Enhancing mRNA-Based Therapeutic Delivery with Coacervate-Encapsulating Polymersomes

Introduction: mRNA-based therapies have gained significant attention in recent years due to their potential to treat a wide range of diseases, including cancers, immune diseases, and neurological disorders. Specifically, the COVID-19 vaccine is an mRNA-based therapy. Compared to traditional genetic modification methods, mRNA therapies offer lower risk of undesired genetic mutations, transient expression, and rapid production. Naked mRNA cannot be delivered effectively to target tissues due to its susceptibility to degradation by enzymes in the bloodstream and its inability to independently cross the cell membrane. However, current non-viral vectors such as liposomes, polymers, and nanoparticles still face limitations in their ability to deliver mRNA including low encapsulation efficiency, potential toxicity, and limited stability. To address these challenges, we investigate the use of droplet microfluidics to synthesize double-emulsion polymersomes that encapsulate complex coacervates of mRNA and spermine. mRNA coacervates are condensed phases (liquid-liquid phase separation) formed by the aggregation of RNA and a polyanionic molecule. This approach aims to capitalize on coacervates’ natural affinity for mRNA molecules to provide protection and facilitate endocytosis, thereby enhancing delivery efficacy and overcoming the current limitations of non-viral vectors. Furthermore, we modify the the polyanionic compartment with a sulfide containing crosslinker to make the system responsive to reactive oxygen species (ROS), allowing for controlled release of mRNA from coacervates. Using this technique, we aim to provide an effective and controlled encapsulation method and drug delivery system for mRNA-based therapeutics.

Materials and Methods: A capillary-based microfluidic device was assembled by connecting two chromatography tees with a central square capillary to align two enclosed tapered capillaries for the breakup zone. An oil phase containing dissolved polyethylene glycol-b-poly lactic acid (PEG-PLA) was simultaneously injected with inner and outer aqueous streams to form submicron polymersomes. Polymersome characteristics were optimized through controlling the degree of capillary tapers as well as varying stream flowrates. Polymersomes were collected, resuspended, and characterized through dynamic light scattering (DLS) to determine size and polydispersity index. In addition, we optimized the coacervation of green fluorescent protein (GFP)-coding mRNA and polycation spermine within the same inner aqueous stream composition to facilitate coacervate encapsulation within polymersomes. Turbidity studies using UV/Vis spectroscopy were conducted to determine coacervate stability and the optimal ratio of coacervate components.
Results and Discussion: Microfluidic synthesis of polymersomes was confirmed via preliminary DLS characterization (Figure 1). The polymersomes were found to have an average size of 306.5 nm +/- 32.6 nm with an average PDI of 0.52 +/- 0.1, which can be further decreased using extrusion. Turbidity studies conducted under the specified inner aqueous stream conditions revealed an optimal concentration of green fluorescent protein-coded mRNA in spermine to ensure desired coacervate size and stability. Given the design of the microfluidic device and the optimized coacervation process, we anticipate the successful encapsulation of mRNA coacervates within polymersomes under optimized inner aqueous conditions. Moreover, the spermine in the coacervates will provide protection to the mRNA, preventing its contact with the oil stream and decreasing mRNA degradation. Based on structural confirmation of the modified spermine and preliminary irradiation studies on the coacervates, we anticipate successful ROS-triggered coacervation within polymersomes.

Conclusions: Using a capillary-based droplet microfluidics approach, PEG-PLA polymersomes can be formed with an inner aqueous phase that not only offers protection to the encapsulated cargo from harsh, degrading solvents, but additionally provides a novel opportunity to encapsulate mRNA/spermine coacervates which could potentially overcome current delivery limitations for mRNA-based therapeutics. Optimization of coacervate formation under inner aqueous conditions provides evidence for future high encapsulation efficiency and protection from mRNA degradation. Further studies need to be conducted to confirm coacervate-maintenance during encapsulation as well as ROS-responsive characteristics. Next steps will involve determining encapsulation efficiency and confirming ROS-responsive mRNA release.


Acknowledgements (Optional): : This work was partially funded through Clemson’s Creative Inquiry Program.

References (Optional): :