A SupraGel for efficient production of cell spheroids

Cell spheroids are markedly more representative of the native tissue and the in vivo environment than traditional two-dimensional(2D)cultured cells,thus offering tremendous potential in cell biology research,tissue engineering,and drug screening.Therefore,it is crucial to develop materials and methods for efficient production of cell spheroids.However,currently developed materials,including natural and synthetic hydrogels,present drawbacks,such as undefined ingredients and imperfect biocompatibility,which hinder their widespread application.In this study,we have rationally designed biotinylated peptides that can self-assemble into supramolecular hydrogels(termed SupraGel)for 3D cell culture.The introduction of one D-amino acid in the peptide may decrease cell-matrix interactions,thus facilitating spontaneous cell spheroid formation.Two cancer cell lines,MCF-7 and 4T1,and intestinal stem cells(ISCs)can efficiently divide into cell spheroids when cultured in SupraGel.The reversible shear-thinning and recovery behavior of SupraGel is highly suitable for live-cell embedding and cell spheroid harvesting.The mechanical properties of SupraGel can be easily tuned by adjusting the peptide concentration,thus enabling its suitability for the 3D culture of diverse cell spheroids.We envision the significant potential of our SupraGel for applications in cell therapy,regenerative medicine,and drug screening.

Seamless and early gap healing of osteochondral defects by autologous mosaicplasty combined with bioactive supramolecular nanofiber-enabled gelatin methacryloyl(BSN-GelMA)hydrogel

Autologous mosaicplasty is a common approach used to treat osteochondral defects in clinical practice.Gap integration between host and transplanted plugs requires bone tissue reservation and hyaline cartilage regeneration without uneven surface,graft necrosis and sclerosis.However,poor gap integration is a serious concern,which eventually leads to deterioration of joint function.To deal with such complications,this study has developed a strategy to effectively enhance integration of the gap region following mosaicplasty by applying injectable bioactive supramolecular nanofiber-enabled gelatin methacryloyl(GelMA)hydrogel(BSN-GelMA).A rabbit osteochondral defect model demonstrated that BSN-GelMA achieved seamless osteochondral healing in the gap region between plugs of osteochondral defects following mosaicplasty,as early as six weeks.Moreover,the International Cartilage Repair Society score,histology score,glycosaminoglycan content,subchondral bone volume,and collagen II expression were observed to be the highest in the gap region of BSN-GelMA treated group.This improved outcome was due to bio-interactive materials,which acted as tissue fillers to bridge the gap,prevent cartilage degeneration,and promote graft survival and migration of bone marrow mesenchymal stem cells by releasing bioactive supramolecular nanofibers from the GelMA hydrogel.This study provides a powerful and applicable approach to improve gap integration after autologous mosaicplasty.It is also a promising off-the-shelf bioactive material for cell-free in situ tissue regeneration.

β-galactosidase responsive AIE fluorogene for identification and removal of senescent cancer cells

The selective identification and removal of senescent cells including senescent cancer cells are very important to prolong life and improve the treatment efficacy of cancer therapy.In this study,we integrated the high selectivity of enzyme-instructed selfassembly(EISA)and efficient reactive oxygen species(ROS)generating property of a novel luminogen with aggregationinduced emission(AIE)character to selectively identify and remove senescent He La(s-He La)cells.The s-He La cells expressed high levels ofβ-galactosidase(β-Gal),which led to the selective accumulation and formation of nanomaterials of Comp.1 in the cells.Upon white light irradiation,the nanomaterials efficiently produced ROS and therefore killed s-He La cells.Our study demonstrated a promising strategy to selectively remove senescent cells and improve the treatment efficacy of cancer therapy.

