Organic Nanoflowers from a Wide Variety of Molecules Templated by a Hierarchical Supramolecular Scaffold

Abstract

Nanoflowers (NFs) are flowered-shaped particles with overall sizes or features in the nanoscale. Beyond their pleasing aesthetics, NFs have found a number of applications ranging from catalysis, to sensing, to drug delivery. Compared to inorganic based NFs, their organic and hybrid counterparts are relatively underdeveloped mostly because of the lack of a reliable and versatile method for their construction. We report here a method for constructing NFs from a wide variety of biologically relevant molecules (guests), ranging from small molecules, like doxorubicin, to biomacromolecules, like various proteins and plasmid DNA. The method relies on the encapsulation of the guests within a hierarchically structured particle made from supramolecular G-quadruplexes. The size and overall flexibility of the guests dictate the broad morphological features of the resulting NFs, specifically, small and rigid guests favor the formation of NFs with spiky petals, while large and/or flexible guests promote NFs with wide petals. The results from experiments using confocal fluorescence microscopy, and scanning electron microscopy provides the basis for the proposed mechanism for the NF formation.

Figure 1

Preparative protocol for f-SHS and the corresponding nanoflowers (NFs). 8ArG self-assembly into SGQs followed by the (i) formation of SHS by LCST, is followed by (ii) decreasing the ionic strength to “fix” (i.e., kinetically stabilized) the SHS (identified as f-SHS). (iii) The encapsulation method relies on an osmotic gradient to the form f-SHS@Guest complexes, and is suitable for the complexation of sensitive guests like proteins, which could be denatured by the initial high ionic strengths. (iv–v) Drop casting the solutions of f-SHS@Guest followed by air-drying leads to the formation of two families of NFs, having either spiky or wide petals as a result of complexing small or large guests, respectively. Some groups in the space filling representation of 1 (e.g., imidazole moiety) are omitted for clarity. We represent complexed guests with an “@”, for example, f-SHS with encapsulated DTR 3 kDa is represented as f-SHS@ DTR-3 where “3” is the molecular weight of the DTR in kDa.

Figure 2

Microscopy images of f-SHS@Guest: (a–h) in solution by CLSM, and in (i–o) the solid state by SEM. The guests corresponding to each image are (a) RhB; (b) Dox; (c) Cyt; (d) DsR; (e) DTR-3; (f) DTR-10; (g) DTR-70; (h) mCh. SEM images correspond to (i) f-SHS alone; (j) f-SHS@ Dox; (k) f-SHS@RhB; (l) lyophilized f-SHS; (m) f-SHS@DTR-3; (n) f-SHS@DTR-10; (o) f-SHS@DTR-70. The SEM images were drop-casted from a solution of 8ArG (0.303 mM, 121 mM KI, in 1X PBS, pH 7.4) and air-dried at 36 °C.

Figure 3

SEM images of 0.303 mM polystyrene beads (PSBs) used for control experiments. (a) PSBs alone (X14000); (b) PSBs + DTR-3 (5 equiv; 0.07 mM; X7000); (c) PSBs + RhB (10 equiv; 3.4 mM; X6000). (d–f) Zooms of a, b, and c at (d) X60000; (e) X30000; and (f) X30000, respectively. All the samples were air-dried at 36 °C after incubating for 1 h with the indicated guest molecule.

Figure 4

SEM images of different organic NFs after air-drying at 36 °C (a–f) and 65 °C (g–i). Protein guests: (a) f-SHS@Cyt; (b) f-SHS@mCh; (c) f-SHS@Ova; (d) f-SHS@DsR. pDNA guests: (e) f-SHS@pCri, (f) f-SHS@pGFP; (g) f-SHS alone. Protein guest: (h) f-SHS@Ova; small molecule guest: (i) f-SHS@RhB. The NFs from f-SHS@Ova (c) were observed primarily on the carbon section of the SEM grid, in contrast to the rest of NFs studied, which formed on the copper section of the grid.

Figure 5

Mechanistic hypothesis for the formation of organic NFs from the f-SHS@Guest complexes: (a) For small/rigid guests like RhB, the guest diffuses throughout the porous f-SHS by osmotic gradient until it fills the gel-like interior. Upon removing the solvent (e.g., 36 or 65 °C), the concentration of guest increases, which lead to enhanced noncovalent interactions resulting in the spiky NFs from f-SHS@RhB. This seems to promote a crystallization growth perpendicular to the particle’s surface leading to the formation of spiky-petalled NFs. (b) Larger or more flexible guests like DsR are concentrated on or near the surface of the f-SHS favoring the formation of the wide-petalled NF surface in f-SHS@DsR.

Figure 6

Time course CLSM images of f-SHS incubated with DTR-3 (5 equiv., 0.07 mM). Images at 0 s, 600 s (10 min), 1300 s (21.7 min), 2400 s (40 min), 2810 s (46.8 min), and 3990 s (1.1 h). The images were taken with an EC Plan-Neofluar 40X/0.75 objective, excitation wavelength of 561 nm and an emission filter of LP 575 (25 °C). The f-SHS were prepared from following the conditions described in Figures 2 and 3.