Self-Sorting and Co-Assembly of Fluorinated, Hydrogenated, and Hybrid Janus Dendrimers into Dendrimersomes

Abstract

The modular synthesis of a library containing seven self-assembling amphiphilic Janus dendrimers is reported. Three of these molecules contain environmentally friendly chiral-racemic fluorinated dendrons in their hydrophobic part (R_F), one contains achiral hydrogenated dendrons (R_H), while one denoted hybrid Janus dendrimer, contains a combination of chiral-racemic fluorinated and achiral hydrogenated dendrons (R_HF) in its hydrophobic part. Two Janus dendrimers contain either chiral-racemic fluorinated dendrons and a green fluorescent dye conjugated to its hydrophilic part (R_F-NBD) or achiral hydrogenated and a red fluorescent dye in its hydrophilic part (R_H-RhB). These R_F, R_H, and R_HF Janus dendrimers self-assembled into unilamellar or onion-like soft vesicular dendrimersomes (DSs), with similar thicknesses to biological membranes by simple injection from ethanol solution into water or buffer. Since R_F and R_H dendrons are not miscible, R_F-NBD and R_H-RhB were employed to investigate by fluorescence microscopy the self-sorting and co-assembly of R_F and R_H as well as of phospholipids into hybrid DSs mediated by the hybrid hydrogenated-fluorinated R_HF Janus dendrimer. The hybrid R_HF Janus dendrimer co-assembled with both R_F and R_H. Three-component hybrid DSs containing R_H, R_F, and R_HF were formed when the proportion of R_HF was higher than 40%. With low concentration of R_HF and in its absence, R_H and R_F self-sorted into individual R_H or R_F DSs. Phospholipids were also co-assembled with hybrid R_HF Janus dendrimers. The simple synthesis and self-assembly of DSs and hybrid DSs, their similar thickness with biological membranes and their imaging by fluorescence and ¹⁹F-MRI make them important tools for synthetic biology.

Graphical Abstract

INTRODUCTION

Synthetic lipids¹ such as phospholipids and glycolipids self-assemble into vesicles that mimic biological membranes. The amphiphilic structure of the lipids contains a hydrophilic head and hydrophobic tails. Lipophilic groups such as alkyl chains and fluorophilic groups such as fluorocarbon fragments have been successfully incorporated into lipids as their hydrophobic tails^1b and therefore form hydrogenated and fluorinated vesicles with different stabilities and permeabilities.² The immiscibility of hydrogenated and fluorinated components induced the phase separation of these domains.^2h Fluorinated amphiphiles have been employed for biomedical applications including for drug and gene delivery.^2d,e Fluorinated components including fluoropolymer nanoparticles also present potential functions such as ¹⁹F magnetic resonance imaging (MRI) agents.³ This requires stable and biocompatible fluorinated vesicles, which can be synthesized and self-assembled by simple methods. The miscibility of hybrid structures with both hydrogenated and fluorinated chains have been preliminarily investigated in phospholipids and glycolipids.⁴ However, very little is known about their assemblies.

Amphiphilc Janus dendrimers⁵ containing both hydrophobic (hydrogenated lipophilic chains) and hydrophilic dendrons self-assemble into nanoscale vesicular dendrimersomes (DSs) by simple injection of their solution in a water miscible organic solvent into water and buffer and into micrometer-scale giant DSs by film hydration. The sizes of DSs can be predicted from the thickness of their bilayer in the bulk state and from the concentration of the Janus dendrimer.^5b It is expected that hydrogenated chains can be replaced with fluorinated chains leading to a new family of fluorinated vesicles named fluorinated DSs that will follow the same self-assembly principles as hydrogenated DSs.

Hybrid vesicles co-assembled from lipids and either block copolymers⁶ or Janus dendrimers^5c provided a platform for the incorporation of biological cell membrane components into polymersomes and DSs.^5c Polymersomes⁷ generate bilayers with a thickness of 8–50 nm and therefore are not compatible with biological membranes that have a thickness of ~4 nm,^6c while DSs overcame this biocompatibility issue.^5c Hence the design and synthesis of stable and biocompatible fluorinated DSs and of their hybrid structures with biological membranes including phospholipids and glycolipids became achievable.

