer of an organic copolymer solution and an aqueous solution containing solubilized 3pRNA, uniform 3pRNA-loaded nanocarriers were produced. Carriers were analyzed for size, polydispersity (PDI), and surface charge using a Malvern Nano ZetaSizer, morphology using cryogenic electron microscopy (cryoEM), and encapsulation efficiency using a Quanti-it RiboGreen assay (Thermo Fisher Scientific). Dose-dependent cytotoxicity was assessed in vitro using a CellTiter-Glo luminescent cell viability assay (Promega), and endosomolytic activity was measured using a galectin (Gal) reporter assay. To examine activation of RIG-I in vitro, reporter cells were treated with formulations and a QUANTI-Luc assay was performed to measure the amount of Lucia luciferase secreted from cells, a readout which is directly proportional to interferon production. Finally, therapeutic efficacy was assessed in a murine EMT6 breast cancer model by monitoring tumor volume, weight loss, and survival after systemic administration of 3pRNA-loaded nanocarriers.

Results and Discussion: Upon turbulent mixing and spontaneous self-assembly, drug-loaded nanocarriers were collected in an aqueous reservoir where they were then characterized as previously described. A schematic of the FNP process, experimental design, and a cryoEM image of nanocarriers are displayed in Fig. 1. Preliminary analysis indicated uniform nanocarriers with variable morphologies depending on second block MW and alkyl chain length. After conducting a thorough screening of particle properties using empty carriers, a potential ‘lead’ nanocarrier was chosen to proceed with further testing. Preliminary data indicated that the ‘lead’ nanocarrier with a DMAEMA pH-responsive component allowed for higher encapsulation efficiency (EE) and induced further RIG-I activation in vitro compared to its nanocarrier counterpart with a DEAEMA pH-responsive component, previously characterized in our lab.¹ Interestingly, increasing the inlet concentration of 3pRNA appeared to decrease EE for both nanocarriers (Fig. 1). In a similar manner, an initial screening with our galectin reporter system indicated increased endosomolytic activity for DMAEMA-based nanocarriers compared to DEAEMA-based nanocarriers. Future work will seek to determine polymer properties and FNP process conditions (aqueous buffer, impingement number, etc.) for 3pRNA-loaded nanocarriers of optimal physical properties capable of inducing potent RIG-I activation. We expect the optimized formulation to significantly increase RIG-I activation, mitigate tumor progression, and prolong survival in mice compared to free 3pRNA and PBS controls.

Conclusions: In this work, we report the fabrication of a library of RNA-loaded polymeric nanocarriers via a simple and versatile formulation method capable of enhancing cytosolic delivery of drug cargo and inducing potent RIG-I activation. With this FNP platform, we highlight the potential for tailored production of a variety of polymeric nanocarriers for potential translatable use in other drug delivery and immunomodulatory applications.

References: [1] Shae, D. et al. Endosomolytic polymersomes increase the activity of cyclic dinucleotide STING agonists to enhance cancer immunotherapy. Nat Nanotechnol 14, 269–278 (2019).

Figure 1. Flash nanoprecipitation (FNP) allows for the self-assembly of 3pRNA-loaded polymeric nanocarriers under turbulent mixing conditions. After mixing, nanocarriers are collected in a scintillation vial where they can then be characterized. It was determined that the DMAEMA-based copolymer utilized in this work was able to significantly encapsulate more 3pRNA within carriers compared to the DEAEMA-based copolymer studied in our previous work.¹