Modification of primary amines to higher order amines reduces in vivo hematological and immunotoxicity of cationic nanocarriers through TLR4 and complement pathways

Randall Toy; Pallab Pradhan; Vijayeetha Ramesh; Nelson C. Di Paolo; Blake Lash; Jiaying Liu; Emmeline L. Blanchard; Philip J. Santangelo; Dmitry M. Shayakhmetov; Krishnendu Roy

A wide variety of nanoparticles have been developed over the past decades to facilitate the delivery of nucleic acid-based therapies, such as plasmid DNA, oligonucleotides, shortinterfering RNAs (siRNA), micro RNA (miRNA) and messenger RNA (mRNA). [1, 2] To form nanoparticles, anionic nucleic acids are often complexed with cationic polymers or cationic lipids -a process that results in nanoparticles with efficient nucleic acid loading and favorable physical characteristics for intracellular delivery of the cargo in vitro. [3] , [4] Despite several desirable attributes, cationic nanoparticles induce significant in vivo toxicity, especially when delivered systemically, which has prevented their clinical translation even after decades of research. For lipid-based particles, toxicity profiles have been improved by using ionizable and neutral lipids with other excipients. In fact, most current clinical trials for systemic delivery using nanoparticles use lipid-based particles. [5] [6] [7] Meanwhile, a whole class of materials (cationic polymers), despite demonstrating promising in vitro efficacy results for decades, has not advanced into the clinic. In some cases nanoparticles consisting of modified cyclodextrin or protamine were evaluated in Phase I clinical trials, but further studies were not pursued. [8, 9] An ideal nucleic acid delivery agent must ensure a balance between low toxicity and high therapeutic efficacy in vivo -two attributes that have been difficult to achieve simultaneously using cationic polymer nanoparticles. [10] Chemists and material scientists have empirically optimized physicochemical properties (e.g. size, surface functional groups, charge) to improve nanoparticle safety and transfection efficiency [11] [12] [13] [14] [15] [16] [17] These studies identified formulations with lower in vitro toxicity, and in some cases, the potential for in vivo efficacy in animal models, but detailed studies on the relationship between nanoparticle design and mechanisms of in vivo systemic toxicity have been scarce or very limited in scope. [18] A widely studied modification to reduce systemic toxicity of nanoparticles is the addition of polyethylene glycol (PEG) to the formulations. A major drawback of PEG-modified formulations, however, is that anti-PEG antibodies can be generated, which activate the complement cascade and further decrease therapeutic efficacy. [19] [20] [21] [22] This shortcoming of PEG, which could not be predicted in vitro, highlight the importance of studies that investigate the detailed in vivo mechanisms by which nanoparticles induce systemic toxicity.