Intracellular delivery of messenger RNA (mRNA) has the potential to induce protein production for many therapeutic applications. Although lipid nanoparticles have shown considerable promise for the delivery of small interfering RNAs (siRNA), their utility as agents for mRNA delivery has only recently been investigated. The most common siRNA formulations contain four components: an amine-containing lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency. Here, we develop a generalized strategy to optimize lipid nanoparticle formulations for mRNA delivery to the liver in vivo using Design of Experiment (DOE) methodologies including Definitive Screening and Fractional Factorial Designs. By simultaneously varying lipid ratios and structures, we developed an optimized formulation which increased the potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles 7-fold relative to formulations previously used for siRNA delivery. Key features of this optimized formulation were the incorporation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and increased ionizable lipid:mRNA weight ratios. Interestingly, the optimized lipid nanoparticle formulation did not improve siRNA delivery, indicating differences in optimized formulation parameter design spaces for siRNA and mRNA. We believe the general method described here can accelerate in vivo screening and optimization of nanoparticle formulations with large multidimensional design spaces.

The induction of a strong cytotoxic T cell response is an important prerequisite for successful immunotherapy against many viral diseases and tumors. Nucleotide vaccines, including mRNA vaccines with their intracellular antigen synthesis, have been shown to be potent activators of a cytotoxic immune response. The intracellular delivery of mRNA vaccines to the cytosol of antigen presenting immune cells is still not sufficiently well understood. Here, we report on the development of a lipid nanoparticle formulation for the delivery of mRNA vaccines to induce a cytotoxic CD 8 T cell response. We show transfection of dendritic cells, macrophages, and neutrophils. The efficacy of the vaccine was tested in an aggressive B16F10 melanoma model. We found a strong CD 8 T cell activation after a single immunization. Treatment of B16F10 melanoma tumors with lipid nanoparticles containing mRNA coding for the tumor-associated antigens gp100 and TRP2 resulted in tumor shrinkage and extended the overall survival of the treated mice. The immune response can be further increased by the incorporation of the adjuvant LPS. In conclusion, the lipid nanoparticle formulation presented here is a promising vector for mRNA vaccine delivery, one that is capable of inducing a strong cytotoxic T cell response. Further optimization, including the incorporation of different adjuvants, will likely enhance the potency of the vaccine.

Advances in biomaterials for drug delivery are enabling significant progress in biology and medicine. Multidisciplinary collaborations between physical scientists, engineers, biologists, and clinicians generate innovative strategies and materials to treat a range of diseases. Specifically, recent advances include major breakthroughs in materials for cancer immunotherapy, autoimmune diseases, and genome editing. Here, strategies for the design and implementation of biomaterials for drug delivery are reviewed. A brief history of the biomaterials field is first established, and then commentary on RNA delivery, responsive materials development, and immunomodulation are provided. Current challenges associated with these areas as well as opportunities to address long-standing problems in biology and medicine are discussed throughout.

One of the most significant challenges in the development of clinically-viable delivery systems for RNA interference therapeutics is to understand how molecular structures influence delivery efficacy. To this end, we synthesized 1400 degradable lipidoids and evaluated their transfection ability and structure function activity. Here we show that lipidoid nanoparticles mediate potent gene knockdown in hepatocytes and immune cell populations upon IV administration to mice (siRNA EC50 values as low as 0.01 mg/kg). Surprisingly, we identify four necessary and sufficient structural and pKa criteria that robustly predict the ability of nanoparticles to mediate greater than 95% protein silencing in vivo. Because these efficacy criteria can be dictated through chemical design, this discovery could eliminate our dependence on time-consuming and expensive cell culture assays and animal testing. Herein, we identify promising degradable lipidoids and describe new design criteria that reliably predict in vivo siRNA delivery efficacy without any prior biological testing.

Therapeutic nucleic acids hold great promise for the treatment of disease but require vectors for safe and effective delivery. Synthetic nanoparticle vectors composed of poly(β-amino esters) (PBAEs) and nucleic acids have previously demonstrated potential utility for local delivery applications. To expand potential utility to include systemic delivery of mRNA, here we develop hybrid polymer-lipid nanoformulations for systemic delivery to the lung. Through co-formulation of PBAEs with lipid-polyethylene glycol (PEG), mRNA formulations were developed with increased serum stability and increased in vitro potency. The formulations were capable of functional delivery of mRNA to the lungs following intravenous administration in mice. To our knowledge, this is the first description of systemic administration of mRNA for delivery to the lungs using a degradable polymer-lipid nanoparticle.