Enzyme-instructed Self-assembly Research Articles

Intranuclear assembly of leucine-rich peptides for selective death of osteosarcoma cells.

Herein, we show a pair of leucine-rich L- and D-phosphopeptides which self-assemble into twisting nanofibers, whose secondary structures contain a strong β-sheet component after being dephosphorylated by alkaline phosphatase (ALP). While being incubated with ALP overexpressing osteosarcoma cells, both of the peptides self-assemble in the nuclei and induce cell death. The cell death involves multiple cell death modalities and occurs along with the disruption of cell membranes. Enzyme-instructed self-assembly (EISA) inhibits osteosarcoma cells and shows no side effect to other cells. In addition, the cancer cells hardly gain drug resistance after repeated treatment. This work reports a pair of EISA-based nanofibers to target cell nuclei, and also provides a novel chemotherapeutic agent to inhibit osteosarcoma cells without side effects and drug resistance.

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.

Enzyme-Instructed Intracellular Peptide Assemblies.

Higher-order or supramolecular protein assemblies, usually regulated by enzymatic reactions, are ubiquitous and essential for cellular functions. This evolutionary fact has provided a rigorous scientific foundation, as well as an inspiring blueprint, for exploring supramolecular assemblies of man-made molecules that are responsive to biological cues as a novel class of therapeutics for biomedicine. Among the emerging man-made supramolecular structures, peptide assemblies, formed by enzyme reactions or other stimuli, have received most of the research attention and advanced most rapidly.In this Account, we will review works that apply enzyme-instructed self-assembly (EISA) to generate intracellular peptide assemblies for developing a new kind of biomedicine, especially in the field of novel cancer nanomedicines and modulating cell morphogenesis. As a versatile and cell-compatible approach, EISA can generate nondiffusive peptide assemblies locally; thus, it provides a unique approach to target subcellular organelles with exceptional cell selectivity. We have arranged this Account in the following way: after introducing the concept, simplicity, and uniqueness of EISA, we discuss the EISA-formed intracellular peptide assemblies, including artificial filaments, in the cell cytosol. Then, we describe the representative examples targeting subcellular organelles, such as mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, and the nucleus, by enzyme-instructed intracellular peptide assemblies for potential cancer therapeutics. After that, we highlight the recent exploration of the transcytosis of peptide assemblies for controlling cell morphogenesis. Finally, we provide a brief outlook of enzyme-instructed intracellular peptide assemblies. This Account aims to illustrate the promise of EISA-generated intracellular peptide assemblies in understanding diseases, controlling cell behaviors, and developing new therapeutics from a class of less explored molecular entities, which are substrates of enzymes and become building blocks of self-assembly after the enzymatic reactions.

Evaluating Alkaline Phosphatase-Instructed Self-Assembly of d-Peptides for Selectively Inhibiting Ovarian Cancer Cells.

Cancer is a major public health concern requiring novel treatment approaches. Enzyme-instructed self-assembly (EISA) provides a unique approach for selectively inhibiting cancer cells. However, the structure and activity correlation of EISA remains to be explored. This study investigates new EISA substrates of alkaline phosphatase (ALP) to hinder ovarian cancer cells. Analogues 2-8 were synthesized by modifying the amino acid residues of a potent EISA substrate 1 that effectively inhibits the growth of OVSAHO, a high-grade serous ovarian cancer (HGSOC) cell line. The efficacy of 2-8 against OVSAHO was assessed, along with the combination of substrate 1 with clinically used drugs. The results reveal that substrate 1 displays the highest cytotoxicity against OVSAHO cells, with an IC50 of around 8 μM. However, there was limited synergism observed between substrate 1 and the tested clinically used drugs. These findings indicate that EISA likely operates through a distinct mechanism that necessitates further elucidation.

An enzyme-instructed self-assembly system induces tumor acidosis via sequential-dual effect for cancer selective therapy.

