Controlling DNA-nanoparticle serum interactions

Abstract

Understanding the interaction of molecularly assembled nanoparticles with physiological fluids is critical to their use for in vivo delivery of drugs and contrast agents. Here, we systematically investigated the factors and mechanisms that govern the degradation of DNA on the nanoparticle surface in serum. We discovered that a higher DNA density, shorter oligonucleotides, and thicker PEG layer increased protection of DNA against serum degradation. Oligonucleotides on the surface of nanoparticles were highly resistant to DNase I endonucleases, and degradation was carried out exclusively by protein-mediated exonuclease cleavage and full-strand desorption. These results enabled the programming of the degradation rates of the DNA-assembled nanoparticle system from 0.1 to 0.7 h^-1 and the engineering of superstructures that can release two different preloaded dye molecules with distinct kinetics and half-lives ranging from 3.3 to 9.8 h. This study provides a general framework for investigating the serum stability of DNA-containing nanostructures. The results advance our understanding of engineering principles for designing nanoparticle assemblies with controlled in vivo behavior and present a strategy for storage and multistage release of drugs and contrast agents that can facilitate the diagnosis and treatment of cancer and other diseases.

Keywords: DNA nanostructures; controlled cargo release; nanoparticle assembly; serum resistance; serum stability.

Fig. 1.

(A) Schematics of serum degradation. DNA linkers mediate assembly of GNP-DNA into supramolecular structures. Incubation with serum causes assembly breakdown into individual component nanoparticles followed by the degradation of surface-immobilized DNA. Various parameters and mechanisms determine the rate of this process. (B) Experimental setup. PAGE band patterns corresponding to different degradation mechanisms are indicated (A).

Fig. 2.

Effect of DNA density, length, and PEG-layer thickness on serum degradation. (A) Degradation of nanoparticles with different DNA grafting ratios. Degradation rates (densitometry curves are in SI Appendix, Fig. S2D) plotted against DNA loading (quantified in SI Appendix, Fig. S2A). The number above each data point indicates the grafting ratio. All nanoparticles are backfilled with 5 kDa PEG. (B) Serum degradation of nanoparticles with ssDNA or dsDNA of variable length. Degradation rates based on densitometry curves are in SI Appendix, Fig. S4A. All nanoparticles are backfilled with 5 kDa PEG. Statistical significance between ssDNA and dsDNA for each oligonucleotide length was determined by unpaired t test. *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001. (C) Serum degradation of nanoparticles with ssDNA and 5, 10, or 20 kDa PEG backfill (grafted at 16,000 PEG GNP). Densitometry curves are in SI Appendix, Fig. S4B. Error bars are 95% confidence intervals of curve fitting based on three experimental replicates; representative PAGE gel data are shown.

Fig. 3.

Mechanisms of serum DNA degradation on nanoparticle surface. (A) 3′ Exonucleases play a major role in degradation. Degradation of DNA45 compared with end-protected DNA45-invT, DNA45-HP, and inverted DNA45-5′. Densitometry curves are in SI Appendix, Fig. S7. (B) DNase I contribution to serum degradation of GNP-DNA is not significant. Nanoparticles with ssDNA45-5′ or dsDNA45 are subjected to degradation by DNase I (at 500× serum concentration) or serum with and without DNase I inhibitor actin (densitometry curves are in SI Appendix, Fig. S10). Actin inhibited activity of concentrated DNase I but had no effect on serum degradation. ****P ≤ 0.0001. (C) Dithiol linker does not prevent serum-induced DNA desorption. Comparison of serum degradation of DNA45-5′ strands adsorbed onto nanoparticle with a monothiol or dithiol linker (densitometry curves are in SI Appendix, Fig. S13). (D) Proteins and not small molecules carry out serum degradation. Nanoparticles incubated with complete serum or its protein and small molecule fractions (SI Appendix, Fig. S16). Error bars are 95% confidence intervals of curve fitting based on three experimental replicates; representative PAGE gel data are shown. Statistical significance was determined by (A) ordinary one-way ANOVA or (B and C) unpaired t test. All nanoparticles had 5 kDa PEG backfill.

Fig. 4.

Controlling serum degradation rates of GNP-DNA assemblies. (A) Representative TEM (zoomed out TEM is shown in SI Appendix, Figs. S22–S26) of five assembled structures (details are in SI Appendix, Fig. S18) incubated in serum. Two-layer structure: 5 kDa PEG; all other structures: 10 kDa PEG. (B) Associated serum degradation rates. Number of satellites per core nanoparticle quantified by direct TEM counting. Degradation rates are based on single-phase decay fits of these data (SI Appendix, Fig. S19A). Increase in satellite density correlates with improved protections against serum breakdown. (C) Degradation rates of the two-layer structures; 5-nm satellites in the outer layer were quantified separately from the inner-layer 3-nm satellites. Degradation of the outer layer occurs much more rapidly that of the inner layer. Degradation rates are based on single-phase decay fits of these data (SI Appendix, Fig. S19B); error bars are 95% fitting confidence intervals. Statistical significance was determined by (B) ordinary one-way ANOVA or (C) unpaired t test. ****P ≤ 0.0001.

Fig. 5.

Controlling the rate of serum breakdown of two-layer assemblies backfilled with 5 kDa PEG as the mechanism of multistage cargo release. (A) Fluorescent dyes FAM and Cy5 loaded onto the outer and inner layers of the structure, respectively (details are in SI Appendix, Fig. S18). Fluorescence plotted against wavelength (λ) shows peaks corresponding to emissions of both dyes. (B) Representative TEM images (zoomed out TEM is shown in SI Appendix, Fig. S27) of structure breakdown in serum. (C) Quantification of breakdown by separate TEM counting of outer-layer 5-nm satellites and inner-layer 3-nm satellites. Data are normalized to 0-h counts. The outer layer breaks down much faster than the inner layer. (D) Decrease in the fluorescence of assemblies over time as a result of dye release from the surface. Data are normalized to 0 h and fitted with single-phase decay curves. (E) Dye release rates are based on decay curves in D. FAM is released significantly faster than Cy5. Error bars are (C and D) SDs or (E) 95% fitting confidence intervals. Statistical significance was determined by unpaired t test based on three experimental replicates. ****P ≤ 0.0001.