Organelle-inspired supramolecular nanomedicine to precisely abolish liver tumor growth and metastasis

Organelles are responsible for the efficient storage and transport of substances in living systems.A myriad of extracellular vesicles(EVs)acts as a bridge to exchange signaling molecules in cell-cell communication,and the highly dynamic tubulins and actins contribute to efficient intracellular substance transport.The inexhaustible cues of natural cargo delivery by organelles inspire researchers to explore the construction of biomimetic architectures for“smart”delivery carriers.Herein,we report a 10-hydroxycamptothecin(HCPT)-peptide conjugate HpYss that simulates the artificial EV-to-filament transformation process for precise liver cancer therapy.Under the sequential stimulus of extracellular alkaline phosphatase(ALP)and intracellular glutathione(GSH),HpYss proceeds via tandem self-assembly with a morphological transformation from nanoparticles to nanofibers.The experimental phase diagram elucidates the influence of ALP and GSH contents on the self-assembled nanostructures.In addition,the dynamic transformation of organelle-mimetic architectures that are formed by HpYss in HepG2 cells enables the efficient delivery of the anticancer drug HCPT to the nucleus,and the size-shape change from extracellular nanoparticles(50-100 nm)to intracellular nanofibers(4-9 nm)is verified to be of key importance for nuclear delivery.Nuclear targeting of HpYss amplifies apoptosis,thus significantly enhancing the inhibitory effect of HCPT(>10-fold)to HepG2 cells.Benefitting from the spatiotemporally controlled nanostructures,HpYss exhibited deep penetration,enhanced accumulation,and long-term retention in multicellular spheroid and xenograft models,potently abolishing liver tumor growth and preventing lung metastasis.We envision that our organelle-mimicking delivery strategy provides a novel paradigm for designing nanomedicine to cancer therapy.

Supramolecular protein glue to boost enzyme activity

Proteins possess many biological functions.However, they can easily degrade or aggregate, thus losing their bioactivity. Therefore, it is very important to develop materials capable of interacting with proteins and forming nanostructures for protein storage and delivery. In this study,we serendipitously found a novel peptide-based supramolecular protein glue(Nap-GFFYK(γE)2-NH2, compound 1) that could co-assemble with proteins into nanofibers and hydrogels. We found that compound 1 rapidly folded into a β-sheet conformation upon contact with many proteins but not with polymers. Total internal reflection fluorescence microscopy(TIRFM) images clearly show the formation of co-assembled nanofibers by proteins and the peptide. The supramolecular protein glue could improve the dispersion of enzymes(lipase and lysozyme) and therefore enhance their catalytic activity,especially at high temperatures. More importantly, the supramolecular protein glue could co-assemble with two enzymes, glucose oxidase/horseradish peroxidase(GOx/HRP)and GOx/cytochrome c(cyt c), to form nanofibers that significantly enhanced the catalytic activity of tandem enzymatic reactions. We envisioned the great potential of our supramolecular protein glue for protein storage, delivery, and bioactivity manipulation.

An amelogenin-based peptide hydrogel promoted the odontogenic differentiation of human dental pulp cells

Amelogenin can induce odontogenic differentiation of human dental pulp cells(HDPCs),which has great potential and advantages in dentine-pulp complex regeneration.However,the unstability of amelogenin limits its further application.This study constructed amelogenin self-assembling peptide hydrogels(L-gel or D-gel)by heating-cooling technique,investigated the effects of these hydrogels on the odontogenic differentiation of HDPCs and explored the underneath mechanism.The critical aggregation concentration,conformation,morphology,mechanical property and biological stability of the hydrogels were characterized,respectively.The effects of the hydrogels on the odontogenic differentiation of HDPCs were evaluated via alkaline phosphatase activity measurement,quantitative reverse transcription polymerase chain reaction,western blot,Alizarin red staining and scanning electron microscope.The mechanism was explored via signaling pathway experiments.Results showed that both the L-gel and D-gel stimulated the odontogenic differentiation of HDPCs on both Day 7 and Day 14,while the D-gel showed the highest enhancement effects.Meanwhile,the D-gel promoted calcium accumulation and mineralized matrix deposition on Day 21.The D-gel activated MAPK-ERK1/2 pathways in HDPCs and induced the odontogenic differentiation via ERK1/2 and transforming growth factor/smad pathways.Overall,our study demonstrated that the amelogenin peptide hydrogel stimulated the odontogenic differentiation and enhanced mineralization,which held big potential in the dentine-pulp complex regeneration.