Here we report the design and synthesis of a library containing hydrogenated, environmentally friendly chiral-racemic fluorinated and hybrid hydrogenated-fluorinated amphiphilic Janus dendrimers. Fluorinated and hydrogenated Janus dendrimers conjugated in their hydrophilic part with complementary fluorescent dyes were also elaborated. This library allowed for the first time to investigate the self-sorting and the co-assembly of fluorinated, hydrogenated and hybrid hydrogenated-fluorinated Janus dendrimers into fluorinated, hydrogenated and hybrid hydrogenated-fluorinated DSs as well as hybrid vesicles co-assembled from phospholipids and fluorinated Janus dendrimers.

RESULTS AND DISCUSSION

Design and Modular Synthesis of Amphiphilic Janus Dendrimers with Fluorinated, Hydrogenated, and Hybrid Fluorinated-Hydrogenated Chains

The chiral-racemic fluorinated dendrons employed in this study provide access to less ordered structures than those resulting from achiral hydrogenated or perfluorinated dendrons.^8,9 Perfluoropropyl vinyl ether (PPVE) used in their synthesis is an environmentally tolerable fluorinated component that has been incorporated into chiral-racemic fluorinated dendrons in one step reaction performed under very mild reaction conditions (Scheme 1).⁹

Scheme 1. Synthesis of R_F Janus Dendrimers and Hybrid R_HF Janus Dendrimer with Both R_F and R_H Chains.^a.

^aReagents and conditions: (i) t-BuOK (cat.), DMF, 0 °C for 2 h, then 23 °C for 24 h; (ii) H₂, Pd/C, DCM, methanol, 23 °C, 12 h; (iii) 4-(Dimethylamino)pyridinium 4-toluenesulfonate, DCC, DCM, 23 °C, 12 h.

With a catalytic amount of potassium tert-butoxide (t-BuOK) as base in anhydrous dimethylformamide, PPVE chains were introduced as side groups to benzyl (3,5)-, (3,4)-, and (3,4,5)-hydroxyl benzoates (Scheme 1). Previous studies have demonstrated that linear perfluorooctyl alkyl groups enhance the stability of vesicles² and of other supramolecular assembles.⁸ However all linear perfluorooctyl based compounds degrade to the toxic and biopersistent perfluorooctanoic acid that was prohibited by the Environmental Protection Agency.⁹ The chiral-racemic fluorinated PPVE based dendrons employed in this study are prepared in one step, are soluble in ethanol rather than Freon-113 as the perfluorooctyl groups are, and degrade to environmental friendly natural hydroxybenzoic acids and perfluoropropyl chains that are nontoxic and nonbiopersistent.⁹

Subsequent hydrogenation with palladium on carbon in a solvent mixture of dichloromethane and methanol gave the respective fluorinated (R_F) –COOH containing first generation dendrons (3,5)PPVE-G1 (3), (3,4)PPVE-G1 (6), and (3,4,5)PPVE-G1 (9) (Scheme S1). These R_F dendrons were conjugated to form half of a Janus dendrimer via esterification with half protected pentaerythritol (PE) as the core and 3,4,5-tris(methyl triethylene glycol)benzoic acid [(3,4,5)-3EO-G1-(OCH₃)₃] (10) as the hydrophilic fragments, which has been previously developed for the modular synthesis of amphiphilic Janus dendrimers that formed stable but soft DSs with narrow size distribution (Scheme 1).⁵ A stepwise esterification strategy (Scheme 1) was employed for the synthesis of a hybrid Janus dendrimer containing both (3,5)PPVE-G1 (3) (R_F) and 3,5-bis(dodecyloxy)benzoic acid [(3,5)12G1] (14) (R_H) fragments. This hybrid compound was denoted as hybrid R_HF Janus dendrimer.

In order to elucidate the self-assembly and co-assembly/self-sorting of R_F, R_H, and hybrid R_HF Janus dendrimers as individual and as a multi-component system and to investigate their miscibility/immiscibility, two Janus dendrimers with R_F or R_H chains, and with different dyes attached to their hydrophilic parts, were designed and synthesized (Scheme 2, and Scheme S4–5).

Scheme 2. Synthesis of NBD Conjugated Janus Dendrimer with R_F Chains and Rhodamine B Labeled Janus Dendrimer with R_H Chains.^a.

^aReagents and conditions: (i) CuSO₄·5H₂O, sodium ascorbate, THF, water, 23 °C, 24 h.

