key: cord-0029430-1xrlfrji
authors: Chagri, Sarah; Ng, David Y. W.; Weil, Tanja
title: Designing bioresponsive nanomaterials for intracellular self-assembly
date: 2022-04-01
journal: Nat Rev Chem
DOI: 10.1038/s41570-022-00373-x
sha: 754b8b0792c18abef555b4b4e6eb85caa13cd1d0
doc_id: 29430
cord_uid: 1xrlfrji

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. [Image: see text]

In nature, the formation of functional structures through the assembly of smaller units can be observed over sev eral structural hierarchies at the macroscopic, micro scopic and nanoscopic scales. While spiderwebs are tangible examples of natural constructs built for specific function, eukaryotic cells reveal that similar phenomena also exist on a molecular level: for instance, the cytoskele ton is made from various dynamic protein assemblies that form intricate networks inside the cell, ensuring cel lular stability, motility and division 1 . Mimicking these naturally occurring structures by integrating synthetic self assembled nanostructures in a biological context is an important milestone in supramolecular chemistry, as these systems expand the boundaries of both nanomedi cine and synthetic biology. Additionally, the bottom up approach of creating non natural assemblies inside cells could help elucidate molecular mechanisms of naturally occurring cellular processes and provide the foundation to unravel the origin of life 2 .

Regarding the potential of synthetic supramolec ular nanostructures for therapeutic applications 3 , a major appeal is the circumvention of limitations that small molecule drugs face, such as drug resistance in cancer cells 4 . In contrast to small molecules, systems capable of intracellular self assembly can form large aggregates inside cells, which forces their accumulation at the target site 5 , resulting in improved pharmacoki netics. Additionally, the process of self assembly can cause mechanical stress due to the disruption of cellular structures, thus, triggering cell death 6 . By instilling bio responsiveness -in which molecules react to physio logical cues -in the design of the assembly precursor, its conversion to an active self assembling monomer can be precisely tailored to a specific nanoenvironment or microenvironment in the cell. Targeted intracellular stimuli include those that have been widely exploited in state of the art prodrugs and delivery systems, such as pH (refs 7-11 ), redox 6,12-15 or enzymes [16] [17] [18] [19] [20] [21] [22] , in which the respective stimulus is often characteristic for a certain subcellular location ( fig. 1 ). As the considerations to program nanostructure formation within cells are multi faceted, we aim to streamline the design concepts to create greater accessibility and understanding for the sci entific community. In this Review, we showcase success ful molecular design principles to control the assembly of functional nanostructures within mammalian cells, which are triggered by endogenous stimuli. Assemblies in bacteria are excluded, as the conditions of transport Designing bioresponsive nanomaterials for intracellular self-assembly Sarah Chagri , David Y. W. Ng ✉ and Tanja Weil ✉ 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. and the available physiological stimuli are different, thus, requiring a distinct set of molecular considerations 23, 24 . Recent advances in this field have been summarized in other review articles 25, 26 .

Furthermore, while other reviews on synthetic assemblies inside mammalian cells have focused more on potential biological applications for bioimaging or chemotherapy [27] [28] [29] [30] , our main goal is to examine the under lying design principles, considerations and challenges of creating bioresponsive precursors for the formation of supramolecular nanostructures, with an emphasis on the spatiotemporal control over the intracellular assembly.

The eukaryotic cell is an intricate machinery made of lipids, proteins and genetic material, all forming supra molecular structures in an aqueous environment, while dynamic chemical crosstalk between organelles and the extracellular space governs internal processes 31 . The living cell is a non equilibrium system characterized by compartmentalization and the energy dependent main tenance of concentration gradients that are essential for proliferation and function 32 . As cellular compartments fulfil different tasks within the cell, they often provide distinct (bio)chemical environments, such as a certain pH, redox environment, level of molecular crowding or the presence of specific enzymes 31 (fig. 1) .

From a chemist's perspective, cellular compartments with their distinct characteristics are analogous to a sys tem of interconnected reaction vessels, each offering dif ferent reaction conditions for chemical transformations. When designing synthetic materials for intracellular self assembly, a knowledge of cell biology is essential to understand and exploit structure-(bio)activity relation ships. In terms of molecular design, this means carefully choosing the respective functional groups and other ele ments, such as enzyme recognition sites, to implement selectivity towards a biological or chemical stimulus associated with a target subcellular location. For exam ple, the decrease in pH associated with the endocytic pathway can be exploited by using pH sensitive mate rials that can undergo supramolecular transformations inside the acidic endosomal or lysosomal environments due to protonation or isomerization [7] [8] [9] [10] [11] . Similarly, the reducing medium of the cytosol can induce the chem ical conversion of reduction sensitive precursors into active monomers upon cell entry 12, 15 , while the higher concentration of reactive oxygen species (ROS) near the mitochondria can lead to oxidation driven self assembly in the vicinity of these organelles 14 103 , as well as, to a lesser degree, by the membrane-bound NADPH oxidase (NOX) 96 , the cellular reducing agent glutathione is present in high concentration (10 mM) throughout the cytosol 55 . 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. 83 ). The localization of enzymes within the cells depends on their respective biocatalytic function, meaning that they are often associated with specific organelles.

Besides the influence of different reaction condi tions associated with intracellular location, the cell type also affects the efficiency of chemical transformations, as cells from different tissues have evolved to behave in specific ways that contribute to the survival of the whole organism. For example, liver cells, which are in charge of detoxification, display a comparatively high cytosolic concentration of glutathione, a cellular anti oxidant and nucleophile essential for scavenging harm ful electrophilic compounds and ROS 33 . Moreover, the gene expression profile of cells also varies depending on the tissue, which includes the expression levels of certain enzymes, such as proteases, that catalyse biochemical transformations.

Alterations in the genetic code can cause cells to cease following expected patterns and instead start displaying an opportunistic, self serving behaviour tar geted at sustained growth and evasion of cell death 34 . These cancerous cells can emerge from different tissues and often show an upregulation of pro-proliferative and pro-angiogenic factors, as well as an elevated cellular meta bolism 35 . While cancer cells are not uniform in their biological and chemical characteristics, there are some common properties of aggressively growing cancers, for example: the acidification of the tumour extracellular microenvironment because of their enhanced glycolytic rate, a tendency to develop low oxygen level in tissue, called hypoxia, due to limited oxygen supply inside the tumour and the overexpression of certain proteases that aid cell invasion and metastasis 34 . Regarding the great challenges associated with drugresistant cancers, upregu lated efflux pumps, such as Pglycoprotein 36 , and elevated glutathione levels in the cytosol 37 play critical roles in treatment failure due to drug inactivation and efficient removal. Therefore, in addition to using targeting moie ties that are recognized by specific cell surface receptors, the aforementioned distinct features of cancerous cells can facilitate the design of novel cancer specific drugs or imaging agents with less toxic side effects on healthy cells or tissue 38 . Consequently, these considerations are also relevant for controlling the bioresponsive formation of synthetic nanostructures within cancer cells, which offers entirely new avenues to potentially revolutionize bioimaging and cancer therapy.

