(558h) Scalable Fabrication of Endosomolytic Polymersomes for Cytosolic Delivery of Immunostimulatory Oligonucleotides

Introduction: Cancer immunotherapy has displayed immense potential to overcome the limited therapeutic efficacy of traditional cancer treatments such as surgery, radiation, and chemotherapy. One major clinical obstacle that many cancer immunotherapies seek to overcome is the presence of a ‘cold’, weakly immunogenic tumor microenvironment (TME). Enhancing the immunogenicity of the TME to a more tumoricidal, ‘hot’ phenotype can be extremely beneficial in enhancing therapeutic responses. Specifically, delivery of immunostimulatory nucleic acid therapeutics can lead to downstream signaling cascades resulting in the production of various antiviral interferons and proinflammatory cytokines, in turn allowing for a more robust antitumor immune response. Unfortunately, the delivery of these therapeutics in vivo is significantly hindered due to their susceptibility to nuclease degradation, rapid clearance from the circulation, and inability to freely cross the cell membrane where they are required to activate cytosolic cellular pathways. To overcome this challenge, our lab has designed pH-responsive polymeric nanoparticles capable of encapsulating and enhancing delivery of nucleic acid therapeutics to the tumor site. We have previously synthesized a library of diblock copolymers consisting of a polyethylene glycol (PEG) first block and a second block comprised of pH-responsive 2-(diethylamino)ethyl methacrylate (DEAEMA) and variable hydrophobic alkyl chain groups.¹ The goal of our work is to optimize the loading of various drug cargo into polymeric nanocarriers via a scalable flash nanoprecipitation (FNP) process. To accomplish this, we have analyzed the effect of polymer properties and FNP process parameters on nanocarrier self-assembly, drug loading, and in vitro behavior in relevant cell lines. As copolymer hydrophilic weight fraction is a critical variable in influencing particle self-assembly, toxicity, and endosomal escape, we primarily sought to fix the hydrophilic first block molecular weight (MW) while varying the hydrophobic second block MW and examine the effect on particle properties.

Materials and Methods: Reversible addition-fragmentation chain transfer (RAFT) polymerization was utilized to synthesize a library of [PEG]_2kDa-bl-[DEAEMA-co-A_nMA]_y copolymers with a PEG first block fixed at a MW of 2 kDa and a second block MW of ‘y’ kDa where ‘A_nMA’ refers to alkyl methacrylate monomer chains ranging from n =2-12 carbons. By inducing rapid mixing of a copolymer solution with an aqueous solution of hydrophilic drug cargo within a central impingement jet (CIJ), uniform nanoparticles encapsulating the drug were produced. Particles were characterized for size and zeta potential using a Malvern Nano ZetaSizer, morphology using an Osiris transmission electron microscope (TEM), and encapsulation efficiency using high-performance liquid chromatography (HPLC). Further, an erythrocyte hemolysis assay was utilized to characterize the membrane-destabilizing capabilities of the system. Particles were characterized for cytotoxicity using a CellTiter-Glo Cell Viability assay in A549 adenocarcinomic human alveolar basal epithelial cells, and in vitro stimulator of interferon genes (STING) activation was determined in THP1-Dual reporter cells using cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) as a model drug cargo.

Results and Discussion: Turbulent mixing of aqueous hydrophilic drug solution and organic copolymer solution allowed for the formation of nanoparticles that were collected in an aqueous reservoir where they were then characterized as described. TEM images verified a spherical, well-defined polymersome particle morphology (Fig. 1A). Particle size distributions displayed an increase in both particle diameter and sample polydispersity (PDI) with increasing second block MW (Fig. 1B). Similarly, an erythrocyte hemolysis assay (Fig. 1C) indicated enhanced membrane destabilization was prevalent at higher second block MWs, and a cell viability assay determined that cytotoxicity on a relevant cell line increased in a dose-dependent manner (Fig. 1D). Encapsulation of cGAMP, an endogenous STING agonist, was quantified, and in vitro STING activation in THP1-Dual cells was found to increase in a dose-dependent manner for all nanoparticle drug carriers (Fig. 1E).

Conclusions: In this work, we report the fabrication of a repertoire of polymeric nanoparticles via a simple and versatile formulation method and examine the effect of copolymer MW on nanoparticle properties. With the facility of this platform, we highlight the potential for the tailored production of a variety of nanoparticles. Such a system could prove extremely beneficial in a large-scale pharmaceutical manufacturing process of polymeric nanoparticles for immunotherapeutic applications.

References: [1] Jacobson, M. E. et al., ACS Cent. Sci. 2020, 6, 2008-2022.

Figure 1. (A) Flash nanoprecipitation (FNP) allows for the spontaneous self-assembly of polymeric nanoparticles under turbulent mixing conditions. Particles were collected in an aqueous reservoir and further characterized as described in the above schematic. (B) An increase in copolymer second block MW corresponded with an increase in nanoparticle diameter and sample PDI. (C) Nanoparticles effectively disrupted erythrocyte cell membranes at endosomal pH. (D) An increase in second block MW resulted in increased cytotoxicity toward A549 cells. Corresponding EC₅₀ values are shown. (E) cGAMP encapsulation within polymeric nanoparticles is shown. Induced cGAMP activity in THP1-Dual cells increased in a dose dependent manner. Schematic created using Biorender. Error bars denote standard deviation.