Significance The effectiveness of nucleic acid drugs is limited by inefficient delivery to target tissues and cells and by unwanted accumulation in off-target organs. Although thousands of chemically distinct nanoparticles can be synthesized, nanoparticles designed to deliver nucleic acids in vivo were first tested in cell culture, yielding poor predictions for delivery in vivo. To facilitate testing of many nanoparticles in vivo, we designed and optimized a high-throughput DNA barcoding system to simultaneously measure nucleic acid delivery mediated by dozens of distinct nanoparticles in a single mouse. This nano-barcoding system can be used to study hundreds, or even thousands, of nanoparticles directly in vivo and could dramatically accelerate the discovery and understanding of nanoparticle drug delivery systems.

Abstract Nucleic acid therapeutics are limited by inefficient delivery to target tissues and cells and by an incomplete understanding of how nanoparticle structure affects biodistribution to off-target organs. Although thousands of nanoparticle formulations have been designed to deliver nucleic acids, most nanoparticles have been tested in cell culture contexts that do not recapitulate systemic in vivo delivery. To increase the number of nanoparticles that could be tested in vivo, we developed a method to simultaneously measure the biodistribution of many chemically distinct nanoparticles. We formulated nanoparticles to carry specific nucleic acid barcodes, administered the pool of particles, and quantified particle biodistribution by deep sequencing the barcodes. This method distinguished previously characterized lung- and liver- targeting nanoparticles and accurately reported relative quantities of nucleic acid delivered to tissues. Barcode sequences did not affect delivery, and no evidence of particle mixing was observed for tested particles. By measuring the biodistribution of 30 nanoparticles to eight tissues simultaneously, we identified chemical properties promoting delivery to some tissues relative to others. Finally, particles that distributed to the liver also silenced gene expression in hepatocytes when formulated with siRNA. This system can facilitate discovery of nanoparticles targeting specific tissues and cells and accelerate the study of relationships between chemical structure and delivery in vivo.

The clinical and scientific potential of nucleic acid therapies is limited by inefficient drug delivery to target cells. Drug delivery vehicles must avoid clearance by the immune and reticuloendothelial systems, access the correct organ, and enter specific cells within a complex tissue microenvironment (1⇓–3). At each of these steps, anatomical structures and biological molecules can actively engage the vehicles and influence their final destination. For example, fenestrations in endothelial cells lining the liver may improve access to hepatocytes, tight junctions in brain endothelial cells inhibit delivery across the blood–brain barrier, the basement membrane in renal tubules can disassemble cationic delivery vehicles, and serum proteins can bind nanoparticles in the blood and affect their interactions with target cells (4⇓⇓–7). It is not currently possible to recapitulate the totality of this complex process in cell culture.

Thousands of nanoparticles with distinct chemical structures and properties have been synthesized to overcome drug delivery obstacles and control nanoparticle biodistribution (8⇓⇓⇓⇓⇓⇓–15). Due to the expensive and laborious nature of in vivo experiments, the current practice is to characterize these diverse nanoparticle “libraries” in cell culture before selecting a small number to test in vivo (8⇓⇓⇓⇓⇓⇓–15). However, in vitro transfection can be a poor predictor of in vivo transfection, and in vitro screens cannot predict whole-body biodistribution, which influences off-target effects (16, 17).

We sought to develop a system that increases the number of nanoparticles testable in vivo. To increase the throughput of in vivo studies, we used a rapid microfluidic mixing system to encapsulate nucleic acid barcodes inside nanoparticles and administered them as a single pool to mice (Fig. 1 A and B). We recovered the barcodes from tissues and cells and used deep sequencing to obtain counts for those barcodes in each sample of interest (18). Deep sequencing is a high throughput, cost-effective method to precisely quantitate nucleic acid species; it has led to the identification of molecules or peptides with specific biological activities and enabled pooled screening with shRNAs, cDNAs, and labeled pools of RNA (19⇓–21). By associating specific nanoparticles with unique DNA barcode sequences, we can now reliably measure the biodistribution of many nanoparticles in a single animal (Fig. 1C).

Fig. 1. DNA barcoded nanoparticles for high throughput in vivo nanoparticle delivery. (A) Using high-throughput fluidic mixing, nanoparticles are formulated to carry a DNA barcode. (B) Many nanoparticles can be formulated in a single day; each nanoparticle chemical structure carries a distinct barcode. Particles are then combined and administered simultaneously to mice. Tissues are then isolated, and delivery is quantified by sequencing the barcodes. In this example, nanoparticle 1 delivers to the lungs, nanoparticle 2 delivers to the liver, and nanoparticle N delivers to the heart. (C) This DNA barcode system enables multiplexed nanoparticle-targeting studies in vivo, improving upon the current practice, which relies on in vitro nanoparticle screening to identify lead candidates.