Nanomaterials for intracellular self-assembly Creating synthetic supramolecular assemblies in a bio logical environment requires molecular design strategies that fulfil necessary criteria regarding biocompatibility, environmental responsiveness and the propensity for self assembly within crowded and complex environ ments. For instance, the assembly precursor must enter cells without premature disruption of cellular functions or causing cell death, since a lack of biocompatibility at this stage would impede the formation of nanostructures at the desired cellular location. Efficient cell uptake of the precursor can be achieved by including structural elements that promote cell entry in the molecular design, such as cell penetrating peptides (CPPs) 6, 8 or ligands of certain cell surface receptors 5, 39, 40 . CPPs comprise 40 or less amino acids and they can be conjugated covalently or non covalently to cargo to facilitate their delivery into the cell 41, 42 . Besides direct translocation into the cyto sol as a result of attractive electrostatic interaction with the negatively charged plasma membrane 42 , CPPs and their conjugates can also be internalized through vari ous endocytic pathways 43 . While some aspects of the cell uptake mechanisms remain elusive, prominent examples of cationic CPPs such as the HIV derived peptide trans activator of transcription or polyarginine peptides have been shown to enter cells through a variety of pathways, depending on the cell type and the conjugated cargo [44] [45] [46] . However, using CPPs for intracellular delivery of assem bly precursors can come with the limitation of lacking cell selectivity, whereas the introduction of certain lig ands for receptor mediated endocytosis can tailor the system towards a more targeted effect 5, 39, 40 . For example, the tripeptide RGD serves as a recognition motif for integrin cell surface receptors, such as αvβ3, which are overexpressed in many cancer cells 47 . Therefore, assem bly precursors that are modified with the linear or cyclic version of the targeting peptide have been employed for cancer specific targeting both in vitro 10, 39, 40 and in vivo 5 .

For the in vivo application of nanomaterials for intra cellular structure formation, achieving tissue selectivity by active or passive targeting is one of the key objec tives. Besides the use of tumour specific recognition motifs 48 , the so called enhanced permeability and reten tion effect in solid tumours with high vascular density and high endothelial permeability 49 contributes to the passive accumulation of macromolecular precursors at the desired location 12 . In general, the administration of compounds for intracellular self assembly comes with similar challenges in terms of pharmacokinetics and immunogenicity as observed for small molecule drugs 48 ; however, these pro assembling systems can possess greater selectivity and avoid affecting healthy cells. This is due to the bioresponsive mode of action of the assem bly precursors, which require the presence of a specific stimulus inside the cells and confines the assembly formation to the intracellular space 48, 50 .

Within the cell, the activated self assembling mon omer resulting from intracellular chemical conversion needs to display a critical aggregation concentration that is sufficiently low for self assembly under physiological conditions, which is usually in the micromolar range 9,51 . The critical aggregation concentration of the active monomer is determined by the self assembly propensity of the material, which is based on attractive intermo lecular interactions. Since the intracellular environment displays a high degree of macromolecular crowding due to, for example, soluble proteins, densely packed fila mentous structures and phase separated components, this increases diffusional obstacles and further enhances the inherent aggregation tendency of the monomers 52,53 . However, this abundance of biomacromolecules with reactive groups, such as proteins with surface exposed cysteines 54 , as well as other ubiquitous molecules such as glutathione 55 or ROS 56 , mandates the need for biorthog onality of the assembly precursor to prevent unwanted interactions. This biorthogonality is also important for achieving spatial control over the subcellular structure formation, which can additionally be aided by introducing 0123456789();: organelle specific targeting groups to the precursor that ferry the pro assembling material through the cellular environment to the desired compartment 57 .

Generally, there are four material classes that are commonly used for intracellular self assembly: pep tides, polyaromatic compounds, polymers and metal nanoparticles ( fig. 2 ). While there are only a few exam ples of polymers 14, 58 or metal nanoparticles 59-61 that have shown intracellular structure formation, many peptide based and polyaromatic materials readily undergo stimuli responsive self assembly in cellular environments, providing new perspectives for diagnostic or therapeutic applications.

Peptides, more specifically, β sheet forming so called amyloid like peptides, constitute one of the most popu lar scaffold materials: they are characterized by amphi philicity caused by an alternating sequence pattern of polar and nonpolar amino acids, which supports the intermolecular hydrogen bonding necessary for adopting β sheet secondary structures 62 . Moreover, they usually display a high content of aromatic amino acids, such as phenylalanine, required for self assembly due to π-π stacking and van der Waals interactions 53, 63 . Often, an aromatic moiety or fluorophore at the amino terminus further adds to their π-π stacking tendency, supporting their assembly into stable nanostructures 62 . For instance, certain peptides can form ordered β sheet rich nanofibres of a few nanometres in width and micrometres in length that show similarities to naturally occurring β amyloid aggregates present in various neurodegenerative illnesses, such as Alzheimer disease 62 . Amphiphilic peptide conjugates are another subcategory of peptide based nanomaterials consisting of a peptide sequence prone to hydrogen bonding and a nonpolar alkyl chain (fatty acid) engaging in hydro phobic interactions. Because of their fast assembly into dynamic but stable nanostructures and their gelation propensity 64 , these biocompatible amphiphilic conju gates have been studied extensively for various medical applications, including tissue regeneration 65 and wound healing 66 . Besides extracellular applications, their poten tial for intracellular structure formation and gelation has been used for enzyme responsive and pH responsive self assembly inside cancer cells 67, 68 .

Polyaromatic compounds can form intracellular aggregates because of π-π aromatic interactions within the aqueous cytoplasm, which has been exploited for the creation of supramolecular imaging agents 15 and drugs for photodynamic therapy 40 . Prominent exam ples include bispyrene based systems that rely on the formation of highly fluorescent J-aggregates 69, 70 and luminogens that display aggregation induced emission (AIE) properties [71] [72] [73] [74] . Aside from fluorophores and AIE luminogens that display a constant number of aromatic rings both in the precursor and in the self assembling monomer, there are also systems that rely on the intra cellular formation of new aromatic units due to chemical transformation. For instance, the intracellular condensa tion reaction between a deprotected aminothiol and an aromatic nitrile can be used to generate self assembling aminoluciferin based macrocycles inside cells 9, 75 . This approach relies on the intracellular creation of poly aromatic cyclic oligomers that can self assemble into fluorescent nanoaggregates via π-π stacking 9 .

