Intrahippocampal Supramolecular Assemblies Directed Bioorthogonal Liberation of Neurotransmitters to Suppress Seizures in Freely Moving Mice.

The precise delivery of anti-seizure medications (ASM) to epileptic loci remains the major challenge to treat epilepsy without causing adverse drug reactions. The unprovoked nature of epileptic seizures raises the additional need to release ASMs in a spatiotemporal controlled manner. Targeting the oxidative stress in epileptic lesions, here the reactive oxygen species (ROS) induced in situ supramolecular assemblies that synergized bioorthogonal reactions to deliver inhibitory neurotransmitter (GABA) on-demand, are developed. Tetrazine-bearing assembly precursors undergo oxidation and selectively self-assemble under pathological conditions inside primary neurons and mice brains. Assemblies induce local accumulation of tetrazine in the hippocampus CA3 region, which allows the subsequent bioorthogonal release of inhibitory neurotransmitters. For induced acute seizures, the sustained release of GABA extends the suppression than the direct supply of GABA. In the model of permanent damage of CA3, bioorthogonal ligation on assemblies provides a reservoir of GABA that behaves prompt release upon 365 nm irradiation. Incorporated with the state-of-the-art microelectrode arrays, it is elucidated that the bioorthogonal release of GABA shifts the neuron spike waveforms to suppress seizures at the single-neuron precision. The strategy of in situ supramolecular assemblies-directed bioorthogonal prodrug activation shall be promising for the effective delivery of ASMs to treat epilepsy.

A Dual-Mechanism Targeted Bioorthogonal Prodrug Therapy.

Bioorthogonal prodrug therapies offer an intriguing two-component system that features enhanced circulating stability and controlled activation on demand. Current strategies often deliver either the prodrug or its complementary activator to the tumor with a monomechanism targeted mechanism, which cannot achieve the desired antitumor efficacy and safety profile. The orchestration of two distinct and orthogonal mechanisms should overcome the hierarchical heterogeneity of solid tumors to improve the delivery efficiency of both components simultaneously for bio-orthogonal prodrug therapies. We herein developed a dual-mechanism targeted bioorthogonal prodrug therapy by integrating two orthogonal, receptor-independent tumor-targeting strategies. We first employed the endogenous albumin transport system to generate the in situ albumin-bound, bioorthogonal-caged doxorubicin prodrug with extended plasma circulation and selective accumulation at the tumor site. We then employed enzyme-instructed self-assembly (EISA) to specifically enrich the bioorthogonal activators within tumor cells. As each targeted delivery mode induced an intrinsic pharmacokinetic profile, further optimization of the administration sequence according to their pharmacokinetics allowed the spatiotemporally controlled prodrug activation on-target and on-demand. Taken together, by orchestrating two discrete and receptor-independent targeting strategies, we developed an all-small-molecule based bioorthogonal prodrug system for dual-mechanism targeted anticancer therapies to maximize therapeutic efficacy and minimize adverse drug reactions for chemotherapeutic agents.

[Advances in targeted delivery of proteolysis targeting chimeras in cancer therapy].

Small-molecule anticancer drugs inhibited tumor growth based on targeted inhibition of specific proteins, while most of oncogenic proteins are "undruggable". Proteolysis targeting chimeras (PROTAC) is an attractive and general strategy for treating cancer based on targeted degradation of oncogenic proteins. This review briefly describes the peptide-based PTOTAC and small molecule-based PROTAC. Subsequently, we summarize the development of targeted delivery of PROTAC, such as targeting molecule-mediated targeted delivery of PROTAC, nanomaterial-mediated targeted delivery of PROTAC and controllable activation of small-molecular PROTAC prodrug. Such strategies show potential application in improving tumor selectivity, overcoming off-target effect and reducing biotoxicity. At the end, the druggability of PROTAC is prospected.

Reductase-Labile Peptidic Supramolecular Hydrogels Aided in Oral Delivery of Probiotics.

