Encapsulation of hydrophobic components in dendrimersomes and decoration of their surface with proteins and nucleic acids

Abstract

Reconstructing the functions of living cells using nonnatural components is one of the great challenges of natural sciences. Compartmentalization, encapsulation, and surface decoration of globular assemblies, known as vesicles, represent key early steps in the reconstitution of synthetic cells. Here we report that vesicles self-assembled from amphiphilic Janus dendrimers, called dendrimersomes, encapsulate high concentrations of hydrophobic components and do so more efficiently than commercially available stealth liposomes assembled from phospholipid components. Multilayer onion-like dendrimersomes demonstrate a particularly high capacity for loading low-molecular weight compounds and even folded proteins. Coassembly of amphiphilic Janus dendrimers with metal-chelating ligands conjugated to amphiphilic Janus dendrimers generates dendrimersomes that selectively display folded proteins on their periphery in an oriented manner. A modular strategy for tethering nucleic acids to the surface of dendrimersomes is also demonstrated. These findings augment the functional capabilities of dendrimersomes to serve as versatile biological membrane mimics.

Keywords: Janus dendrimer vesicles; biological membrane mimic; folded protein; nucleic acid; onion-like vesicles.

Fig. 1.

Chemical structures of amphiphilic Janus dendrimers used for vesicle assembly, fluorescent labeling, and chelating.

Fig. 2.

Enhanced loading of hydrophobic small molecules in onion-like dendrimersomes. (A–C) Superior loading of hydrophobic small molecule, BODIPY, into dendrimersome vesicles compared with stealth liposomes. All vesicles formed by hydration. Onion-like dendrimersome vesicles load significantly higher concentrations of cargo than unilamellar vesicles. Representative confocal images of (A) multilamellar vesicles, and (B) unilamellar vesicles. (C) Image quantitation: R_H-12 dendrimersomes load >10-fold higher concentration of BODIPY cargo relative to either form of stealth liposome. (D and E) A hydrophilic small molecule, HPTS, loads more efficiently into stealth liposomes than dendrimersome vesicles; it is largely excluded from R_H-10 and R_F dendrimersomes. (F and G) Chemotherapeutic drug, doxorubicin, loads much more efficiently in R_H-10 dendrimersome onion-like vesicles compared with stealth liposomes. (H) Summary of max fold enhancement in small molecule loading, comparing dendrimersome to stealth liposome. Mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001; ns, nonsignificant.

Fig. 3.

Retention of hydrophobic cargo in vesicles and dendrimersomes. Amount of BODIPY loaded in (A) vesicles or (B) dendrimersomes before and after incubation with PBS + 10% FBS mixture at 37 °C for 2 h. Mean ± SD. ***P < 0.001; ns, nonsignificant; a.u., arbitrary units.

Fig. 4.

(A) Synthesis of a Janus dendrimer conjugated to a nitrilotriacetic acid (NTA) ligand (R_H-NTA) and (B) scheme of R_H-NTA binding to histidine (His) residues from proteins.

Fig. 5.

Protein recruitment to dendrimersomes without NTA ligand. (A) Schematic of protein internal loading and surface binding to dendrimersome and liposome vesicles. (B) Dendrimersomes display higher spontaneous loading of protein cargos compared with stealth liposomes in their onion-like structures. (C) Confocal images show higher loading of His-GFP cargo to multilamellar dendrimersomes and stealth liposomes. Mean ± SD. ***P < 0.001.

Fig. 6.

Selective protein recruitment to dendrimersomes with NTA ligand. (A) Confocal images show selective recruitment of His-GFP to dendrimersomes containing R_H-NTA. (B) Quantitation of cargo recruitment to dendrimersomes: surface recruitment is specific to presence of R_H-NTA and does not depend on extent of lamellarity. Mean ± SD. *P < 0.05, **P < 0.01; ns, nonsignificant.

Fig. 7.

Modular tethering of DNA to SNAP-tagged dendrimersomes. (A) Schematic showing layering of a protein and DNA coat to dendrimersome vesicle. (B) His-SNAP proteins binds to R_H-NTA to form the initial protein layer. SNAP binds to a BG conjugated to the DNA, allowing for modular formation of a second layer, composed of nucleic acids. The DNA aptamer is labeled with FAM to enable imaging. (C) Schematic of multilayer dendrimersome containing DNA and protein coat. (D) Binding of DNA aptamer to liposomes, stealth liposomes, or dendrimersome vesicles is dependent on presence of NTA group. (E) Quantitation of DNA aptamer recruitment to vesicle types. R_H-NTA dendrimersomes demonstrate elevated DNA recruitment compared with NTA liposomes. Mean ± SD. *P < 0.05, **P < 0.01; ns, nonsignificant.