Stimuli induced chemical transformations yielding a self assembling monomer can be grouped by different

Nanofibres, nanoparticles Nanoparticle aggregates Spherical aggregates, nanofibres

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 39 . In the case of peptide amphiphiles, hydrophobic interactions of alkyl chains also contribute to the self-assembly propensity 67, 68 . Polyaromatic compounds form fibrous structures or nanoparticles because of aromatic interactions between the monomers 9,69-74 . Polymers, such as polyvinyl alcohol, transform into spherical aggregates or fibres 14, 58 , whereas metal nanoparticles can aggregate into larger assemblies due to covalent crosslinking of their coating 59-61 .

Aggregates of fluorescent dyes characterized by a narrow absorption band that is shifted to a longer wavelength (bathochromic shift) in comparison with the monomer and a small stokes shift with a narrow band.

Compounds that exhibit an increase in luminescence or fluorescence upon aggregation instead of self-quenching.

www.nature.com/natrevchem levels of complexity, depending on the length of the reac tion sequence ( fig. 3 ). Generally, the molecular design concepts for bioresponsive self assembly can be divided into three categories: structures that undergo a purely morphological transformation ( fig. 3a ), pro assembling monomers that transform into active monomers due to the removal of a hydrophilic group ( fig. 3b ) and mol ecules that rely on the bioresponsive deprotection of reactive groups causing a multistep reaction cascade ( fig. 3c ). While the macrocyclization based systems fit the last category, the most common strategy for con trolled intracellular self assembly uses the chemical or 84 . b | Removal of a hydrophilic group in a phospho-tyrosine-containing peptide 18 . c | Deprotection of a bioresponsive cyclization precursor and following reaction cascade 9 . FITC, fluorescein isothiocyanate.

Nature reviews | Chemistry enzymatic removal of a hydrophilic unit to trigger the aggregation of the remaining less polar fragment. By contrast, purely morphological transformations com prise systems that do not require the breakage of chemi cal bonds to transform into active monomers but instead rely on pH induced isomerization or protonation. Apart from materials that rely on bioresponsive transformations for self assembly, local accumulation can also lead to aggregation, which does not depend on any specific endogenous stimulus 76 . A subcellular accumulation occurs by integrating organelle specific targeting groups into the nanomaterial design, which can be exploited to locally exceed the critical aggrega tion concentration. For example, the positively charged targeting group triphenylphosphonium (TPP) has been used to induce the mitochondria specific accu mulation of short aromatic peptides, which leads to cellular dysfunction and cancer cell death [77] [78] [79] . Besides TPP, methylpyridinium substituted materials also tar get the mitochondria: a methylpyridinium containing oligothiophene conjugate revealed time dependent and temperature dependent accumulation and self assembly first in the mitochondria, followed by the perinuclear region. The aggregation process was indicated by changes in fluorescence due to a bathochromic shift upon formation of superstructures 80 . For targeting the nucleus, lysine rich peptides that bind to RNA can direct the self assembling material to the desired organelle 81 .

Although a high local concentration of active mon omers is the basis for assembly, the specificity and control over structure formation can be enhanced by leveraging physiological cues. The next sections are ded icated to the exploration of strategies for bioresponsive stimuli induced self assembly and the ways in which it allows for spatiotemporal control over the formation of intracellular nanostructures. Thus, the following sec tions are divided according to the respective chemical or biological stimulus that triggers the conversion of a pro assembling precursor into an active monomer that can form supramolecular structures inside living cells.

The physiological pH in tissues and inside cellular organelles depends on a multitude of factors and repre sents an important parameter for biological processes. Moreover, compared with healthy tissue, the tumour microenvironment is often characterized by a slightly acidic pH (6.7-7.1), which is attributed to the enhanced glycolytic rate of cancer cells that results in hypoxia and acidification of the immediate extracellular matrix 82 . Inside cells, the endocytic pathway is accompanied by a pH gradient ranging from 6.3 in the early endosomes to 5.5 in the late endosomes and 4.7 in the lysosome 83 . In these cellular vesicles, a unique acidic environment is created, in which proteolysis and recycling of unneeded cellular components and waste take place.

These changes in pH during the entry of a certain cellular compartment can serve as an endogenous trigger for the self assembly or the morphological transformation of a pH responsive nanomaterial 8, 10, 11, 84 . Functional groups that are susceptible to protonation or deprotonation within physiological pH ranges can be found in the side chain residues of amino acids, making peptides a suitable platform for controlling pHdriven selfassembly. For example, a block copolypeptide com posed of a hydrophilic oligoarginine (R 12 ) and an amphi philic peptide with a repeating FKFE motif undergoes a morphological transformation upon endocytosis 8 . Inside the acidic late endosomes and lysosomes (pH ≤ 5), the protonation of glutamic acid residues (E) increases the hydrophobicity of the β sheet forming peptide and, therefore, its propensity for self assembly. This causes a pH driven morphology transition of the amphi philic peptide -from unstable aggregates outside of the cell to defined vesicular structures inside the acidic compartments. As this supramolecular process corre lates with a spatially confined increase in positive sur face charge density of the nanostructures only within the acidic endosomal and lysosomal environments, the pHresponsive peptide exhibits a low cytotoxicity 8 . The high positive surface charge density of the locally formed vesicles is also responsible for the therapeutic effect of the nanomaterial as a supramolecular anti prion drug because it suppresses the misfolding of proteins through electrostatic interactions.

While the acidification of extracellular pH and a slightly elevated cytosolic pH are seen as typical for metastatic cancer cells 82 , the core of solid tumours usually displays hypoxic cells with a slightly lowered intracellular pH caused by the accumulation of acidic metabolites such as lactate 85 . To exploit this particular phenomenon of pH dysregulation, a peptide amphiphile was designed that can self assemble below pH 7 (ref. 68 ) ( fig. 4a ). The palmitoylated hexapeptide contains three carboxy terminal glutamic acid residues that render the molecule sensitive to protonation induced transforma tion at only slightly acidic pH levels, which is likely due to pK a variations of the side chain carboxylic acids 86 . Consequently, the formation of cytotoxic fibres of the peptide amphiphile was observed exclusively inside cells with decreased intracellular pH, for example, HeLa cer vical cancer cells (in vitro and in vivo) or HEK293 cells at the core of spheroids 68 .

Besides incorporating ionizable groups into the chemical design of a bioresponsive material, the use of structural elements prone to pH dependent isomer ization can also lead to the controlled formation of intracellular assemblies 11, 84 . The relationship between bioactivity and pH dependent morphology transfor mation is exemplified by a self assembling pentapeptide with a central 4 amino proline capable of pH sensitive cis/trans amide isomerization in a physiological envi ronment ( fig. 4b ). Without the need for enzyme catalysis, which is a prerequisite for the isomerization of proline in native proteins 87 , the 4 amino proline based scaffold can switch from β sheet dominated helices at neutral pH to random coil peptide nanoparticles at pH 6.5. Acidification inside the tumour microenvironment causes the dominance of the trans isomer and, therefore, the formation of globular assemblies, whereas endo cytosis and endosomal escape into the neutral cytosol www.nature.com/natrevchem triggers a trans-cis isomerization, resulting in a second morphology transformation to superhelical structures inside the cell 11, 84 .