A Janus dendrimer with R_H chains was labeled with a rhodamine B (RhB) red fluorescent dye, and a Janus dendrimer with R_F chains was labeled with a 7-nitrobenzofurazan (NBD) green fluorescent dye. These azido triethylene glycol conjugated dyes were conjugated with the alkyne groups from the tris(hydroxymethyl)aminomethane (Tris) core via copper-catalyzed click chemistry¹⁰ which was previously developed for the synthesis of carbohydrates (glycan) conjugated Janus glycodendrimers.¹¹

The molecular structures of three R_F Janus dendrimers (3,5)PPVEG1-PE-(3,4,5)-3EO-G1-(OCH₃)₃, (3,4)PPVEG1-PE-(3,4,5)-3EO-G1-(OCH₃)₃, and (3,4,5)PPVEG1-PE-(3,4,5)-3EO-G1-(OCH₃)₃, one hybrid R_HF Janus dendrimers [(3,5)12G1+(3,5)PPVEG1]-PE-(3,4,5)-3EO-G1-(OCH₃)₃ together with a R_H Janus dendrimer (3,5)12G1-PE-(3,4,5)-3EO-G1-(OCH₃)₃^5a–5c are presented in Scheme 3a, in which magenta denotes R_F chains, green denotes R_H chains, and blue denotes hydrophilic parts. The fluorescent R_F Janus dendrimer with NBD label (3,5)PPVEG1-Tris-(3,4,5)-3EO-G1-(OCH₃)₃-NBD and R_H Janus dendrimer with RhB label (3,5)12G1-Tris-(3,4,5)-3EO-G1-(OCH₃)₃-RhB were presented in Scheme 3b, with abbreviations as R_F-NBD and R_H-RhB.

Scheme 3. The Library Containing (a) Three Janus dendrimers with R_F, One Hybrid with R_HF, and One with R_H chains.^a (b) Green Fluorescent Probe (NBD) Labeled Janus Dendrimer with R_F Chains and Red Fluorescent Probe (RhB) Labeled Janus Dendrimer with R_H Chains.

^aDiameters (D_DLS) and polydispersities (in between parentheses) indicated were obtained by DLS and refer to dendrimersomes obtained by injection of their ethanol solution into water (final concentration: 0.5 mg·mL⁻¹).

The structures of the newly synthesized R_F and R_HF Janus dendrimers were confirmed by NMR, in particular a ¹H NMR chemical shift at 6.0–6.2 ppm, with a hydrogen–fluorine coupling ²J_(HF) = 53 Hz (Figure 1c). The five different fluorine atoms in the PPVE chain were observed by ¹⁹F NMR as five groups of signals, from F-CF₂ at the lowest field to F-C(O)H at the highest field (Figure 1a).

Figure 1.

Excerpt of (a–b) ¹⁹F NMR spectra (470 MHz) (a) R_HF Janus dendrimer [(3,5)12G1+(3,5)PPVEG1]-PE-(3,4,5)-3EO-G1-(OCH₃)₃ in CDCl₃, and (b) self-assembled dendrimersomes prepared by injection of an ethanol solution of R_HF Janus dendrimer [(3,5)12G1+(3,5)PPVEG1]-PE-(3,4,5)-3EO-G1-(OCH₃)₃ into D₂O. (c) Excerpt of ¹H NMR spectrum (CDCl₃, 500 MHz) of R_HF Janus dendrimer [(3,5)12G1+(3,5)PPVEG1]-PE-(3,4,5)-3EO-G1-(OCH₃)₃.

Self-Assembly of Amphiphilic Janus Dendrimers with Fluorinated, Hydrogenated, and Hybrid Chains into Nanoscale Unilamellar or Onion-Like DSs