The acidosis anti-tumor therapy, based on the altered energy metabolism pathway of tumor cells, has been proposed as an attractive method for cancer selective treatment. However, the strategy of inducing tumor acidosis by using a single drug to simultaneously inhibit both lactate efflux and consumption has not been reported yet. Herein, an in situ enzyme-instructed self-assembly (EISA) system was rationally fabricated to induce tumor acidosis apoptosis for cancer selective therapy. Depending on the sequential effect of the in situ EISA system, the targeted drug was successively distributed on the membrane and intracellular, inhibiting MCT4 mediated lactate efflux and mitochondrial tricarboxylic acid (TCA) cycle mediated lactate consumption, respectively. Through the dual obstruction of lactate metabolism to trigger tumor acidosis, the in situ EISA nanomedicine showed selective growth and migration inhibition against cancer cells. In addition, the nanomedicine also displayed a radio-sensitization effect in vitro due to causing the mitochondrial dysfunction, and exhibited a prominent synergistic chemo-radiotherapy anti-tumor performance in vivo. Accordingly, this work demonstrated that the in situ EISA system could endow the LND with sequential-dual effects to induce tumor acidosis, which may provide an enlightening strategy for anticancer drug delivery and cancer selective therapy. STATEMENT OF SIGNIFICANCE: With the help of the sequential effect of in situ EISA , the serial attack of LND against different targets was effectively realized to induce tumor acidosis and combined chemo-radiotherapy, implying the importance of the relationship between structure and function, which could offer a distinctive inspiration for future drug delivery system design and anti-tumor application.

A Concerted Enzymatic and Bioorthogonal Approach for Extra‐ and Intracellular Activation of Environment‐Sensitive Ruthenium(II)‐Based Imaging Probes and Photosensitizers

AbstractIn this article, we report a novel targeting strategy involving the combination of an enzyme‐instructed self‐assembly (EISA) moiety and a strained cycloalkyne to generate large accumulation of bioorthogonal sites in cancer cells. These bioorthogonal sites can serve as activation triggers in different regions for transition metal‐based probes, which are new ruthenium(II) complexes carrying a tetrazine unit for controllable phosphorescence and singlet oxygen generation. Importantly, the environment‐sensitive emission of the complexes can be further enhanced in the hydrophobic regions offered by the large supramolecular assemblies, which is highly advantageous to biological imaging. Additionally, the (photo)cytotoxicity of the large supramolecular assemblies containing the complexes was investigated, and the results illustrate that cellular localization (extracellular and intracellular) imposes a profound impact on the efficiencies of photosensitizers.

A Concerted Enzymatic and Bioorthogonal Approach for Extra- and Intracellular Activation of Environment-Sensitive Ruthenium(II)-Based Imaging Probes and Photosensitizers.

In this article, we report a novel targeting strategy involving the combination of an enzyme-instructed self-assembly (EISA) moiety and a strained cycloalkyne to generate large accumulation of bioorthogonal sites in cancer cells. These bioorthogonal sites can serve as activation triggers in different regions for transition metal-based probes, which are new ruthenium(II) complexes carrying a tetrazine unit for controllable phosphorescence and singlet oxygen generation. Importantly, the environment-sensitive emission of the complexes can be further enhanced in the hydrophobic regions offered by the large supramolecular assemblies, which is highly advantageous to biological imaging. Additionally, the (photo)cytotoxicity of the large supramolecular assemblies containing the complexes was investigated, and the results illustrate that cellular localization (extracellular and intracellular) imposes a profound impact on the efficiencies of photosensitizers.

Enzyme-Activatable Polypeptide for Plasma Membrane Disruption and Antitumor Immunity Elicitation.

Enzyme-instructed self-assembly of bioactive molecules into nanobundles inside cells is conceived to potentially disrupt plasma membrane and subcellular structure. Herein, an alkaline phosphatase (ALP)-activatable hybrid of ICG-CF4 KYp is facilely synthesized by conjugating photosensitizer indocyanine green (ICG) with CF4 KYp peptide via classical Michael addition reaction. ALP-induced dephosphorylation of ICG-CF4 KYp enables its transformation from small-molecule precursor into rigid nanofibrils, and such fibrillation in situ causes severe mechanical disruption of cytomembrane. Besides, ICG-mediated photosensitization causes additional oxidative damage of plasma membrane by lipid peroxidation. Hollow MnO2 nanospheres devote to deliver ICG-CF4 KYp into tumorous tissue through tumor-specific acidity/glutathione-triggered degradation of MnO2 , which is monitored by fluorescent probing and magnetic resonance imaging. The burst release of damage-associated molecular patterns and other tumor antigens during therapy effectively triggers immunogenetic cell death and improves immune stimulatory, as demonstrated by the promotion of dendritic cell maturation and CD8+ lymphocyte infiltration, as well as constraint of regulatory T cell population. Taken together, such cytomembrane injury strategy based on peptide fibrillation in situ holds high clinical promise for lesion-specific elimination of primary, abscopal, and metastatic tumors, which may enlighten more bioinspired nanoplatforms for anticancer theranostics.