Glutathione is the most abundant low molecular weight peptide in eukaryotic cells and consists of three amino acids: glutamate, cysteine and glycine 37 . Due to the reactive thiol group of its cysteinyl moiety, it acts as a reducing agent, a nucleophile and a cell protective anti oxidant, ensuring redox homeostasis by scavenging free radicals, such as ROS, lipid peroxides and heavy metals 88 . Normal levels of glutathione in human tissues range from 0.1 mM to 10 mM (ref. 55 ), in which 85% to 90% of reduced glutathione is localized in the cytosolic-nuclear compartment 89 , transforming it into a reducing environ ment for both endogenous compounds and xenobiotics. Many cancers exhibit elevated levels of intracellular glutathione that correlate with higher cellular prolif eration and metastatic activity 90, 91 , as well as multidrug resis tance caused by an increased efflux of glutathio nylated chemotherapeutics 92 . Glutathione, therefore, constitutes an attractive endogenous stimulus for con trolled intracellular self assembly, due to its ubiquity in cells, its capability of efficiently reducing disulfide bonds and the significant difference in its distribution inside and outside of the cell 93 .

The reduction and cleavage of a disulfide bond by glutathione can cause the removal of a hydrophilic aggregation inhibitive unit, thereby, releasing the selfassembling component to form the designated nanostructures inside the cell 12,15,58,94 . An example for this strategy is the glutathione responsive fluor escence probe that is composed of a bispyrene linked to a positively charged cyanine dye via a disulfide bridge 15 . Glutathione induced cleavage of the disulfide in the cytosol causes the separation of the two fluoro phores, thereby, allowing the free bispyrene to form large, highly fluorescent J aggregates 69 in vitro and in vivo 15 . Aside from this small molecule probe for the imaging of glutathione levels, glutathione responsive polymer-peptide conjugates have also been synthe sized 58 . These conjugates consist of a thermorespon sive poly(N isopropylacrylamide) backbone covalently linked to a hydrophilic cell penetrating peptide by a disulfide bond ( fig. 5a) Nature reviews | Chemistry which causes a transition from a random coil state into spherical aggregates inside the cell. In addition, this system can be tuned to respond to different endo genous stimuli, including the proteolytic cleavage of the hydrophilic peptide by various enzymes, for example, caspase3 (ref. 58 ).

ROS are generated during mitochondrial oxidative metabolism 56 and as a cellular response to xenobiotics, cytokines and bacterial invasion 95, 96 . Controlled ROS production inside the cell fulfils many biological pur poses, such as the regulation of signal transduction 56 www.nature.com/natrevchem with regards to angiogenesis 96 and insulin metabolism 98 . However, overproduction of ROS leads to oxidative stress and is linked to various pathological phenomena 99 , including abnormal cell growth that can perpetuate cancer initiation and progression 100, 101 . Targeting cancer cells with high levels of ROS 102 for oxidation driven self assembly is a relatively new strategy to create supramolecular architectures inside cells 6, 14 . The mitochondria produce ROS by means of the electron transport chain 103 , thereby, influencing the immediate redox environment of the organelle and mak ing it an attractive subcellular target for ROSsensitive materials. Thioketals are an example of a ROSsensitive functionality that can be degraded in the presence of sufficient amounts of cellular oxidants 104 . For instance, using a thioketal linker to attach a hydrophilic poly ethylene glycol (PEG) chain to a self assembling pep tide on a polymer backbone, that is also decorated with a mitochondria targeting peptide (KLAK), gives rise to a multifunctional platform for mitochondria specific accumulation, morphology transformation and assem bly 14 (fig. 5b ). This polymer-peptide conjugate con sisting of two different polymers (PEG side chain and polyvinyl alcohol backbone), as well as two different peptides (KLAK and β sheet forming peptide), can enter the cells as a nanoparticle and undergoes a trans formation into a fibrous network in the proximity of ROS overproducing mitochondria. The morphological transformation is designed in a way that a lipophilic β sheet forming sequence is flanked by two hydrophilic polymers, KLAK functionalized polyvinyl alcohol and PEG. In this initial state, the chains collapse into spheri cal aggregates, where the steric component of two bulky hydrophilic polymers prevents molecular interaction between the β sheet peptides. Upon oxidation by high amounts (μM) of ROS, the PEG segment is cleaved, transforming the molecule into an amphiphilic struc ture. Hence, the steric hindrance is relieved and chain flexibility promotes intermolecular interactions between interchain β sheet peptides to afford a fibrillar morpho logy. The ROS sensitive supramolecular system exhibits significantly higher cytotoxicity towards HeLa cervical cancer cells than towards normal cells, both in vitro and in vivo 14 .

Biocatalytic reactions carried out by enzymes enrich the chemical depth of cellular pathways, allowing molecules and biopolymers to be processed within the environmen tal limitations of a living system. Besides converting nat ural substrates, many enzymes have also been exploited for the intracellular transformation of synthetic precur sors into self assembling monomers 18, 50, 105, 106 (TAble 1) . For this purpose, enzyme specific recognition motifs can be included in the molecular design of the precursor to tailor its bioresponsiveness towards a certain enzyme within the cell (fig. 6 ). The following sections highlight the use of various types of intracellular enzymes, namely, proteases, phosphatases and other kinds of enzymes, to instruct targeted structure formation at different subcellular locations.

Proteases. Proteolytic enzymes catalyse the hydrolysis of peptide bonds 107 , making them an attractive bio logical tool for the activation of bioresponsive nano materials. The diversity of proteases in terms of their subcellular localization, level of expression in different cell types, as well as their specificity for a certain cleav age site, facilitates spatiotemporal programming of enzyme instructed self assembly.