Oral delivery of probiotics has been a promising method for treatment of inflammatory bowel diseases (IBDs). However, probiotics always suffer from substantial loss of viability due to the harsh gastrointestinal conditions, especially the highly acidic environment in the stomach and bile salts in the intestine. In addition, to overcome the challenging conditions, an ideal delivery of probiotics requires the on-demand release of probiotics upon environmental response. Herein, a novel nitroreductase (NTR) labile peptidic hydrogel based on supramolecular self-assembly is demonstrated. The efficient encapsulation of typical probiotic Escherichia coli Nissle 1917 (EcN) into supramolecular assemblies yielded a probiotic-loaded hydrogel (EcN@Gel). Such a hydrogel adequately protected EcN to improve its viability against harsh acid and bile salt environments during oral delivery. The upregulated NTR in the intestinal tract triggered the disassembly of the hydrogel and accomplished the controlled release of EcN locally. In ulcerative colitis (UC)-bearing mice, EcN@Gel showed significantly enhanced therapeutic efficacy by downregulating proinflammatory cytokines and repairing the intestinal barrier. Moreover, EcN@Gel remolded the gut microbiome by increasing the diversity and abundance of indigenous probiotics, contributing to ameliorated therapies of IBDs. The NTR-labile hydrogel provided a promising platform for the on-demand delivery of probiotics into the intestinal tract.

Nitroreductase-instructed supramolecular assemblies for microbiome regulation to enhance colorectal cancer treatments.

The development of human microbiome has collectively correlated the sophisticated interactions between Fusobacterium nucleatum and colorectal cancers (CRCs). However, the treatment of CRC via disruption of gastrointestinal flora remains less explored. Aiming at the up-regulated activity of nitroreductase in F. nucleatum-infected tumors, here, we developed the nitroreductase-instructed supramolecular self-assembly. The designed assembly precursors underwent enzymatic transformation to form assemblies, which agglutinated F. nucleatum and eradicated the targeted bacteria. These assemblies with anti-F. nucleatum activity could further alleviate the bacteria-induced drug resistance effect, thus sensitizing CRC cells against chemo-drugs. Eventually, in mice bearing F. nucleatum-infected CRC, the local introduction of nitroreductase-instructed assemblies could efficiently inhibit the tumor growth. Overall, this study incorporated nitroreductase to broaden the toolbox of enzyme-instructed supramolecular self-assembly. The local introduction of nitroreductase-instructed assemblies could target F. nucleatum to eliminate its contribution to CRC drug resistance and ameliorate chemotherapy outcomes.

Intracellular fluorogenic supramolecular assemblies for self-reporting bioorthogonal prodrug activation.

A visual drug delivery system (DDS) is urgently needed for precision medicine. DDS-mediated bioorthogonal prodrug activation strategies have demonstrated remarkable advantages in enlarging a therapeutic index via the alleviation of adverse drug reactions. However, the events of bioorthogonal prodrug activation remain inaccessible. Here, we construct a self-reporting bioorthogonal prodrug activation system using fluorescence emission to interpret prodrug activation events. In designed reactive oxygen species (ROS)-instructed supramolecular assemblies, the bioorthogonal reaction handle of tetrazine carries a dual role as fluorescence quencher and prodrug activator. The subsequent inverse-electron-demand Diels-Alder (IEDDA) reaction simultaneously liberates fluorescence and active drugs, which form a linear relationship. Differentiated by their cellular redox status, ROS-instructed supramolecular assemblies form selectively in both tumor cells and cell spheroids. Upon prodrug treatment, the brightness of fluorescence reflects the liberation of active drugs, which further correlates with the cell survival rate. Therefore, a fluorescence-based visualizable DDS (VDDS) for bioorthogonal prodrug activation is demonstrated, which should be useful to elucidate the multi-step processes in drug delivery and determine prodrug activation efficacy.

Bioorthogonal Disassembly of Tetrazine Bearing Supramolecular Assemblies Inside Living Cells.

Supramolecular assemblies are an emerging class of nanomaterials for drug delivery systems (DDS), while their unintended retention in the biological milieu remains largely unsolved. To realize the prompt clearance of supramolecular assemblies, the bioorthogonal reaction to disassemble and clear the supramolecular assemblies within living cells is investigated here. A series of tetrazine-capped assembly precursors which can self-assemble into nanofibers and hydrogels upon enzymatic dephosphorylation are designed. Such an enzyme-instructed supramolecular assembly process can perform intracellularly. The time-dependent accumulation of assemblies elicits oxidative stress and induces cellular toxicity. Tetrazine-bearing assemblies react with trans-cyclooctene derivatives, which lead to the disruption of π-π stacking and induce disassembly. In this way, the intracellular self-assemblies disassemble and are deprived of potency. This bioorthogonal disassembly strategy leverages the biosafety aspect in developing nanomaterials for DDSs.

