Mechanism of endosomal escape by $p\mathrm{H}$-responsive nucleic-acid vectors

Successful intracellular delivery of nucleic acids (NAs) hinges on many factors, one of them being NAs’ efficacious escape from endosomes. As competent NA vectors, $p\mathrm{H}$-responsive gemini surfactants (GSs) might achieve high efficacy by facilitating endosomal escape. However, how the GSs assist the escape remains debated as many proposed mechanisms still lack experimental support, which hinders replication and further improvement of the efficient delivery. Here, via UV, fluorescence spectroscopy, and small-angle neutron scattering (SANS), we examined a $p\mathrm{H}$-responsive GS's and a $p\mathrm{H}$-unresponsive GS's capabilities to compact DNA and withstand binding competition, and their interactions with model endosomal and lysosomal membranes, at varied $p\mathrm{Hs}$. Acidification-driven enhancement of DNA-compaction capability and of stability against binding competition were found specific to the $p\mathrm{H}$-responsive GS. Alongside the $p\mathrm{H}$-responsive GS's structural perturbation to the membranes as observed with SANS, the features suggest that $p\mathrm{H}$-responsive GSs facilitate endosomal escape by releasing excess GS molecules from DNA-GS complexes upon acidification in endosome maturation, with the released GS molecules disrupting endosomal and lysosomal membranes and thereby assisting the escape. A general design principle for NA vectors is proposed on the basis of this experimental finding.

Figure 1

UV absorption assay for DNA complexation with (a) 12-6-12 or (b) $p\mathrm{HC}12$ at varied $p\mathrm{Hs}$. Reduction in Abs reflects complexation between DNA and GSs. Efficient vectors are expected to reach large Abs reduction with low vector-to-DNA molar ratios. Dashed lines are to guide the eyes. Chemical structures of the GSs are also shown. Data are expressed as mean ± S.D. and were collected from at least three independently prepared samples.

Figure 2

UV absorption assay for binding competition between the anionic surfactant SDS and (a) DNA-12-6-12 or (b) DNA-$p\mathrm{HC}12$ complexes at varied $p\mathrm{Hs}$. The competition can result in the GS binding to SDS and re-disperse DNA to make it UV detectable. Stable complexes are expected to require high SDS-to-GS molar ratios to be destabilized and reach certain Abs elevation. Dashed lines are to guide the eyes. The GS-to-${\mathrm{DNA}}_{\mathrm{bp}}$ molar ratios were (a) ∼2.3 mol/mol for all $p\mathrm{Hs}$; and (b) ∼2.4 mol/mol, ∼2.0 mol/mol, ∼1.5 mol/mol, and ∼1.3 mol/mol for $p\mathrm{H}=7$, 6, 5, and 4, respectively. Data are expressed as mean ± S.D. and were collected from at least three independently prepared samples.

Figure 3

Fluorescence assay for binding competition between endosome- and lysosome-mimicking LUVs and (a) DNA-12-6-12 or (b) DNA-$p\mathrm{HC}12$ complexes at varied $p\mathrm{Hs}$. The competition can strip DNA base pairs of the GSs and allow the fluorescence tag, EtBr, to bind to the GS-free base pairs, which enhances EtBr fluorescence. Stable complexes are expected to have low proportions of GS-free base pairs at a given lipid concentration. Dashed lines are fits against a mass-balance-based equation. Data are expressed as mean ± S.D. and were collected from at least three independently prepared samples.

Figure 4

SANS profiles of absolute scattering intensity $I$ against momentum transfer $|\vec{Q}|$ for endosome- and lysosome-mimicking LUVs with or without adding the dGSs at (a) $p\mathrm{H}=7$ and (b) $p\mathrm{H}=4$. Dashed lines are fittings against the structural models of hollow vesicle (for GS-free samples) and micelle (for GS-containing samples). The scattering data for the dGSs alone (diamonds) confirm the matching of coherence scattering length densities of the dGSs and the buffer.