Furin is an endoprotease that is associated with the Golgi apparatus in eukaryotic cells. Belonging to the enzyme family of proprotein convertases, it catalyses downstream peptide cleavage of the polybasic motif RX(R/K)R for the bioactivation of certain proteins 108 . This enzymatic cleavage reaction plays an essential role in pathogenesis, as the enzyme activity of furin has been linked to cancer progression 109, 110 , as well as viral diseases 111 , including SARS CoV2 infection 112 . Therefore, the imaging of furin activity inside cells or tumour tissue is an attractive target that can be addressed using bioresponsive supramolecular chem istry. For example, the intracellular condensation reaction between an aminothiol and a cyanobenzothi azole for the formation of self assembling macrocyclic dimers was first realized using a furin sensitive pre cursor ( fig. 3c ). By adding the four amino acids RVRR to the amino terminal cysteine, they serve not only as an enzyme cleavable protecting group ( fig. 7a) for the amino function but also facilitate cell entry of the pre cursor due to the positively charged side chain residues 9 . Further investigation of the intracellular reaction cas cade showed that the furin induced proteolytic cleavage near the Golgi apparatus constitutes the rate limiting step for the formation of active monomers, which cor relates to the subcellular localization of the resulting nanostructures at this site 21 . This enzyme instructed intracellular aggregation also enables in vivo imaging of furin activity inside furin overexpressing tumour tis sue, since it can promote the accumulation and reten tion of an imaging agent that is covalently linked to the 117 . In all these studies, the imaging agent was introduced to the precursor molecule via side chain modification of a lysine positioned between the cyanobenzothiazole and the cysteine. The use of this strategy for the chemical modification of the condensation precursor has also been used for the conjugation of the chemotherapeu tic drug, Taxol, which helps to prevent undesired drug efflux and promotes sustained drug release due to the furin induced self assembly of Taxol nanoparticles 118 . Aside from the attachment of small organic molecules, metal based nanoparticles can also be decorated with the bioresponsive precursor by using the amino group of the lysine side chain for amide coupling reactions on a carboxyl modified particle surface 60 Caspases are proteolytic enzymes that play an essen tial role in the initiation and execution of apoptosis, a mode of programmed cell death 119 . These endopro teases contain a characteristic nucleophilic cysteine as a central part of a polybasic substrate binding pocket 120 . The chemical structure of their active site enables them to catalyse the hydrolytic cleavage of proteins for the controlled dismantling of the cell. Within the intricate activation cascade of different caspases, the so called 'effector' caspases3, 6 and 7 carry out most of the pro teolysis during apoptosis and can, therefore, be consid ered biomarkers for programmed cell death 121 . Since caspase3 and caspase7 both exhibit similar preferential downstream cleavage of the motif DEVD in synthetic substrates 122 , many supramolecular systems have been designed to include this sequence as a bioresponsive moiety for intracellular self assembly 16, 22, 39, 48, 50, 58, 59, [123] [124] [125] ( fig. 6 ). For example, a fluorescent probe for the real time imaging of apoptosis in drug treated cancer cells was combined with the hydrophobic tetraphe nylethene (TPE) with the hydrophilic peptide motif DEVD_K via azide-alkyne click chemistry 123 . After enzymatic cleavage by caspase3/7, the polyaromatic fragment TPE K can self assemble inside cells, which results in AIE caused by the restriction of intramolec ular rotation of its phenyl rings 126 . These so called AIE luminogens 127 -which exhibit a turn on fluorescence upon aggregation -can be useful reporters of enzyme activity in cancer cells, especially when they are part of a multifunctional theranostic nanomaterial 39 .

A sequential approach to the caspase induced for mation of intracellular nanostructures uses systems containing tumour specific recognition motifs able to trigger apoptosis 22, 48 . A peptide based precursor, containing a biomimetic sequence that can bind and inactivate the X linked inhibitor of apoptosis pro tein (XIAP) 128 , promotes the activation of caspase3/7 through the manipulation of intracellular signal transduction 129 . The activated pro apoptotic proteases can then remove the non assembling XIAP recognition motif, thereby, inducing the self assembly of the remain ing β sheet forming peptide-drug conjugate 48 or pep tide-cyanine dye conjugate 22 . This sophisticated system selectively causes assembly formation in cancerous tissue both in animal models and in ex vivo human bladders that were taken from patients with late stage bladder cancer 48 . The latter experiment shows the potential clinical use of the caspase sensitive assembly precur sor, as it could help detect tumour boundaries during image guided surgery.

For quantitative imaging of caspase3/7 activity in live animals, a condensation driven system was devel oped, in which the enzymatic activation of the pre cursor is decoupled from a subsequent imaging tag immobilization 50 . The pro assembling molecule con sists of the amino terminal protecting group DEVD ( fig. 7a) linked to a cysteine, a lysine with a clickable trans cyclooctene (TCO) on its side chain, as well as a pyrimidinecarbonitrile as the aromatic nitrile for the enzyme instructed macrocyclization. Since this caspase3/7 responsive molecule can form stable intracellular nanoaggregates, subsequent addition or injection of a tetrazine functionalized fluorophore leads to an immobilization of the imaging probe due to inverse electron demand Diels-Alder reactions between the tetrazine and the aggregated TCO bearing monomers.

Enterokinase (ENTK) (also known as enteropepti dase) 130 is a membrane bound protease that is essen tial for digestion 131 . Its main physiological purpose is the cleavage of the acidic motif DDDDK in the pan creatic proenzyme trypsinogen, which leads to the activation of trypsin 132 . There are multiple reasons as to why ENTK is an interesting enzyme candidate for programming cell specific and organelle specific self assembly 106, [133] [134] [135] : firstly, its role in the activation of trypsin has been linked to matrix degradation and migration of lung, colorectal and glioblastoma cancer cells 136 ; secondly, it allows cell specific targeting of the mitochondria in cancer cell lines, such as HeLa (cervical cancer), Saos2 (bone cancer) and HepG2 (liver can cer) 134 ; and, thirdly, it enables the enzyme driven release of cargo from a supramolecular vehicle at the organelle surface 133 . The ENTK sensitive systems consist of the cleavable FLAG tag DYKDDDDK covalently linked to a self assembling peptide sequence 106, 133, 134 or a lipid like moiety 135 . The chemical design of the peptide based precursor is a branched structure that is important for mitochondria targeting 133 . After entering the cell in a micellar form by clathrin mediated endocytosis 134 , the supramolecular precursors escape the endosome facil itated by pH buffering of the carboxylic acid groups 135 . The subsequent subcellular accumulation at the mito chondria is promoted by the increased mitochondrial membrane potential in cancer cells and the electrostatic interaction between the negatively charged FLAG tag and voltage dependent anion channels at the orga nelle surface 135 . The enzymatic cleavage of the hydro philic tag by ENTK initiates the local conversion from micelles to peptide nanofibres 106, 133, 134 (or more lipo philic micelles in the case of the peptide-lipid conju gates) 135 . This supramolecular transformation causes an increase in perimitochondrial viscosity and enables the organelle specific delivery of drugs 133,135 , proteins 106 or gene vectors 134 .

Trypsin is a potent serine protease that has been the subject of ongoing biomedical research due to its role in the invasion and metastasis of various cancers, such as ovarian 137 , colorectal 138, 139 , lung 140 and prostate cancer 141 . The overexpression of different trypsin iso forms by tumours leads to the amplified degradation of extracellular proteins 142 and the increased activation of other matrix associated proteases 137, 143, 144 . The preferen tial cleavage of trypsin at the carboxy terminal of lysine or arginine can be exploited as an enzymatic trigger for the self assembly of synthetic monomers, as shown by a trypsin responsive precursor molecule 19 . Similar to the ENTK responsive systems, the branched pro assembling peptide consists of an aromatic tetrapeptide backbone and a hydrophilic lysine rich sequence KYDKKKKDG that can be cleaved in trypsin 1 overexpressing ovar ian cancer cells (OVSAHO). As these particular cells exhibit an abnormally high activity of the protease at the endoplasmic reticulum (ER), the resulting formation Theranostic Combining therapeutic and diagnostic properties within a single material platform.

www.nature.com/natrevchem of cytotoxic nanofibres is not only cell specific but also organelle specific, despite high concentrations of the supramolecular precursor being necessary.