Self-Assembly, Bioactivity, and Nanomaterials Applications of Peptide Conjugates with Bulky Aromatic Terminal Groups

The self-assembly and structural and functional properties of peptide conjugates containing bulky terminal aromatic substituents are reviewed with a particular focus on bioactivity. Terminal moieties include Fmoc [fluorenylmethyloxycarbonyl], naphthalene, pyrene, naproxen, diimides of naphthalene or pyrene, and others. These provide a driving force for self-assembly due to π-stacking and hydrophobic interactions, in addition to the hydrogen bonding, electrostatic, and other forces between short peptides. The balance of these interactions leads to a propensity to self-assembly, even for conjugates to single amino acids. The hybrid molecules often form hydrogels built from a network of β-sheet fibrils. The properties of these as biomaterials to support cell culture, or in the development of molecules that can assemble in cells (in response to cellular enzymes, or otherwise) with a range of fascinating bioactivities such as anticancer or antimicrobial activity, are highlighted. In addition, applications of hydrogels as slow-release drug delivery systems and in catalysis and other applications are discussed. The aromatic nature of the substituents also provides a diversity of interesting optoelectronic properties that have been demonstrated in the literature, and an overview of this is also provided. Also discussed are coassembly and enzyme-instructed self-assembly which enable precise tuning and (stimulus-responsive) functionalization of peptide nanostructures.

An Exploration of Multiple Component Peptide Assemblies by Enzyme-Instructed Self-Assembly.

Based on the motifs (RNISY (M) and DEEVELILGDT (D)) in the protein crystal structures of Merlin and CRL4DCAF-1, we phosphorylated the tyrosine residue in M and conjugated M to a self-assembling motif to produce a phosphopeptide (1P) and examined enzyme-instructed self-assembly (EISA) of 1P with and without the presence of D (4). Our results show that EISA of 1P forms a hydrogel at exceedingly low volume fraction (~ 0.03%) even with the presence of the hydrophilic peptide, 4. Unlike 1P, 2P (a diastereomer of 1P) or 3P (the enantiomer of 1P) forms a hydrogel via EISA when their concentration is four or three times that of 1P, respectively. Circular dichroism (CD) spectra show that increasing the concentration of the phosphopeptides lowers the CD signals of the mixtures, and the magnitudes of the CD signals depends on the interaction between M and D. This work contributes insight for understanding multi-component hydrogels formed by self-assembly that involves both specific intermolecular interaction and enzymatic reactions.

A Time of Change

A Time of Change

Intracellular Enzyme-Instructed Self-Assembly of Peptides (IEISAP) for Biomedical Applications.

Despite the remarkable significance and encouraging breakthroughs of intracellular enzyme-instructed self-assembly of peptides (IEISAP) in disease diagnosis and treatment, a comprehensive review that focuses on this topic is still desirable. In this article, we carefully review the advances in the applications of IEISAP, including the development of various bioimaging techniques, such as fluorescence imaging, photoacoustic imaging, magnetic resonance imaging, positron-emission tomography imaging, radiation imaging, and multimodal imaging, which are successfully leveraged in visualizing cancer tissues and cells, bacteria, and enzyme activity. We also summarize the utilization of IEISAP in disease treatments, including anticancer, antibacterial, and antiinflammation applications, among others. We present the design, action modes, structures, properties, functions, and performance of IEISAP materials, such as nanofibers, nanoparticles, nanoaggregates, and hydrogels. Finally, we conclude with an outlook towards future developments of IEISAP materials for biomedical applications. It is believed that this review may foster the future development of IEISAP with better performance in the biomedical field.

Protease-Responsive Peptide-Conjugated Mitochondrial-Targeting AIEgens for Selective Imaging and Inhibition of SARS-CoV-2-Infected Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to human health and lacks an effective treatment. There is an urgent need for both real-time tracking and precise treatment of the SARS-CoV-2-infected cells to mitigate and ultimately prevent viral transmission. However, selective triggering and tracking of the therapeutic process in the infected cells remains challenging. Here, we report a main protease (Mpro)-responsive, mitochondrial-targeting, and modular-peptide-conjugated probe (PSGMR) for selective imaging and inhibition of SARS-CoV-2-infected cells via enzyme-instructed self-assembly and aggregation-induced emission (AIE) effect. The amphiphilic PSGMR was constructed with tunable structure and responsive efficiency and validated with recombinant proteins, cells transfected with Mpro plasmid or infected by SARS-CoV-2, and a Mpro inhibitor. By rational construction of AIE luminogen (AIEgen) with modular peptides and Mpro, we verified that the cleavage of PSGMR yielded gradual aggregation with bright fluorescence and enhanced cytotoxicity to induce mitochondrial interference of the infected cells. This strategy may have value for selective detection and treatment of SARS-CoV-2-infected cells.