Enzymatic non-covalent synthesis of supramolecular assemblies as a general platform for bioorthogonal prodrugs activation to combat drug resistance.

Multi-drug resistance (MDR) is one of the leading causes of the anticancer failures. Besides the blockage of the MDR pathways, the development of more potent drugs is with urgent needs, but has been postponed mainly due to an imbalance between safety and efficacy. The recent development of the bioorthogonal prodrug activation strategy has shown immense potential to balance safety and efficacy, while recent studies only focused on few drug entities such as doxorubicin and monomethyl auristatin E, leaving the vast collection of toxins undetermined. Here we have enumerated typical molecular entities ranging from food and drug administration (FDA) approved drugs to a heated antibody drug conjugates (ADC) warhead and a trichothecene toxin to demonstrate that the bioorthogonal caging and specific activation could serve as a general design to increase the therapeutic index of bioactive molecules. These prodrugs can be efficiently activated on-demand by the bioorthogonal activators whose distribution was regulated by the cancer cell specific enzymatic non-covalent synthesis of supramolecular self-assemblies. The prodrug activation not only enhanced the synergistic therapeutic effect within a broad range of dose ratios but also allowed the convenient switching of drug identities to successfully combat MDR tumor in vivo. In general, this strategy might serve as a general platform, which can be readily applicable to enlarge the therapeutic window for various bioactive molecules. We envision that the spatiotemporal controlled bioorthogonal prodrug activation would facilitate the discovery of anticancer drugs.

Enzyme-instructed supramolecular assemblies promote intracellular boron accumulation for boron neutron capture therapy.

Selective accumulation of boron agents in cancer cells is of critical importance for BNCT. Here we involve enzyme-instructed supramolecular assembly (EISA) to facilitate the accumulation of a typical boron agent borylphenylalanine (BPA) in cancer cells. By covalently conjugating BPA to the phosphorylated assembly precursor, the boron-bearing precursors undergo phosphatase-catalyzed dephosphorylation to yield assembly molecules, which then self-assemble to form nanomaterials. Due to the up-regulated phosphatase activity of cancer cells, kinetic preference allows the EISA to accumulate boron in HeLa cells selectively. Interestingly, by attaching BPA on the backbone or side-chain of precursor, the boron-bearing isomers show different assembly propensity with time-dependent morphology change, which leads to the differentiated accumulation of boron inside cells. Overall, the optimized boron-bearing assembly precursor could significantly improve the boron accumulation compared with BPA in cancer cells. In this study, we have demonstrated a convenient method to introduce boron agents to cancer cells. We envision that the EISA-mediated accumulation of boron will be helpful in the design of boron agents to facilitate BNCT treatment.

Redox-Mediated Reversible Supramolecular Assemblies Driven by Switch and Interplay of Peptide Secondary Structures.

The construction of reversible supramolecular self-assembly in vivo remains a significant challenge. Here, we demonstrate the redox-triggered reversible supramolecular self-assembly governed by the "check and balance" of two secondary conformations within a brushlike peptide-selenopolypeptide conjugate. The conjugate constitutes a polypeptide backbone whose side chain contains selenoether functional moieties and double bonds to be readily grafted with ß-sheet-prone short-peptide NapFFC. The backbone of the conjugate initially assumes a robust and rigid α-helical conformation, which inhibits the supramolecular assembly of the short peptide in the side chain and yields an overall irregular aggregate morphology under native/reduced conditions. Upon oxidation of the selenoether to more hydrophilic selenoxide, the backbone helix switches to a flexible and disordered conformation, which unleashes the side-chain NapFFC self-assembly into nanofibrils via the adoption of ß-sheet conformation. The reversible switch of the supramolecular morphology enables efficient loading and tumor-microenvironment-triggered release of anticancer drugs for in vivo cancer treatment with satisfactory efficacy and biocompatibility. The interplay and interaction between two well-defined secondary structures within one scaffold offer tremendous opportunity for the design and construction of functional supramolecular biomaterials.

Pathological environment directed in situ peptidic supramolecular assemblies for nanomedicines.