Cathepsin B is a lysosomal protease that has been linked to the invasiveness of several types of cancer 145 , including colorectal 146 and breast cancer 147 . This pro angiogenic enzyme 148 recognizes and cleaves the sequence GFLG in synthetic substrates, which has made it a useful biological tool for cancer specific drug delivery 149 . Combining the technology of AIE luminogens with a cathepsin B cleavable peptide led to a light up imaging probe and photosensitizer for the detection and ablation of breast cancer cells 40 . The precursor consists of a tetraphenylethylene derivative with two azido groups that are each 'clicked' to a bio active peptide composed of the cathepsin B responsive sequence GFLG, a hydrophilic linker (DDD) and the targeting moiety cyclo RGD. The double substituted AIE imaging probe can form fluorescent aggregates in the lysosomes of breast cancer cells (MDA MB 231), which facilitates the quantification of enzyme activity, as well as photodynamic therapy 40 . To study the auto catalytic growth of intracellular nanofibres, a cathep sin B responsive prodrug was developed that could undergo a morphology transformation from nano particles to fibrous networks in vitro and in vivo: after shedding a hydrophilic PEG RGD unit, the remaining self assembling peptide-drug conjugate undergoes β sheet driven fibrillation inside HeLa cells, which is accelerated by the presence of previously formed fibrils 5 .

Matrix metalloproteinase 7 (MMP7) is a protease involved in the degradation of extracellular matrix proteins 150 , contributing to cancer cell migration 151 . To initiate cancer cell death by intracellular self assembly, a peptide-lipid conjugate susceptible to cleavage by MMP7 was developed, an enzyme that is secreted by HeLa cells 67 . The palmitoylated peptide consists of a glycine rich nonpolar sequence, an MMP7 cleavage site (PLG_L) and a hydrophilic carboxy terminal fragment (_LARK) that inhibits unspecific aggregation before enzymatic conversion. After incubation with the pre cursor, HeLa cells exhibited decreased viability caused by the cancer cell specific formation of intracellular peptide-lipid nanofibres 67 .

ATG4B is a cysteine protease that contributes to autophagy, during which protein aggregates and dam aged organelles are digested and recycled 152 . While it is essential for homeostasis in healthy tissue, dysregu lated autophagy is associated with pathogenesis, such as tumorigenesis 153,154 and neurodegeneration 155 . Since ATG4B is crucial for the formation of autophagosomes 156 , its activity was monitored using a bioresponsive supra molecular probe 157 . The chemical design of this imaging agent combines a self assembling bispyrene with a pep tide displaying the ATG4B cleavage site TFG_F 157 , as well as a highly cationic poly(amidoamine) dendrimer that enables cell entry and provides water solubility. After the enzymatic cleavage in MCF7 breast cancer cells, the free bispyrene units selfassemble into fluorescent nano particles due to the formation of J aggregates 69 , before transforming into cytosolic nanofibres over time 157 .

Phosphorylation and dephosphorylation of protein substrates play a vital role in signal transduction and the regulation of enzyme and receptor activity 158 . Since dysregulation of these processes is associated with cel lular dysfunction and tumorigenesis, phosphatases, which are the enzyme family responsible for the removal of phosphate groups 159 , are interesting candi dates for controlling enzyme instructed intracellular self assembly. Alkaline phosphatase (ALP) is a prom inent example of a ubiquitous enzyme that catalyses dephosphorylation 160 . Regarding subcellular localiza tion, ALP as a membrane bound phosphatase is asso ciated with the cell membrane 160 , while PTP1B, another important protein tyrosine phosphatase, is located at the surface of the ER 161 . By exploiting the overexpres sion of both ALP and PTP1B as known tumour mark ers, phosphatase instructed self assembly of short phospho tyrosine (Yp) containing peptides can be used for bioimaging 18 and combinational cancer therapy 162 .

A general structure of the precursor for phosphatase instructed self assembly was established: a tetrapep tide containing two phenylalanines, one Yp that can be dephosphorylated, as well as a basic amino acid, such as lysine, which can be coupled to a fluorophore or a targeting unit. The amino terminus of the short pep tide is substituted with an additional aromatic moiety, for example, a naphthyl group, that contributes to the propensity for self assembly due to intermolecular aro matic interactions of the dephosphorylated monomer. For example, the fluorescent aromatic tetrapeptide naphthyl FFK(nitrobenzoxadiazole (NBD)) Yp was used as a phosphatase sensitive precursor for the formation of fluorescent nanofibres inside HeLa cells 18 (fig. 3b) . Interestingly, the fibrous structures were observed to grow within minutes from the ER towards the cell membrane, which can be explained by the high enzy matic activity of PTP1B at the surface of the organelle 18 . Besides using NBD as the dye, introducing other fluoro phores revealed that the supramolecular behaviour and the spatial distribution of the intracellular assemblies vary drastically depending on the fluorescent unit 163, 164 ; NBD containing monomers can form intracellular nanofibres that protect the cell and its cytoskeleton against an F actin toxin, whereas dansyl substituted peptide localizes in the cell membrane and exhibits a cytotoxic effect after dephosphorylation 124 . These differ ences in the bioactivity of the precursors and the respec tive activated monomers can be explained by variations in hydrophobicity of the fluorophore dictating both their interactions with cellular membranes and their self assembly propensity. For example, the dansyl group exhibits a higher solubility in phospholipid membranes, partly due to its compatibility with the headgroup of phosphatidylcholine 165 , while the peptide precursor substituted with positively charged rhodamine cannot produce a hydrogel even after dephosphorylation by ALP, leading to an unspecific fluorescence distribution inside and outside of the cells 124 .

Aside from modulating the intracellular assembly behaviour by changing the fluorophore, exchanging amino acids with different stereochemical properties in Autophagy Ordered degradation and recycling of dysfunctional cellular components, which is important for homeostasis. Tumorigenesis formation or emergence of cancerous cells.

Nature reviews | Chemistry the precursor can also change the morphology of the nanostructures resulting from dephosphorylation 17 . By attaching an l homoarginine ( L hR) at the carboxyl terminus of the d tripeptides, naphthyl D F D F D Yp and NBD D F D F D Yp precursors were generated for phosphatase instructed self assembly that, after dephos phorylation, could form cytotoxic crescent shaped assemblies targeted for accumulation at the ER. The toxic effect of the crescent shaped structures seemed to be cell specific, as it was limited to cancerous cells with high levels of ALP at their cell membrane, such as HeLa cervical cancer cells and OVSAHO ovarian cancer cells, while normal stromal cells (HS5) were not affected 17 .