Nanoscale DSs of R_F, hybrid R_HF and of R_H Janus dendrimers were prepared by injection of their ethanol solution into water. Monodisperse peaks with narrow polydispersity (PDI) obtained from dynamic light scattering (DLS) analysis (Scheme 3a, Figure S1) indicated the formation of nanoscale assemblies. R_F, R_HF and R_H Janus dendrimers shared a similar trend of increasing the size of their assemblies with increasing concentration of the Janus dendrimer (Figure S1).^5b The vesicular morphologies of the assemblies were characterized by cryogenic-transmission electron microscopy (Cryo-TEM) (Figure 2a). (3,4)PPVEG1-PE-(3,4,5)-3EO-G1-(OCH₃)₃ and (3,4,5)PPVEG1-PE-(3,4,5)-3EO-G1-(OCH₃)₃ self-assembled into unilamellar DSs with single bilayers, while (3,5)PPVEG1-PE-(3,4,5)-3EO-G1-(OCH₃)₃ and [(3,5)12G1+(3,5)PPVEG1]-PE-(3,4,5)-3EO-G1-(OCH₃)₃ formed multilamellar DSs.¹² In samples of hybrid R_HF Janus dendrimer [(3,5)12G1+(3,5)PPVEG1]-PE-(3,4,5)-3EO-G1-(OCH₃)₃, well-defined onion-like vesicles were observed, with numbers of bilayers from 2 to 11 (Figure 2b). The unilamellar vesicular assemblies from R_H Janus dendrimers was reported before.^7a The thickness of the bilayers of DSs was calculated from cryo-TEM data (Figure 3).^8a,13 Cryo-TEM together with molecular modeling (Figure S2), demonstrated that R_F Janus dendrimers with (3,5)- and (3,4)-substituted PPVE chains and the hybrid R_HF Janus dendrimer showed interdigitation of their fluorinated fragments, with a membrane thickness in the range of 4.5 to 5 nm. These values are comparable to those those of the biological phospholipid-derived membranes (~4 nm) and thinner than that of the interdigitated R_H Janus dendrimer (6.1 nm).^5a This is due to the shorter PPVE chain compared to the dodecyl alkyl chain and/or the perfluorooctyl-based linear chains of the same length in the dendrons.^8a In contrast, the (3,4,5)-substituted derivative showed a thicker bilayer (6.3 nm) indicative of no interdigitation. These results are consistent with the structures reported in a previous report on R_H based Janus dendrimers.^5b

Figure 2.

(a) Cryo-TEM images of DSs assembled by 0.5 mg·mL⁻¹ (final concentration) of R_F and R_HF Janus dendrimers. (b) Selected cryo-TEM images of onion-like DSs self-assembled from 0.5 mg·mL⁻¹ [(3,5)12G1+(3,5)PPVEG1]-PE-(3,4,5)-3EO-G1-(OCH₃)₃ with hybrid R_HF chains and their 3D intensity-plotting images with different numbers of bilayers and diameters. Scale bar = 100 nm.

Figure 3.

Molecular models of the bilayer thickness with and without interdigitation of fluorinated or hydrogenated chains calculated from cryo-TEM images.

A representative ¹⁹F NMR spectrum of the DSs self-assembled from hybrid R_HF Janus dendrimers exhibited a clear fluorine signal (Figure 1b) and suggests potential applications of these or related fluorinated DSs as ¹⁹F MRI imaging agents capable of being loaded with drugs, proteins, nucleic acids, or even with other imaging agents to generate hybrid imaging tools.^5f

The non-crystallization nature of R_F chains of these Janus dendrimers was also confirmed by a combination of differential scanning calorimetry (DSC) (Figure S3) and X-ray diffraction (XRD) (Figure S4) experiments. Below the phase transition temperature at −60 °C, all R_F and hybrid R_HF Janus dendrimers present a glassy phase. The R_H Janus dendrimer exhibited a 2D hexagonal columnar phase as previously reported.^5b These non-crystallizable R_F chains help their self-assembly into soft vesicles.

Self-Sorting and Co-assembly of Janus Dendrimers with R_F, R_F, and Hybrid R_HF chains