Peptidic self-assembly provides a powerful method to build biomedical materials with integrated functions. In particular, pathological environment instructed peptidic supramolecular have gained great progress in treating various diseases. Typically, certain pathology related factors convert hydrophilic precursors to corresponding more hydrophobic motifs to assemble into supramolecular structures. Herein, we would like to review the recent progress of nanomedicines based on the development of instructed self-assembly against several specific disease models. Firstly we introduce the cancer instructed self-assembly. These assemblies have exhibited great inhibition efficacy, as well as enhanced imaging contrast, against cancer models both in vitro and in vivo. Then we discuss the infection instructed peptidic self-assembly. A number of different molecular designs have demonstrated the potential antibacterial application with satisfied efficiency for peptidic supramolecular assemblies. Further, we discuss the application of instructed peptidic self-assembly for other diseases including neurodegenerative disease and vaccine. The assemblies have succeeded in down-regulating abnormal Aß aggregates and immunotherapy. In summary, the self-assembly precursors are typical two-component molecules with (1) a self-assembling motif and (2) a cleavable trigger responsive to the pathological environment. Upon cleavage, the self-assembly occurs selectively in pathological loci whose targeting capability is independent from active targeting. Bearing the novel targeting regime, we envision that the pathological conditions instructed peptidic self-assembly will lead a paradigm shift on biomedical materials.

Gag Protein Oriented Supramolecular Nets as Potential HIV Traps.

For HIV/AIDS treatment, the cocktail therapy which uses a combination of anti-retroviral drugs remains the most widely accepted practice. However, the potential drug toxicity, patient tolerability, and emerging drug resistance have limited its long-term efficiency. Here, we design a HIV Gag protein-targeting redox supramolecular assembly (ROSA) system for potential HIV inhibition. An assembling precursor was constructed through conjugation of an oxidation-activatable fluorogenic compound BQA with a selected tetrapeptide GGFF. Since BQA shares a similar structure with the known Gag inhibitor, the precursor could bind to HIV Gag protein with moderate affinity. Moreover, after oxidation, the corresponding nanofibers could bind to Gag protein and trap HIV to realize virus control, thus providing a potential anti-HIV strategy.

Supramolecular assemblies mimicking neutrophil extracellular traps for MRSE infection control.

Neutrophil extracellular traps (NETs) stick to bacteria and prevent infections in vivo, whose activation is upon inflammatory stimuli along with the sudden increase of reactive oxygen species (ROS). Nevertheless, the risky over activation in NETosis may result in deleterious outcome. A big challenge in using NETs for therapeutics is to synthesize an artificial system that can function as NETs in vivo. Here, we developed an in vivo supramolecular assembly system to imitate the innate immune process of NETs to inhibit methicillin-resistant staphylococcus epidermidis (MRSE) infection. Our synthesized small molecules undergo oxidation to form supramolecular nanofibers at inflammatory loci. The in situ formed nanofibers network efficiently traps MRSE cells and prevent them from aggressive dissemination. The extended interactions between nanofibers and bacteria directly result in the death of MRSE via the transcriptomes alterations. In clinically relevant models (intraperitoneal infection and catheter implantation), our supramolecular nets show significant antibacterial activity, yielding a three times efficacy comparing to vancomycin. The spontaneous consumption of ROS and the formation of antibacterial networks create a steady negative feedback system to combat bacterial infections.

Dynamic Detection of Active Enzyme Instructed Supramolecular Assemblies In Situ via Super-Resolution Microscopy.

Inspired by the self-assembly phenomena in nature, the instructed self-assembly of exogenous small molecules in a biological environment has become a prevalent process to control cell fate. Despite mounting examples of versatile bioactivities, the underlying mechanism remains less understood, which is in large hindered by the difficulties in the identification of those dynamic assemblies in situ. Here, with direct stochastic optical reconstruction microscopy, we are able to elucidate the dynamic morphology transformation of the enzyme-instructed supramolecular assemblies in situ inside cancer cells with a resolution below 50 nm. It indicates that the assembling molecules endure drastically different pathways between cell lines with different phosphatase activities and distribution. In HeLa cells, the direct formation of intracellular supramolecular nanofibers showed slight cytotoxicity, which was due to the possible cellular secretory pathway to excrete those exogenous molecules assemblies. In contrast, in Saos-2 cells with active phosphatase on the cell surface, assemblies with granular morphology first formed on the cell membranes, followed by a transformation into nanofibers and accumulation in cells, which induced Saos-2 cell death eventually. Overall, we provided a convenient method to reveal the in situ dynamic nanomorphology transformation of the supramolecular assemblies in a biological environment, in order to decipher their diverse biological activities.

Enzyme-Instructed Self-assembly in Biological Milieu for Theranostics Purpose.

