Self-assembly of photoresponsive azo-containing phospholipids with a polar group as the tail

Vesicles or micelles prepared from amphiphiles with azobenzene (Az) moieties and long alkyl chains have attracted much attention in drug delivery systems. To induce release behavior from smart carriers via trans–cis photoisomerization of the Az groups, UV light exposure is typically used, but it can damage DNA and hardly penetrates cells. In this paper, Az-containing phospholipids without long alkyl tails were designed and synthesized; in these compounds, the end group of the Az moiety was substituted with a –NO2 and –OCH3 group (abbreviated N6 and M6, respectively). N6 self-assembled into H-aggregates with an interdigitated bilayered structure in water through the antiparallel orientation due to π–π interactions of the Az group, the attractive van der Waals forces, and the interactions and bending behavior of the phosphocholine groups. Vesicles showing visible light stimuli-responsive behavior were obtained by mixing N6 and M6, and the release of encapsulated calcein was triggered by visible light.


Characterization of N6, M6, and N3
NMR spectra were measured in CD 3 OD solutions. 1 H NMR spectra were recorded on a JEOL JNM-LA400 at 400 MHz. The chemical shifts of the 1 H NMR signals were referenced to Me 4 Si as the internal standard ( = 0.00) and are expressed as chemical shifts in ppm (), multiplicity, coupling constant (Hz), and relative intensity. Elemental

Crystal data and X-ray single crystal structure of N3
For the evaluation of the self-assembling structure of N6 and N6/M6 mixtures, xray single crystal structure analysis of N3 was carried out. The single crystal was obtained by slow cooling of aqueous hot solution of N3 containing acetonitrile. The crystal data were collected by a R-AXIS RAPID diffractometer using multi-layer mirror monochromated Cu-K radiation at 90 K, and the crystal structure was analyzed using the SHELXL within the OLEX-2 GUI for modelling the molecular structures. The   Figure   2(d) shows the part of the crystal structure of N3 focusing on the phosphocholinemoiety to clarify the formation mechanism of the stacked bilayer of N6. The magentacolored part represents the phosphocholine-moiety of N3. The part bended away from the long axis of the Az skeleton to increase the affinity for the water layer through hydrogen bonding. The bending behavior of the phosphocholine moiety might enable phospholipids without a long alkyl tail to form a bilayer-type self-assembled structure through H-aggregation. Actually, it is impossible for the hydrophilic parts amphiphiles with only ammonium moieties but no phosphocholine moiety to bend in this way. Figure S2. An X-ray single crystal structure of N3.

Effect of hydrophilic moiety on the formation of H-aggregates
As shown in Figure S5(a), the blue shift was not observed when the concentration of NAz6TM increased. It suggested that the NAz6TM molecules in water did not assembled into aggregation even at a higher concentration. As shown in Figure S5(b), the same situation was also found in the mixture of NAz6TMA and MAz6TMA in water. Figure S5. Absorption spectra of (a) NAz6TMA; (b) a mixture of NAz6TMA and MAz6TMA (R = 50/50) in water S10 Figure S6(a), an absorption band at approximately 362 nm was observed at lower concentration in water (e.g. c = 0.05 mM). The wavelength of the absorption band was shifted to shorter wavelength (329 nm) by increasing the concentration (e.g. c = 1.0 mM). The blue shift of this band with increasing concentration was due to the formation of H-aggregates, and the driving force is furtherly enhanced by the stacking of the Az moieties. Figure S6

1 HNMR characterization to form self-assembled vesicles in water
The solubility of Az-containing phospholipids is poor at room temperature, which resulted in the concentration of samples too low and signals too weak to be detected. Therefore, 1 H NMR measurements were performed at 60 °C in D 2 O to investigate interactions between N6 and M6 ( Figure S7). When measuring M6 in D 2 O at 60 °C using 1 H NMR, one of sharp peaks was obtained at 7.7 (δ/ppm), corresponding to the symmetrical aromatic hydrogens (-ArH-N=, 4H) (M6-a). Meanwhile, the peak at 7.8 (δ/ppm), was assigned to the aromatic hydrogens (O 2 N-ArH-, 2H) (N6-a) of N6.
However, a relatively broadened and shifted peak was observed at 7.51 (δ/ppm) for the M6-a and N6-a, when incubated N6/M6 mixture in D 2 O at 60 °C. The reason for the chemical shifts and broaden peaks was that electrostatic interactions was introduced between the groups MeO-and O 2 N-. Consequently, the change of hydrogen environment drove the occurrence of chemical shifts and broadened peaks.  Four peaks at 23, 27, 30, 38 Å were mainly observed, and the peak profiles were changed by the change in the N6/M6 mixing ratio. By the comparison with the results shown in Figure 2, the scattering at 38 Å could be assigned as the bilayer thickness of the vesicles. On the other hand, peaks at 30 and 27 Å were due to layer thickness of N6 and M6 crystalline powders. As shown in Figure 2, there was neither phase separation nor recrystallization of N6 and M6. In spite of careful treatment, freeze drying process might influenced the crystal formation. Further, an unassignable broad scattering at 23 Å were sometimes observed.

Changes in the absorption spectra of the vesicles as a function of irradiation time of UV light
The N6/M6 mixture (R = 50/50, c = 0.5 mM) in water are vesicles with an average diameter of ca. 500 nm, which has been presented in Figure S11. Then, the changes in the absorption spectra of the vesicles as a function of irradiation time of UV light was detected. As shown in Figure S12, before UV irradiation, there was an absorption band at 340 nm, corresponding to the H-aggregation. The UV irradiation respectively caused the decrease and increase in absorbance at 340 nm and at 450 nm due to the photoisomerization of the Az groups in N6 and M6 from trans-form to cis-one, and the reverse situation was observed upon visible light irradiation less than 30 s.