In order to be analyzable by fluorescence microscopy, giant DSs (diameter > 1 μm) in phosphate-buffered saline (PBS) from R_F, R_H, and hybrid R_HF Janus dendrimers were prepared by film hydration on a Teflon sheet (Figure 4),⁵ via co-assembly with 1% of the dye-labeled Janus dendrimers R_H-RhB with red fluorescence, and R_F-NBD with green fluorescence. (3,5)PPVEG1-PE-(3,4,5)-3EO-G1-(OCH₃)₃ was selected as the fluorinated R_F molecule based on its structural similarity with the R_H and hybrid R_HF Janus dendrimers (Scheme 3). Due to the expected immiscibility between R_H and R_F, the R_H Janus dendrimer preferentially co-assemble with R_H-RhB (Figure 4a), while the R_F Janus dendrimer co-assemble with R_F-NBD (Figure 4e), leading to bright red or green fluorescence along the boundary of the vesicles. By contrast, since R_F and R_H are not miscible and therefore attempts to co-assemble R_F with R_H-RhB (Figure 4b) and R_H with R_F-NBD (Figure 4d) produced vesicles that exhibit much weaker fluorescence. This miscibility problem was alleviated with the help of R_HF which enabled both dye-labeled Janus dendrimers R_H-RhB (Figure 4c) and R_F-NBD (Figure 4f) to be incorporated into the giant DSs formed from R_HF Janus dendrimers, resulting in intense red or green fluorescence (Figure 4c and 4f). A representative confocal scanning laser tomograph (Movie S1) of a giant DS formed from R_F Janus dendrimer and a reconstructed 3D projection (Movie S2) demonstrate their self-assembly into vesicular structures.

Figure 4.

Representative microscopy images of giant unilamellar vesicles assembled from R_H, R_F and hybrid R_HF Janus dendrimers with an additional 1% (wt/wt) R_H-RhB, or R_F-NBD. Exposure time for fluorescence = 5 ms. Phase-contrast images and fluorescence images under red fluorescence channel or green fluorescence channel were taken by successive exposures on the same vesicle. Images in the same fluorescence channel were normalized to have the same values for the darkest and brightest pixels.

When both R_H-RhB and R_F-NBD were co-assembled with R_HF Janus dendrimers, their giant DSs showed an expected decrease in green emission (Figure 5) (significant p-value < 0.001) due to intermolecular fluorescent resonance energy transfer (FRET)¹⁴ from the green dye to the red dye over a short distance (1–10 nm). This FRET experiment indicates that immiscible R_H-RhB and R_F-NBD Janus dendrimers mix well in giant DSs with the help of R_HF Janus dendrimers without phase separation.

Figure 5.

(a) Representative microscopy images of giant unilamellar vesicles assembled from hybrid R_HF Janus dendrimer with an additional 1% (wt/wt) R_H-RhB, and 1% (wt/wt) R_F-NBD. Exposure time for fluorescence = 5 ms. Phase-contrast images and fluorescence images under red fluorescence channel and green fluorescence channel were taken by successive exposures on the same vesicle. Images in the same fluorescence channel were normalized to have the same values for the darkest and brightest pixels. (b) Relative fluorescence intensity of giant vesicles in Figure 4c, 4f, and 5a, respectively. Green and red columns represent green and red fluorescence intensities of vesicles generated from hybrid R_HF Janus dendrimer with additional R_H-RhB or R_F-NBD (left two columns), and of vesicles obtained from hybrid R_HF Janus dendrimer with both R_H-RhB and R_F-NBD (right two columns). Relative intensity was calculated from the brightness histogram analysis by dividing the maximum value of each vesicle by the background. Error bars indicate standard error (SEM) of the mean based on data derived from five vesicles. *** denotes p-value < 0.001.

The phase diagram of the three-component assemblies generated from R_H, R_F, and hybrid R_HF Janus dendrimers was investigated by confocal microscopy performed on giant DSs (Figure 6). The ratio of R_H and R_F was maintained 1:1. The proportion of R_HF could be decreased to 50% and subsequently to 40% without phase isolation between the R_H and R_F components (Figure 6a–6c).

Figure 6.

Representative confocal fluorescent microscopy images of giant vesicles assembled from mixtures of R_H, R_F and hybrid R_HF Janus dendrimers with proportions (in wt%) of (a) R_H = 0%, R_F = 0%, R_HF = 100%; (b) R_H = 25%, R_F = 25%, R_HF = 50%; (c) R_H = 30%, R_F = 30%, R_HF = 40%; (d) R_H = 33%, R_F = 33%, R_HF = 34%; (e) R_H = 50%, R_F = 50%, R_HF = 0%. All mixtures contain an additional 1% (wt/wt) of R_H-RhB and 1% (wt/wt) R_F-NBD. Red and green fluorescent images were merged into one to demonstrated their co-assembly or self-sorting.