To further control and exploit the subcellular localization of phosphatase instructed assemblies, the structure of the precursor can be adapted for organellespecific targeting 57, 166, 167 . Integrating posi tively charged TPP via lysine side chain modification gives rise to mitochondria targeted tetrapeptides that can undergo dephosphorylation by ALP at the cell membrane of Saos2 osteosarcoma cells 57 . The resulting polymorphic aggregates can enter the cells and trigger the release of cytochrome c from the mitochondria, causing cell death only in ALP overexpressing can cer cells. The in situ generation of the self assembling monomers seems to be essential for the toxicity towards the cells, since incubating them directly with the final product of the enzymatic conversion has no effect on their viability 57 . Aside from the use of well known tar geting moieties such as TPP, incorporating ligands of enzymes that are specific for a certain organelle can also influence the accumulation and structure forma tion at the desired subcellular location. An example for this is the naproxen (Npx) substituted tetrapeptide Npx D F D F D Yp L hR used for intracellular enzyme seques tration on the surface of the ER 166 . Npx is a nonsteroidal anti inflammatory drug and a ligand of COX2, which is an enzyme located at the surface of the ER. By binding to COX2 via Npx and to PTP1B via Yp, the precursor and the resulting fibrous assemblies can sequester the two enzymes on the organelle surface, thereby, induc ing their co localization and potential protein-pro tein interaction. Another recent discovery regarding organelle specific phosphatase instructed self assembly was that short thiophosphopeptides can form nanofibres specifically at the Golgi apparatus 167 . This phenomenon seems to be due to the rapid conversion of thiophos phopeptides into thiopeptides by Golgi associated ALP followed by disulfide bond formation with cysteine rich proteins in the oxidative environment of the organelle.

Other enzymes. β Galactosidase is a lysosomal sugar hydrolase that is overexpressed in senescent cells 168 . As the accumulation of non proliferating cells can nega tively impact tissue function 169 , detecting and clearing senescent cells is of therapeutic interest, especially for agerelated diseases such as atherosclerosis 170 . For this reason, a short aromatic peptide was designed with a βgalactosefunctionalized tyrosine (NBDFFY(βGal)G) that is susceptible to enzymatic cleavage inside senescent endothelial cells and HeLa cells 171 . While the viability of non senescent cells was only slightly affected by the administration of the enzyme responsive precursor, it caused the β galactosidase instructed formation of cytotoxic nanofibres in senescent cells due to intermo lecular aromatic interaction. A different approach to implement β galactosidase sensitivity into a precursor for condensation driven self assembly used a benzyl carbamate amino protecting group with a β galactose substituent on the aromatic ring that could undergo self immolative degradation upon enzymatic removal of the sugar 16 (fig. 7a ). This β galactosidase induced depro tection of a reactive amino thiol triggers a subsequent condensation reaction with an aromatic nitrile that results in the intracellular formation of self assembling macrocycles.

Carboxylesterases (CES) are essential for the cleav age of endogenous and xenobiotic esters 172 . Aside from their general importance for lipid homeostasis and drug metabolism, CES were also the first enzymes to be exploited for the intracellular selfassembly of synthetic nanomaterials 173 . The CESresponsive precursors gen erally consist of a diphenylalanine, an aminoterminal naphthyl group or aromatic fluorophore, and a carboxy terminal ethanolamine offering a hydroxyl group for the esterification of a hydrophilic unit (for example, butyric acid or taurine) that prevents aggregation 51, [173] [174] [175] [176] . Inside cells with high CES activity, such as certain ovarian and cervical cancer cells, the enzymatic cleavage of the ester bond leads to the formation of nanofibres that can disrupt actin filaments 175 and boost the activity of the anticancer drug cisplatin 51 .

While the enzymes mentioned so far in this Review are all hydrolases, meaning that they catalyse the breakage of chemical bonds using water, there are also enzymes that create new bonds and that can be used for the programming of nanomaterial aggregation inside cells.

Transglutaminase is an enzyme that catalyses the amide bond formation between glutamine and lysine side chains 177 . This biocatalytic condensation reaction can be exploited for the intracellular polymerization of short peptide monomers into elastin like polypeptides (ELPs) 105 . The topology and the thermoresponsive prop erties of these ELPs is predetermined by the number of lysines and glutamines in the polymerizable monomer: while peptide monomers with only one lysine and glu tamine each yield linear ELPs with upper or lower critical solution temperature behaviour to form globular aggre gates, monomers containing more than one lysine and glutamine yield a three dimensional gel like ELP net work inside the cell. The latter exhibits a dose dependent cytotoxicity, whereas linear ELPs forming random coils or aggregates are biocompatible 105 .

The most common chemical design strategy for bio responsive molecules for supramolecular assembly focuses on the combination of a cleavable hydrophilic unit with a more hydrophobic self assembling moiety, such as a β sheet forming peptide or a polyaromatic group. In this general approach, the stimulus induced separation of the two building blocks directly triggers Senescent Characteristic of cells with arrested cell cycle that can no longer divide but are still metabolically active. Cellular senescence is associated with multiple diseases later in life, such as cancer or atherosclerosis.

www.nature.com/natrevchem the formation of synthetic structures inside cells (fig. 3b ). However, given the complexity of the cellular environ ment and the many potential endogenous stimuli, sys tems for intracellular self assembly can also be made to undergo a more complex multistep transformation to the final monomer. The idea of having a reaction sequence or cascade rather than a single transformative step also offers the opportunity to implement more than one bioresponsive element into the chemical design of the precursor, which can increase spatial control over the assembly formation.

