Copper-Induced Supramolecular Peptide Assemblies for Multi-pathway Cell Death and Tumor Inhibition.

Although self-assembly has emerged as an effective tool for fabricating biomaterials, achieving precise control over the morphologies and functionalities of the resultant assemblies remains an ongoing challenge. Inspired by the copper peptide naturally present in human plasma, in this study, we designed a synthetic precursor, FcGH. FcGH can self-assemble via two distinct pathways: spontaneous and Cu2+-induced. These two assembly pathways enabled the formation of assemblies with tunable morphologies by adjusting the amount of added Cu2+. We found that the nanoparticles formed by Cu2+-induced self-assembly exhibited a significantly higher cellular uptake efficiency than the wormlike fibers formed spontaneously. Moreover, this Cu2+-induced assembly process occurred spontaneously at a 1:1 molar ratio of Cu2+ to FcGH, avoiding the excessive use of Cu²âº and a tedious preparation procedure. By co-assembling with FcGH-conjugated 10-hydroxycamptothecin (HCPT), Cu2+-induced supramolecular nanodrugs elicited multiple cell death modalities in cancer cells with elevated immunogenicity, enhancing the therapeutic effect compared to free HCPT. This study highlights Cu2+-induced self-assembly as an efficient tool for directing the assembly of nanodrugs and for synergistic tumor therapy.

Enzyme-Instructed Photoactivatable Supramolecular Antigens on Cancer Cell Membranes for Precision-Controlled T-Cell-Based Cancer Immunotherapy.

Cancer immunotherapies based on cytotoxic CD8+ T lymphocytes (CTLs) are highly promising for cancer treatment. The specific interaction between T-cell receptors and peptide-MHC-I complexes (pMHC-I) on cancer cell membranes critically determines their therapeutic outcomes. However, the lack of appropriate endogenous antigens for MHC-I presentation disables tumor recognition by CTLs. By devising three antigen-loaded self-assembling peptides of pY-K(Ag)-ERGD, pY-K(Ag)-E, and Y-K(Ag)-ERGD to noncovalently generate light-activatable supramolecular antigens at tumor sites in different manners, we report pY-K(Ag)-ERGD as a promising candidate to endow tumor cells with pMHC-I targets on demand. Specifically, pY-K(Ag)-ERGD first generates low-antigenic supramolecular antigens on cancer cell membranes, and a successive light pulse allows antigen payloads to efficiently release from the supramolecular scaffold, directly producing antigenic pMHC-I. Intravenous administration of pY-K(Ag)-ERGD enables light-controlled tumor inhibition when combined with adoptively transferred antigen-specific CTLs. Our strategy is feasible for broadening tumor antigen repertoires for T-cell immunotherapies and advancing precision-controlled T-cell immunotherapies.

Supramolecular Nanofibers Ameliorate Bleomycin-Induced Pulmonary Fibrosis by Restoring Autophagy.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal interstitial lung disease, with limited therapeutic options available. Impaired autophagy resulting from aberrant TRB3/p62 protein-protein interactions (PPIs) contributes to the progression of IPF. Restoration of autophagy by modulating the TRB3/p62 PPIs has rarely been reported for the treatment of IPF. Herein, peptide nanofibers are developed that specifically bind to TRB3 protein and explored their potential as a therapeutic approach for IPF. By conjugating with the self-assembling fragment (Ac-GFFY), a TRB3-binding peptide motif A2 allows for the formation of nanofibers with a stable α-helix secondary structure. The resulting peptide (Ac-GFFY-A2) nanofibers exhibit specific high-affinity binding to TRB3 protein in saline buffer and better capacity of cellular uptake to A2 peptide. Furthermore, the TRB3-targeting peptide nanofibers efficiently interfere with the aberrant TRB3/p62 PPIs in activated fibroblasts and fibrotic lung tissue of mice, thereby restoring autophagy dysfunction. The TRB3-targeting peptide nanofibers inhibit myofibroblast differentiation, collagen production, and fibroblast migration in vitro is demonstrated, as well as bleomycin-induced pulmonary fibrosis in vivo. This study provides a supramolecular method to modulate PPIs and highlights a promising strategy for treating IPF diseases by restoring autophagy.