At 34% of the R_HF component, the red and green colors from the dyes started to separate into a core-shell structure (Figure 6d). Without R_HF in the mixture (0% of R_HF), R_F and R_H self-assembled into individual green and red giant vesicles, indicating the self-sorting of R_F and R_H into incompatible perfluorinated and perhydrogenated incompatible DSs (Figure 6e). Self-sorting¹⁵ of immiscible fluorinated and hydrogenated components during their attempts to co-assemble was previously demonstrated with a hydrogenated-fluorinated peptide system.^15a Here we demonstrate and visualize self-sorting of vesicular DSs self-assembled from individual R_F and R_H Janus dendrimers.

Co-assembly of Janus Dendrimers with Phospholipid

Finally, for potential biomedical applications of these R_F and R_HF DSs, the co-assembly of R_H, R_F and R_HF Janus dendrimers with phospholipids was conducted by using a phospholipid labeled with the red dye, Texas Red (TR), denoted phospholipid TR-DHPE, where DHPE = 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Figure 7a).

Figure 7.

(a) Chemical structure of Texas Red (TR)-labeled phospholipid TR-DHPE, DHPE = 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine. (b–e) Representative confocal fluorescent microscopy images of giant unilamellar vesicles assembled from (b) R_H, (c,d) R_F and (e) hybrid R_HF Janus dendrimers with 1% (wt/wt) of Texas Red (TR)-labeled phospholipid TR-DHPE, DHPE = 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine. Images (c) and (d) show the same area, but the intensity has been multiplied by 5-fold in (d) to better display vesicle formation. (f) Fluorescence intensity of giant vesicles. Error bars indicate standard error (SEM) of the mean based on data derived from 25 vesicles of each sample. *** denotes p-value < 0.001.

Confocal images demonstrated that TR-DHPE could be incorporated into vesicles generated from R_HF derived Janus dendrimers (Figure 7e, Movie S3) even more efficiently than in vesicles assembled from R_H Janus dendrimers (Figure 7b, 7f) with higher intensity of fluorescence emission. As expected, giant DSs from R_F Janus dendrimer showed quite weak fluorescence (Figure 7c, 7d, 7f). These co-assembly experiments are consistent with the previous co-assembly with dye-labeled Janus dendrimers and reveal that different fluorinated vesicles, miscible or immiscible with the biological membrane, can be used for different purposes. For example, DSs from R_F Janus dendrimers can be used for remote lipophilic and hydrophilic drug loading due to low diffusion within biological membranes. In contrast, DSs from R_HF Janus dendrimers may co-assemble with multiple components from biological membranes, including glycolipids, glycoproteins, and transmembrane proteins, to build biocompatible hybrids for immunology and targeted delivery. It is expected that these fluorinated components may co-assemble with bacterial and human cell membranes as demonstrated recently with R_H Janus dendrimers.^5c Therefore the building block reported here provide an important new toolbox for the area of synthetic biology.

CONCLUSIONS

A library containing achiral hydrogenated (R_H), chiral-racemic fluorinated (R_F), hybrid hydrogenated-fluorinated (R_HF), hydrogenated conjugated in the hydrophilic part with a red fluorescent dye (R_H-RhB) and a fluorinated conjugated in the hydrophilic part with a green fluorescent dye (R_F-NBD) is reported. These R_H, R_F and R_HF Janus dendrimers are soluble in ethanol and self-assemble into nanometer size unilamellar and multilamellar onion-like DSs by injection of their ethanol solution into water or buffer and by hydration into micrometer size DSs. The thickness of these narrow size distribution DSs is similar to that of biological membranes. Self-assembly and co-assembly of these Janus dendrimers were monitored by fluorescence microscopy to reveal self-sorting and self-assembly of the immiscible R_H and R_F Janus dendrimers into hydrogenated and fluorinated DSs. Co-assembly of R_H with R_F and R_HF as a function of composition provided hybrid hydrogenated-fluorinated DSs when a high concentration (> 40%) of R_HF was employed, and self-sorting into individual R_H or R_F DSs when low proportions of the R_HF component were used. The simple synthesis and self-assembly, the similar thickness to biological membranes and the imaging capabilities via fluorescence and ¹⁹F MRI of these DSs make these building blocks and assemblies interesting tools for supramolecular science, medicine and synthetic biology,¹⁶ including hybrid hydrogenated-fluorinated cell-like assemblies.^5c