The latter can be achieved, for example, by combin ing an enzyme responsive unit on one part of the mole cule with a redox sensitive group on another part of the molecule (fig. 7a ). This combination has been used for the condensation reaction between a deprotected ami nothiol and a cyanobenzothiazole for the formation of self assembling macrocycles 75 . As both the amino group and the thiol group of the amino-terminal aminothiol are necessary for condensation and self assembly, choos ing complementary bioresponsive protecting groups helps to tailor the system towards a specific intracel lular behaviour. For example, the amino group of the aminothiol can be attached to an enzyme responsive peptide sequence that is cleavable by a protease (for example, furin 9,21,113 or caspase3/7 (refs 16,50,125 )), while the thiol group can be protected via a disulfide bond to a small molecule such as ethanethiol ( fig. 7a ). Upon cell entry, this disulfide bond is cleaved by intracellular glutathione in the reducing environment of the cytosol. Consequently, the determining step for the formation of the reactive intermediate, which can transform into the self assembling monomer, is the final deprotection of the N terminal amino group. As this can only occur at the subcellular localization of the targeted enzyme, spatial control over the structure formation is achieved by careful selection of the enzyme responsive moiety in the precursor. This strategy is not only applicable to proteases but also works for other enzymes such as β galactosidase 16 or nitroreductase 178 if a self immolative benzyl carbamate derived protecting group is cho sen for the amino terminus ( fig. 7a ). The cyanobenzo thiazole moiety can also be substituted for other less electrophilic aromatic nitriles, which lowers the risk of potential side reactions with endogenous aminothiols 16 . Aside from implementing bioresponsive groups that control self assembly, additional enzyme cleavage sites can be included in the centre of the molecule that allow the programmed disassembly of intracellularly formed nanostructures by proteolysis later on 16, 17 . For biomed ical purposes, the cyclization precursor can be modi fied to include photothermal agents that are activated by condensation mediated and assembly mediated quenching 13 or chemotherapeutic drugs that can slowly be released inside tumour cells 118 .

For programming peptide fibre formation inside cancer cells, a multiresponsive system was designed that is pH sensitive and ROS sensitive 6 . The peptide based precursors contain a phenylboronic acid trigger group, which serves as both a ROS sensitive cage for the amino group of an esterified serine and as a handle for the attachment of a cell penetrating peptide via an acid labile dynamic covalent bond ( fig. 7b ). In the acidic environment of the endosome, the cell penetrating pep tide is removed. After endosomal escape, cytosolic ROS causes the degradation of the self immolative phenylb oronic acid group, revealing a free reactive amine. In the neutral pH of the cytosol, the nucleophilic amino group can perform an intramolecular attack on the adjacent ester bond of the serine side chain, yielding linearized peptides that can co assemble into cytotoxic peptide fibres inside ROS overproducing cancer cells 6 . These decoupled steps of the reaction cascade -the initial activation by deprotection and the following transfor mation to the monomer -take place under different chemical conditions and display individual reaction kinetics. Therefore, the multistep approach offers more opportunities to modulate the speed of the transforma tion of a precursor into a self assembling monomer, in contrast to most systems for intracellular self assembly that rely on a singular transformative step.

Synthetic supramolecular chemistry in a cellular envi ronment uses a strategic implementation of biorespon siveness in chemical design to achieve control over the formation of nanoscale architectures. In this Review, we discuss various chemical scaffolds, such as peptides, poly aromatic molecules and polymers, that can undergo a triggered transformation into assemblies due to endog enous stimuli. Tailoring the reactivity of the nanomate rial towards a specific intracellular cue can be realized by incorporating redox sensitive or pH sensitive groups, as well as enzyme recognition motifs that allow biocata lytic transformations. These stimulus dependent mech anisms of self assembly come with numerous advantages for therapeutic or diagnostic application. For example, the bioresponsive properties of the materials contribute to cell and tissue specificity in vivo, while also allowing for spatial control over the structure formation inside cells by targeting organelle specific enzymes. Another therapeutic advantage of systems for intracellular self assembly is the circumvention of multidrug resist ance in certain cancers due to an impaired efflux of the intracellularly formed nanostructures compared with small molecules. Moreover, this accumulation effect can also contribute to the concentration of fluorescent agents inside the targeted cells, enabling more efficient bioimaging. Compared with ex situ self assembled mate rials, such as therapeutic nanoparticles, the monomeric assembly precursors generally display an easier clearance from the liver, spleen or kidneys, thereby, reducing their systemic toxicity 179 .

However, there are also several challenges regard ing the potential clinical translation that need to be addressed. For example, a detailed pharmacokinetic and pharmacodynamic analysis of nanomaterials for intra cellular self assembly is necessary to ensure a favourable biodistribution and circulation time of precursors, as well as low immunogenicity. The chemical stability of the precursor molecule is crucial for its in vivo applica tion, as, for instance, the proteolysis of peptide based materials prior to cell entry and self assembly needs to be avoided. Additionally, the eventual clearance of the in situ formed nanostructures must be considered thoroughly, since stable assemblies, particularly amyloid like β sheet peptide assemblies, could potentially cause negative side effects over time as a result of their systemic accumulation. Generally, the morphology, stability and overall physiological impact of synthetic nano structures need to be closely monitored and evalu ated, which is still a challenge for current methods of in vivo analysis.

In the future, further exploration of the existing tool box of functionalities that can be reduced by glutathione, oxidized by ROS or that are labile to acidification could broaden the scope of potential precursors for intracel lular self assembly. With systems for enzyme instructed self assembly being prominent in recent years, the inves tigation of other enzymes beyond the commonly used phosphatases and proteases could not only help to diver sify strategies but also contribute to a more cell specific approach that could benefit the targeted therapy of can cer or other diseases with a characteristic dysregulation of enzyme expression. For this purpose, the study of molecular pathology is essential to lay the foundations for the design of novel enzyme responsive materials. Moreover, including more than one bioresponsive group can increase the spatial control over the structure for mation, as well as cell specificity, since multiple endog enous stimuli need to be present for assembly to occur.

Adding other bioactive components to the molecular design, such as signalling peptides 48, 180 or small molecule drugs 162 , can further enhance the impact of the system on cellular processes leading to potentially synergistic therapeutic effects.

Many existing systems rely on thermodynamic control as the primary driving force to construct the intracellular architectures. Advanced methodologies involving non equilibrium assemblies 181, 182 coupled to cellular feedback dynamics would be highly attractive to induce reversible changes in cellular behaviour, as cell death is not always the desired outcome. From this per spective, there is also room to explore intracellular struc tures that boost cellular functions, possibly to address cell senescence (ageing) and environmental adaptation. The creation of such biomimetic materials that imitate reversibly formed biological supramolecular assemblies would be a milestone in synthetic biology and challenge frontiers in biomedical science.

In the upcoming years, we expect the exploration of bioresponsive materials for intracellular assembly to give rise to new sophisticated approaches to create nanostructures in complex biological environments. We hope that the design concepts presented in this Review contribute to these efforts.

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Peptides and Peptide-based Biomaterials and their Biomedical Applications

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Reactive oxygen species in vascular biology: implications in hypertension

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The signaling mechanism of ROS in tumor progression

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The authors acknowledge funding by the Max Planck-Bristol Centre for Minimal Biology and the Max Planck Society. This work was supported by the Max Planck Graduate Center (MPGC) with the Johannes Gutenberg University Mainz.

S.C. researched data for the article, contributed to discussion of content and wrote the article. D.Y.W.N. and T.W. edited the manuscript and contributed substantially to discussion of the content. All authors reviewed the manuscript before submission.

The authors declare no competing interests.

Nature Reviews Chemistry thanks J. Shen (who co-reviewed with R. Liu) and the other, anonymous, reviewers for their contribution to the peer review of this work.

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