Supramolecular Nanofibers Formed by Enzyme‐Instructed Self‐Assembly for SKBR‐3 Cell Selective Inhibition

Cell-targeted peptides are recommended for precision cancer treatment due to their comparable targeting properties, small molecular size, and good biocompatibility. However, unpredictable bioactivity, low penetration rate and poor stability greatly limit its efficacy. Supramolecular self-assembly based on synthetic peptide has great potential to solve related problems and achieve better therapeutic effects. Herein, we report and compare the effects of two different assembly pathway, heating-cooling, and enzyme instruction, on the penetrability of SKBR-3 cell targeted peptides. It was found that enzyme-instructed self-assembly (EISA) resulted in hydrogels composed of uniform supramolecular nanofibers, whereas heating-cooling resulted in solutions and precipitations composed of slightly different nanoparticles. The nanofibers formed by EISA showed enhanced cellular uptake (2.54 μM), which was significantly higher than the 1.06 μM of the nanoparticles formed by temperature regulation. Thus, EISA is a promising strategy to improve the cell penetration rate of targeted peptides and could provide a better solution for precision cancer treatment.

Enhancing the Cellular Uptake of Macromolecules via Enzyme-Instructed Self-Assembly.

Poor solubility, low cellular uptake, and poor cell selectivity are the main obstacles hampering the therapeutic potential and clinic application of macromolecules. To overcome these limitations, here we propose a chemical modification strategy of macromolecules based on enzyme-instructed self-assembly (EISA). By using protoporphyrin IX (PpIX) and its metal complex Zn-PpIX as the modification objects, we demonstrated that the integration of enzymatic transformation and molecular self-assembly of macromolecules successfully improved the solubility of macromolecules, enhancing their intracellular uptake selectively against cancer cells. The proposed strategy is potentially applicable as a general tool for the development of macromolecule-based nanomedicine.

A Self-Assembling Probe for Imaging the States of Golgi Apparatus in Live Single Cells.

Despite the enormous progress in genomics and proteomics, it is still challenging to assess the states of organelles in living cells with high spatiotemporal resolution. Based on our recent finding of enzyme-instructed self-assembly of a thiophosphopeptide that targets the Golgi Apparatus (GA) instantly, we use the thiophosphopeptide, which is enzymatically responsive and redox active, as an integrative probe for revealing the state of the GA of live cells at the single cell level. By imaging the probe in the GA of live cells over time, our results show that the accumulation of the probe at the GA depends on cell types. By comparison to a conventional Golgi probe, this self-assembling probe accumulates at the GA much faster and are sensitive to the expression of alkaline phosphatases. In addition, subtle changes of the fluorophore results in slightly different GA responses. This work illustrates a novel class of active molecular probes that combine enzyme-instructed self-assembly and redox reaction for high-resolution imaging of the states of subcellular organelles over a large area and extended times.

Enzyme-instructed self-assembly of peptide-drug conjugates in tear fluids for ocular drug delivery

In vivo self-assembly of small molecules offers an excellent opportunity for targeted and long-term accumulation of a therapeutic agent at the lesion site. Here we demonstrate the strategy of enzyme-instructed self-assembly (EISA) by designing a phosphorylated peptide-drug (IBF-HYD-GFFpY) precursor through the ester bond to release active drugs at the target site. Meanwhile, the in vivo assembly can be achieved by the catalysis of alkaline phosphatase (ALP) in the tear fluid for ocular drug delivery efficiently. The in vitro enzymatic experiments indicate that the dephosphorylation of IBF-HYD-GFFpY occurs firstly with the yield of IBF-HYD-GFFY which subsequently self-assembles into the supramolecular hydrogel to afford sustained drug release over 96 h. In the treatment of lipopolysaccharide (LPS)-activated Raw 264.7 macrophages, IBF-HYD-GFFpY exerts the more potent anti-inflammatory efficacy than that of free ibuprofen (IBF) at the concentration of 200 μM. Moreover, the aqueous solution of IBF-HYD-GFFpY via topical instillation hardly causes ocular irritation, and displays longer precorneal retention compared to the conventional eye drop formulation. In addition, in the in vivo study, a rabbit model of endotoxin-induced uveitis (EIU) evidences the comparable therapeutic efficacy of IBF-HYD-GFFpY eye drops with that of clinically used 0.1 wt% diclofenac (DIC) sodium eye drops by the reduction of macrophage and leukocyte influx. This work, in situ EISA in the tear microenvironment directing in vivo self-assembly of small molecules, may guide a powerful approach for developing enzymatic self-assembled molecules as an efficient delivery system of ocular drugs.