Precision medicine is in an urgent need for public healthcare. Among the past several decades, the flourishing development in nanotechnology significantly advances the realization of precision nanomedicine. Comparing to well-documented nanoparticlebased strategy, in this review, we focus on the strategy using enzyme instructed selfassembly (EISA) in biological milieu for theranostics purpose. In principle, the design of small molecules for EISA requires two aspects: (1) the substrate of enzyme of interest; and (2) self-assembly potency after enzymatic conversion. This strategy has shown its irreplaceable advantages in nanomedicne, specifically for cancer treatments and Vaccine Adjuvants. Interestingly, all the reported examples rely on only one kind of enzymehydrolase. Therefore, we envision that the application of EISA strategy just begins and will lead to a new paradigm in nanomedicine.

Enzyme-Instructed Supramolecular Self-Assembly with Anticancer Activity.

Cancer remains one of the leading causes of death, which has continuously stimulated the development of numerous functional biomaterials with anticancer activities. Herein is reviewed one recent trend of biomaterials focusing on the advances in enzyme-instructed supramolecular self-assembly (EISA) with anticancer activity. EISA relies on enzymatic transformations to convert designed small-molecular precursors into corresponding amphiphilic residues that can form assemblies in living systems. EISA has shown some advantages in controlling cell fate from three aspects. 1) Based on the abnormal activity of specific enzymes, EISA can differentiate cancer cells from normal cells. In contrast to the classical ligand-receptor recognition, the targeting capability of EISA relies on dynamic control of the self-assembly process. 2) The interactions between EISA and cellular components directly disrupt cellular processes or pathways, resulting in cell death phenotypes. 3) EISA spatiotemporally controls the distribution of therapeutic agents, which boosts drug delivery efficiency. Therefore, with regard to the development of EISA, the aim is to provide a perspective on the future directions of research into EISA as anticancer theranostics.

Synergistic enzymatic and bioorthogonal reactions for selective prodrug activation in living systems.

Adverse drug reactions (ADRs) restrict the maximum doses applicable in chemotherapy, which leads to failure in cancer treatment. Various approaches, including nano-drug and prodrug strategies aimed at reducing ADRs, have been developed, but these strategies have their own pitfalls. A renovated strategy for ADR reduction is urgently needed. Here, we employ an enzymatic supramolecular self-assembly process to accumulate a bioorthogonal decaging reaction trigger inside targeted cancer cells, enabling spatiotemporally controlled, synergistic prodrug activation. The bioorthogonally activated prodrug exhibits significantly enhanced potency against cancer cells compared with normal cells. This prodrug activation strategy further demonstrates high tumour inhibition efficacy with satisfactory biocompatibility, pharmacokinetics, and safety in vivo. We envision that integration of enzymatic and bioorthogonal reactions will serve as a general small-molecule-based strategy for alleviation of ADRs in chemotherapy.

Hydrogen sulfide induced supramolecular self-assembly in living cells.

Here we developed a critical gasotransmitter (H2S) mediated reduction to induce supramolecular self-assembly in multiple living cell lines. Specifically, the reduction converted an azido group to an amine which allowed the formation of intramolecular hydrogen bonds. The hydrogen bonds planarized the assembling molecules and promoted the supramolecular self-assembly.

Redox supramolecular self-assemblies nonlinearly enhance fluorescence to identify cancer cells.

Based on the nonlinear fluorescence enhancement, our H2O2 induced supramolecular self-assembly reveals a H2O2 threshold among multiple cancer and normal cells. Oxidative elimination restores an intramolecular hydrogen bond which can planarize the molecule to generate a fluorophore. The planarization enhances the intermolecular π-π stacking to promote self-assembly.

Determination of the packing model of a supramolecular nanofiber via mass-per-length measurement and de novo simulation.

Herein, we report an example of using scanning transmission electron microscopy to determine the mass-per-length of supramolecular nanofibers. Together with the measurement of the diameter of nanofibers via transmission electron microscopy, we could estimate the packing density of assembling molecules along the nanofibers. A parallel unbiased de novo simulation screens and reveals the most plausible molecular packing pattern of small molecules along the supramolecular nanofibers. Remarkably, the simulated packing patterns and density correlate well with the experimental measurements. Unexpectedly, the naphthalene groups are likely facing outward, creating a hydrophobic surface, which is driven by the geometry of the hydrogelator molecule. Overall, this study establishes a complementary method to determine molecular arrangement along the supramolecular nanofibers, which is potentially useful for the guidance of rational design of biomaterials based on self-assembly.