Supramolecular Hydrogels of Indole-Capped Short Peptides as Vaccine Adjuvants

Many materials as immune adjuvant are researched to help raise immnogenicity of subunit vaccines. Among them, peptide-based hydrogels are gradually coming into notice because of their application in drugs delivery, can- cer cell inhibition, vaccine adjuvants and detection of important analytes. In this work, we introduced a novel aro- matic capping group based on indole to construct short peptide-based supramolecular hydrogelators Indol-GFFY and Indol-GDFDFDY and demonstrated their potential applications as vaccine adjuvants.

Tandem Molecular Self-Assembly Selectively Inhibits Lung Cancer Cells by Inducing Endoplasmic Reticulum Stress

The selective formation of nanomaterials in cancer cells and tumors holds great promise for cancer diagnostics and therapy.Until now,most strategies rely on a single trigger to control the formation of nanomaterials in situ.The combination of two or more triggers may provide for more sophisticated means of manipulation.In this study,we rationally designed a molecule(Comp.1)capable of responding to two enzymes,alkaline phosphatase(ALP),and reductase.Since the A549 lung cancer cell line showed elevated levels of extracellular ALP and intracellular reductase,we demonstrated that Comp.1 responded in a stepwise fashion to those two enzymes and displayed a tandem molecular self-assembly behavior.The selective formation of nanofibers in the mitochondria of the lung cancer cells led to the disruption of the mitochondrial membrane,resulting in an increased level of reactive oxygen species(ROS)and the release of cytochrome C(Cyt C).ROS can react with proteins,resulting in endoplasmic reticulum(ER)stress and the unfolded protein response(UPR).This severe ER stress led to disruption of the ER,formation of vacuoles,and ultimately,apoptosis of the A549 cells.Therefore,Comp.1 could selectively inhibit lung cancer cells in vitro and A549 xenograft tumors in vivo.Our study provides a novel strategy for the selective formation of nanomaterials in lung cancer cells,which is powerful and promising for the diagnosis and treatment of lung cancer.

Supramolecular hydrogels of self-assembled zwitterionic-peptides

Zwitterionic polymer materials have been extensively studied,but zwitterionic peptides supramolecular hydrogel materials are rarely studied.In this study,the preparation of two zwitterionic hydrogels using self-assembled peptides were reported.The hydrogels could be fabricated easily by changing the temperature or enzyme catalysis in a short time.And the differences in structure and function of the zwitterion peptide hydrogels caused by the two preparation methods were also be compared.We found that the hydrogel prepared by enzyme induced self-assembly has better solubility and lower cytotoxicity than that prepared by the heating-cooling process.The result showed the enzyme induced self-assembly way to form zwitterionic peptides supramolecular hydrogel materials could have further biomedical applications.

Preorganization boosts the artificial esterase activity of a self-assembling peptide

The creation of artificial enzymes to mimic natural enzymes remains a great challenge owing to the complexity of the structural arrangement of the essential amino acids in catalytic centers.In this study,we used the phosphatase-based enzyme-instructed self-assembly(EISA)to supervise artificial esterases'final structures and catalytic activities.We reported that peptide precursors containing different phosphorylation sites could preorganize into alternated nanostructures and undergo dephosphorylation in the presence of alkaline phosphatase(ALP)with variation in kinetic and thermodynamic profiles.Although identical self-assembly compositions were formed after dephosphorylation,precursors with more enhanced preorganized states tended to better promote ALP dephosphorylation,facilitate further self-assembly,and strengthen the catalytic activities of the final assemblies.We envisioned that our strategy would be useful for further construction and manipulation of various artificial enzymes with superior catalytic activities.

Enzyme-instructed and mitochondria-targeting peptide self-assembly to efficiently induce immunogenic cell death

Immunogenic cell death(ICD) plays a major role in cancer immunotherapy by stimulating specific T cell responses and restoring the antitumor immune system.However,effective type Ⅱ ICD inducers without biotoxicity are still very limited.Herein,a tentative drug-or photo sensitizer-free strategy was developed by employing enzymatic self-assembly of the peptide F-pY-T to induce mitochondrial oxidative stress in cancer cells.Upon dephosphorylation catalyzed by alkaline phosphatase overexpressed on cancer cells,the peptide F-pY-T self-assembled to form nanoparticles,which were subsequently internalized.These affected the morphology of mitochondria and induced serious reactive oxygen species production,causing the ICD characterized by the release of danger-associated molecular patterns(DAMPs).DAMPs enhanced specific immune responses by promoting the maturation of DCs and the intratumoral infiltration of tumor-specific T cells to eradicate tumor cells.The dramatic immunotherapeutic capacity could be enhanced further by combination therapy of F-pY-T and anti-PD-L1 agents without visible biotoxicity in the main organs.Thus,our results revealed an alternative strategy to induce efficient ICD by physically promoting mitochondrial oxidative stress.