Discussion Genetic therapeutics, including aptamers, antisense oligonucleotides, RNAi, and gene-editing technologies, function through distinct biological mechanisms (28, 29). However, all genetic therapies are limited by the inability to predict delivery to on- and off-target tissues. Although syntheses of chemically distinct nanoparticles can be high throughput, characterization of nanoparticle behavior in vivo is still low throughput (8⇓⇓⇓⇓⇓⇓–15). Rapidly screening chemically distinct nanoparticles in vivo could accelerate preclinical screening and enable efforts to relate chemical structure to biological function. By incorporating deep sequencing, our approach dramatically increases the number of particles that can be simultaneously measured, as well as improves the sensitivity, specificity, and accuracy of those measurements. Our work, as well as DNA barcoded particles that were shown to target tumors, demonstrates the power of unbiased in vivo approaches (30). Notably, this platform is distinct from previous reports, which conjugate nucleic acids to the exterior of particles to fluorescently label them or use them to identify known pathogenic DNA sequences in bodily fluids (31⇓⇓–34). We carefully tested our workflow to identify biases that may arise from particle mixing or differences in barcode sequence. DNA barcode readouts varied linearly with the input across four orders of magnitude encompassing typical doses of effective nucleic acid therapeutics (0.0001–0.5 mg/kg DNA) (25) (Fig. 2D); the ability to measure a single nanoparticle at a dose as low as 0.0001 mg/kg DNA suggests that dozens, or even hundreds, of nanoparticles can be multiplexed in a single experiment. DNA barcode amplification did not vary with barcode sequence (Fig. 2E). We did not observe any evidence of hybrid particle formation with 7C1 and C12-200 LNPs over 24 h (Fig. 2 B and C) or when 10 C12-200–based LNPs were tested simultaneously (Fig. 2D). However, because hybrid particle mixing may occur with other nanoparticles, especially if dozens or hundreds nanoparticles are tested simultaneously, it will be important to control for, and test, particle mixing. Nevertheless, these data demonstrate that the DNA barcoding approach can be used to rapidly study biodistribution and pharmacokinetics. For example, although we did not uncover any structure–function mechanisms or LNPs with new targeting functionality in this study, we did identify a LNP that performed well in many organs, as well as LNPs that distributed inefficiently in all organs (Fig. 3B). We designed this system to be useful in many in vivo contexts. Because this approach can quantify delivery to cells isolated by flow cytometry (Fig. 2F), we anticipate that future studies will simultaneously study delivery to multiple cell types in a complex microenvironment. Similarly, we believe this system may be used to study how nanoparticle delivery changes with an animal disease state. Finally, although this system cannot directly differentiate between delivery to, and into, a cell, future work could use this approach to study nanoparticle delivery to intracellular and subcellular compartments using standard fractionation approaches, with the goal of identifying nanoparticles that evade lysosomes, remain in the cytoplasm, or, alternatively, enter the nucleus (35). By associating DNA barcodes with ligands, this high-throughput nanoparticle barcoding system may also be used to rapidly identify effective targeting sequences via a process that is akin to phage display. It is unlikely that this system will work for every drug delivery vehicle; it will be most effective for well-tolerated nanoparticles that are stable in solution before injection. In future studies, it will be important to characterize nanoparticle stability before using this system to study the activity of different nanoparticles. Even with these constraints, we anticipate that this methodology will facilitate nanoparticle pharmacokinetic studies and will rapidly accelerate the discovery of nanoparticles with wide-ranging therapeutic and research applications.

Acknowledgments We thank the Animal Imaging and Preclinical Testing and Flow Cytometry Cores at the Koch Institute at MIT. J.E.D. was funded by an MIT Presidential Fellowship; National Defense Science and Engineering Graduate Fellowship; and National Science Foundation Graduate Research Fellowship Program Fellowship. K.J.K. was funded by the Marble Center for Cancer Nanomedicine and the Cancer Center Support (core) Grant P30-CA14051. Y.X., T.E.S., and C.C.D. were funded by the MIT Undergraduate Research Opportunities Program. Funding was provided by the Kathy and Curt Marble Cancer Research Fund/Koch Institute Frontier Grant (to E.T.W. and D.G.A.). Funding was also provided by the NIH Grant DP5-OD017865 (to E.T.W.).