Cross-Linked Micellar Spherical Nucleic Acids from Thermoresponsive Templates

Abstract

A one-pot synthesis of micellar spherical nucleic acid (SNA) nanostructures using Pluronic F127 as a thermoresponsive template is reported. These novel constructs are synthesized in a chemically straightforward process that involves intercalation of the lipid tails of DNA amphiphiles (CpG motifs for TLR-9 stimulation) into the hydrophobic regions of Pluronic F127 micelles, followed by chemical cross-linking and subsequent removal of non-cross-linked structures. The dense nucleic acid shell of the resulting cross-linked micellar SNA enhances their stability in physiological media and facilitates their rapid cellular internalization, making them effective TLR-9 immunomodulatory agents. These constructs underscore the potential of SNAs in regulating immune response and address the relative lack of stability of noncovalent constructs.

Scheme 1. Assembly of Cross-Linked Micellar SNAs from Pluronic F127 Templates and Amphiphilic DNA

Figure 1

(A) DLS histograms of the Pluronic F127 templates before DNA insertion (blue) and the micellar SNAs after cross-linking (red). (B) Plot of the amount of free, unincorporated DNA in the solution (i.e., the dispersion of micellar SNAs), showing complete removal after three centrifugal washing steps at 4 °C. The inset is a photograph of the filtrates after the first three washes, showing that the blue color of the Cy-5 labeled DNA visually disappears after the third wash. (C–D) In situ AFM images of the micellar SNAs after being drop-cast on mica, showing the micellar SNAs as bright dots. The inset shows a distribution centering at 22 ± 8 nm, slightly smaller than the DLS data, as expected for embedded materials.

Figure 2

(A) A schematic representation of the hybridization of micellar SNAs with complementary SNAs. (B) Melting profile of micellar SNA conjugates that have hybridized to the complementary nanoconstructs (SI, Section S5). The hybridized micellar SNAs exhibit a sharper melting transition with a higher melting point in comparison to the corresponding hybridized duplex between complementary linear nucleic acids.

Figure 3

(A) A schematic representation of the serum-stability study of cross-linked micellar SNAs. In the non-cross-linked precursor (top branch), dissociation of the BHQ-modified DNA would result in increased DilC₁₈ fluorescence compared to the cross-linked analog (bottom branch). (B) The fluorescent profiles of DilC₁₈-encapsulated micelles that was functionalized with the BHQ-T₂₀-lipid material but not cross-linked (i.e., the non-cross-linked precursor to the micellar SNA), before (black trace) and after (red trace) dissociation of DNA from the micelle template, showing an increase in fluorescence upon disassembly. (C) The fluorescent profiles of the cross-linked micellar SNAs and the non-cross-linked precursor after being incubated at 37 °C in 10% serum-containing media, showing stark contrast in stability. Unlike their non-cross-linked precursors, the cross-linked micellar SNAs show minimal dissociation.

Figure 4

(A) A confocal fluorescent micrograph of HEK-Blue mTLR9 cells that were incubated with Cy5-labeled micellar SNAs ([DNA] = 100 nM) for 4 h. Cell nuclei were stained with Hoechst 33342 (scale bar = 20 μm). (B) Flow cytometry analysis of HEK-Blue mTLR9 cells that have been incubated with free Cy5-labeled DNA (blue bars) and Cy5-labeled micellar SNAs after 16 h (purple bars), showing a higher fluorescence intensity for the latter. (C) A cell-viability assay for HEK-Blue mTLR9 cells after treatment with micellar SNAs for 24 h. (D) Plots of the amounts of secreted alkaline phosphatase (SEAP) by HEK-Blue cells, as visualized by a colorimetric assay, showing enhanced immunostimulatory activity by micellar SNAs in comparison to control micellar SNAs bearing a T₂₀ sequence and unmodified linear nucleic acids.