Designing bioresponsive nanomaterials for intracellular self-assembly

Abstract

Supramolecular assemblies are essential components of living organisms. Cellular scaffolds, such as the cytoskeleton or the cell membrane, are formed via secondary interactions between proteins or lipids and direct biological processes such as metabolism, proliferation and transport. Inspired by nature's evolution of function through structure formation, a range of synthetic nanomaterials has been developed in the past decade, with the goal of creating non-natural supramolecular assemblies inside living mammalian cells. Given the intricacy of biological pathways and the compartmentalization of the cell, different strategies can be employed to control the assembly formation within the highly crowded, dynamic cellular environment. In this Review, we highlight emerging molecular design concepts aimed at creating precursors that respond to endogenous stimuli to build nanostructures within the cell. We describe the underlying reaction mechanisms that can provide spatial and temporal control over the subcellular formation of synthetic nanostructures. Showcasing recent advances in the development of bioresponsive nanomaterials for intracellular self-assembly, we also discuss their impact on cellular function and the challenges associated with establishing structure-bioactivity relationships, as well as their relevance for the discovery of novel drugs and imaging agents, to address the shortfall of current solutions to pressing health issues.

Fig. 1. The eukaryotic cell and its compartments as interconnected reaction vessels.

The conditions for chemical transformation of bioresponsive nanomaterials in terms of pH, redox environment and enzyme catalysis differ widely, depending on the subcellular location. While reactive oxygen species (ROS) are mainly generated in the mitochondria, as well as, to a lesser degree, by the membrane-bound NADPH oxidase (NOX), the cellular reducing agent glutathione is present in high concentration (10 mM) throughout the cytosol. During the endocytic pathway, the pH inside the vesicles decreases significantly from 6.3 in the early endosomes to 5.5 in the late endosomes to 4.7 in the lysosomes, whereas the cytosolic pH is typically neutral, at around 7.2 (ref.). The localization of enzymes within the cells depends on their respective biocatalytic function, meaning that they are often associated with specific organelles.

Fig. 2. Material classes for intracellular self-assembly.

Peptides represent the largest subset of nanomaterials for intracellular structure formation and can self-assemble into peptide nanofibres due to hydrogen bonding and π–π stacking caused by aromatic amino acid residues. In the case of peptide amphiphiles, hydrophobic interactions of alkyl chains also contribute to the self-assembly propensity^,. Polyaromatic compounds form fibrous structures or nanoparticles because of aromatic interactions between the monomers^,–. Polymers, such as polyvinyl alcohol, transform into spherical aggregates or fibres^,, whereas metal nanoparticles can aggregate into larger assemblies due to covalent crosslinking of their coating^–.

Fig. 3. Differences in complexity of the chemical transformation for intracellular self-assembly.

a | Morphology transformation due to trans/cis-amide isomerization. b | Removal of a hydrophilic group in a phospho-tyrosine-containing peptide. c | Deprotection of a bioresponsive cyclization precursor and following reaction cascade. FITC, fluorescein isothiocyanate.

Fig. 4. pH as stimulus for intracellular self-assembly or morphology transformation.

a | Protonation of glutamate residues due to the acidification inside cancer cells with abnormally low cytosolic pH induces supramolecular assembly of a peptide amphiphile, as shown by the transmission electron microscopy images at pH 7.4 and 6.8 (ref.). b | trans/cis-Isomerization of pH-sensitive 4-amino proline causes the morphology transformation of a pentapeptide during cell entry. The transmission electron microscopy images show a transition from nanoparticles at pH 6.5 to nanofibres at pH 7.4 (ref.). Part a (right) is adapted with permission from ref., American Chemical Society. Part b (right) is adapted with permission from ref., American Chemical Society.

Fig. 5. Redox-induced intracellular self-assembly.

a | Glutathione-induced cleavage of a disulfide bond changes the lower critical solution temperature (LCST) behaviour of a polymer conjugate, causing it to form intracellular nanoaggregates. Bio-transmission electron microscopy of MC7 cells shows the presence of intracellular nanoparticle. b | Cleavage of a reactive oxygen species (ROS)-sensitive thioketal group induces fibre formation. Transmission electron microscopy images show ROS-induced transformation from nanoparticles to nanofibres in hydrogen-peroxide-containing phosphate buffer. KLAK, mitochondria-targeting peptide; P₁₈, purpurin 18; PNIPAm, poly(N-isopropylacrylamide); PVA, polyvinyl alcohol. Part a (right) is adapted with permission from ref., American Chemical Society. Part b (bottom left and right) is adapted with permission from ref., American Chemical Society.

Fig. 6. Cellular localization of enzymes for self-assembly and their respective recognition and transformation sites.

γGT, γ-glutamyltransferase; ALP, alkaline phosphatase; ATG4B, autophagy-related 4B cysteine peptidase; ENTK, enterokinase; ER, endoplasmic reticulum; PTP1B, protein tyrosine phosphatase 1B.

Fig. 7. Multistep transformation for intracellular self-assembly.

a | Structures of assembly precursors for macrocyclization-based self-assembly. The amino protecting group determines the sensitivity to a specific enzyme, such as furin^,,, caspase-3/7 (refs^,,), β-galactosidase or nitroreductase. The peptide-based spacer R² between the reactive functionalities can contain fluorophores, drugs or other bioactive compounds. b | Multistep reaction sequence of intracellular conversion pH-sensitive and reactive oxygen species (ROS)-sensitive isopeptides into linear co-assembling peptides. The targeting group contains cell-penetrating peptide TAT (transactivator of transcription) and enables cell entry, upon which the acid-labile dynamic covalent bond between the salicyl hydroxamate of targeting peptide and the phenylboronic acid group of the isopeptides is hydrolysed in the endosomes. The hydrogen-peroxide-induced deprotection via self-immolation of the phenylboronic acid group reveals a reactive amino group that can attack the adjacent ester bond. This rearrangement due to an O,N acyl shift results in the linearized co-assembling peptides. The formation of peptide nanofibres inside A549 cells can be visualized with bio-transmission electron microscopy. Part b (bottom left) is adapted with permission from ref., American Chemical Society.