Enzyme-Instructed Self-Assembly of Peptides: From Concept to Representative Applications.

Enzyme-instructed self-assembly, integrating enzymatic reaction and molecular self-assembly, has drawn noticeable attention over the last decade with the intension of being used in valuable applications. Recent advances in the field make it possible to spatiotemporally control peptide self-assembly in cellular milieu, broadening the potential applications of peptide assemblies to cancer therapy and subcellular delivery. In this review, the concept of enzyme-instructed self-assembly of peptide, containing enzymatic trigger and spatiotemporal control, is described. Representative applications in cells are also discussed, followed by an outlook on the field of enzyme-instructed self-assembly.

Enzyme-instructed self-assembly (EISA) assists the self-assembly and hydrogelation of hydrophobic peptides.

Enzyme-instructed self-assembly (EISA) has several advantages in the preparation of supramolecular self-assembly materials for biomedical applications. In this study, we demonstrated that the enzyme-instructed self-assembly (EISA) strategy could assist the self-assembly and hydrogelation of two hydrophobic and bioactive peptides, tyroservatide (YSV) and laminin pentapeptide (YIGSR). We first synthesized the peptide derivatives of Nap-GFFYSV (peptide 1) and Nap-GFFYIGSR (peptide 2) and found that both peptides could not self-assemble into hydrogels due to their poor solubility. We therefore designed the phosphorylated precursors of the two hydrophobic peptides, Nap-GFFpYSV (precursor 1) and Nap-GFFpYIGSR (precursor 2), respectively, which had good solubility and can be dephosphorylated by alkaline phosphatase (ALP) to form supramolecular hydrogels. In addition, we found that the EISA could also occur on the surface of cells that overexpress ALP. The EISA strategy was a powerful method to generate hydrogels of hydrophobic compounds. We envision the big promise of the strategy in the preparation of biomaterials and nanomaterials of hydrophobic bioactive molecules.

MMP 9-instructed assembly of bFGF nanofibers in ischemic myocardium to promote heart repair.

Background: The only effective treatment for myocardial infarction (MI) is the timely restoration of coronary blood flow in the infarcted area, but further reperfusion exacerbates myocardial injury and leads to distal coronary no-reflow, which affects patient prognosis. Angiogenesis could be an important therapeutic strategy for re-establishing the blood supply to save the ischemic myocardium after MI. Basic fibroblast growth factor (bFGF) has been shown to promote angiogenesis. However, direct intravenous administration of bFGF is not a viable option given its poor half-life in vivo. Methods: Herein, we developed a peptide Lys-Lys-Pro-Leu-Gly-Leu-Ala-Gly-Phe-Phe (K2) to encapsulate bFGF to form bFGF@K2 micelle and proposed an enzyme-instructed self-assembly (EISA) strategy to deliver and slowly release bFGF in the ischemic myocardium. Results: The bFGF@K2 micelle exerted a stronger cardioprotective effect than free bFGF in a rat model of myocardial ischemia-reperfusion (MI/R). In vitro results revealed that the bFGF@K2 micelle could be cleaved by matrix metallopeptidase 9 (MMP-9) to yield bFGF@Nanofiber through amphipathic changes. In vivo experiments indicated that intravenous administration of bFGF@K2 micelle could lead to their restructuring into bFGF@Nanofiber and long term retention of bFGF in the ischemic myocardium of rat due to high expression of MMP-9 and assembly-induced retention (AIR) effect, respectively. Twenty-eight days after MI/R model establishment, bFGF@K2 micelle treatment significantly reduced fibrosis and improved cardiac function of the rats. Conclusion: We predict that our strategy could be applied in clinic for MI treatment in the future.