The protective effects of a D-tetra-peptide hydrogel adjuvant vaccine against H7N9 influenza virus in mice

Repeated waves of influenza virus H7N9 epidemics after 2013 have caused severe influenza in humans,with mortality reaching approximately 40%–50%.To prevent possible pandemics,the development of highly effective vaccines against influenza virus H7N9 is highly desired.In the present study,by taking advantage of the D-tetra-peptide adjuvant(G^(D)F^(D)F^(D)Y),we reported a simple method to prepare H7N9 vaccines.Naproxen(Npx),with good anti inflammatory and broad anti-viral effects,was employed as an N-terminal capping group to construct a hydrogel precursor,Npx-G^(D)F^(D)F^(D)Y.The hydrogel adjuvant was prepared using a routine heating cooling protocol and the final vaccine was ready after mixing with the split A/Zhejiang/DTID-ZJU01/2013(H7N9)antigen by vortexing.Compared with the traditional Al(OH)_(3) adjuvant vaccine and the split vaccine,our hydrogel adjuvant vaccine showed the best preventive effects against H7N9 infection.A mechanistic study illustrated that higher antibody responses and variations in cytokine expression might account for its increased protective effects.Our strategy demonstrated the advantages of a peptide hydrogel adjuvant in the application of vaccines against H7N9 and demonstrated its potential application in vaccines against emerging threats from other viruses.

Imaging cellular distribution of fluorescent supramolecular nanofibers

In this study, we used the 4-nitro-2,1,3-benzoxadiazole （NBD） as an aromatic capping group for a peptide to construct the su- pramolecular nanofibers. Taking the advantage of the fluorescence property of NBD, we could directly observe the cellular distribution of the self-assembled nanofibers. We found that the distributions of the nanofibers of NBD-FFETIGGY are dif- ferent in four mammalian cells and two plant cells. The nanofibers are mainly located at the surface of two mammalian cells and one plant cell, while in the intracellular space of other cells. Different distributions of nanofibers lead to different protein binding patterns o1! the nanofibers in two different cell lines. We believe that a useful and versatile platform has been offered to the image cellular distribution of nanofibers, which can provide useful information to the biological functions of the self-assembled nanostructures.

Chemo-immunotherapy by dual-enzyme responsive peptide self-assembling abolish melanoma

Herein,we designed Comp.1 to simultaneously respond to two enzymes:alkaline phosphatase and matrix metalloproteinase 2,which is commonly found in highly malignant cancer cell lines containing B16-F10 murine melanoma cells and CT26 murine colon carcinoma cells.We used the regional differences in the expression levels of dual-markers to accurately release immune molecule IND into tumor microenvironment for the activation of anti-tumor related immune effects,while in-situ self-assembly occurs.The dual-enzyme response process can further regulate the peptide precursors’self-assembly in the form of short rod-shaped nanofibers,enabling the delivery of the loaded chemotherapeutic drug HCPT into the cancer cells and further allowing the peptide assemblies to escape from lysosomes and return to cytoplasm in the form of tiny nanoparticles to induce apoptosis of cancer cells.This process does not occur in the single-positive breast cancer cell line MCF-7 or the normal hepatocytes cell line LO2,indicating the selectivity of the cancer cells exhibited using our strategy.In vivo studies revealed that Comp.1 can effectively cooperate with chemotherapy to enhance the immunotherapy effect and induce immune responses associated with elevated pro-inflammatory cytokines in vivo to inhibit malignant tumors growth.Our dual-enzyme responsive chemo-immunotherapy strategy feasible in anti-tumor treatment,provides a new avenue for regulating peptide self-assembly to adapt to diverse tumor properties and may eventually be used for the development of novel multifunctional anti-tumor nanomedicines.