A Transformable Supramolecular Bispecific Cell Engager for Augmenting Natural Killer and T Cell-Based Cancer Immunotherapy.

Immune cells are pivotal in cancer immunotherapy, yet their therapeutic effectiveness is often hampered by limited tumor infiltration and inhibitory tumor microenvironments. An alkaline phosphatase (ALP)-responsive and transformable supramolecular bis-specific cell engager (Supra-BiCE) to harness natural killer (NK)/T cells for effective cancer immunotherapy is introduced here. The Supra-BiCE, consisting of both SA-P (a phosphorylated peptide targeting and blocking programmed cell death ligand 1 (PD-L1)) and SA-T (a phosphorylated peptide targeting and blocking T cell immunoglobulin and ITIM domain (TIGIT)) is constructed by a simple co-assembling strategy. Upon intravenous administration, Supra-BiCE self-assembles into nanoribbons and interacts with NK/T cells via TIGIT. Notably, these nanoribbons undergo transformation into long nanofibrils within ALP-overexpressing tumor regions, resulting in enhanced binding affinities of Supra-BiCE to both PD-L1 and TIGIT. Consequently, this leads to the accumulation and retention of NK/T cells within tumor regions. Furthermore, the combinatorial blockade of checkpoints by Supra-BiCE activates infiltrating NK/T cells. Moreover, the adjustable peptide ratio in Supra-BiCE enables customization for optimal therapeutic effects against distinct tumor types. Particularly, Supra-BiCE (T:P = 1:3) achieved 98.27% tumor suppression rate against colon carcinoma model. Overall, this study offers a promising tool for engaging NK and T cells for cancer immunotherapy.

In Situ Construction of Ferrocene-Containing Membrane-Bound Nanofibers for the Redox Control of Cancer Cell Death and Cancer Therapy.

Precise manipulation of cancer cell death by harnessing reactive oxygen species (ROS) is a promising strategy to defeat malignant tumors. However, it is quite difficult to produce active ROS with spatial precision and regulate their biological outcomes. We succeed here in selectively generating short-lived and lipid-reactive hydroxyl radicals (•OH) adjacent to cancer cell membranes, successively eliciting lipid peroxidation and ferroptosis. DiFc-K-pY, a phosphorylated self-assembling precursor that consists of two branched Fc moieties and interacts specifically with epidermal growth factor receptor, can in situ produce membrane-bound nanofibers and enrich ferrocene moieties on cancer cell membranes in response to alkaline phosphatase. Within the acidic tumor microenvironment, DiFc-K-pY nanofibers efficiently convert tumoral H2O2 to active •OH around the target cell membranes via Fenton-like reactions, leading to lipid peroxidation and ferroptosis with good cellular selectivity. Our strategy successfully prevents tumor progression with acceptable biocompatibility through intratumoral administration.

A Cascade-Targeted Enzyme-Instructed Peptide Self-Assembly Strategy for Cancer Immunotherapy through Boosting Immunogenic Cell Death.

Immunogenic cell death (ICD) approaches by encumbering mitochondrial functions provide great promise for the treatment of malignant tumors, but these kinds of ICD strategies are still in their infancy. Here, one multifunctional drug-loaded, cascade-targeted, and enzyme-instructed self-assembling peptide nanomedicine (Comp. 4) for ICD-based cancer therapy is constructed. Comp. 4 consists of 1) lonidamine (LND) that specifically interferes with mitochondrial functions; 2) a programmed death ligand 1 (PD-L1) binding peptide sequence (NTYYEDQG) and a mitochondria-specific motif (triphenylphosphonium, TPP) that can sequentially control the cell membrane and mitochondria targeting capacities, respectively; and 3) a -GD FD FpD Y- assembly core to in situ organize peptide assemblies responsive to alkaline phosphatase (ALP). Comp. 4 demonstrates noticeable structural and morphological transformations in the presence of ALP and produces peptide assemblies in mouse colon cancer cells (CT26) with high expressions of both ALP and PD-L1. Moreover, the presence of PD-L1- and mitochondria-specific motifs can assist Comp. 4 for effective endocytosis and endosomal escape, forming peptide assemblies and delivering LND into mitochondria. Consequently, Comp. 4 shows superior capacities to in vivo induce abundant mitochondrial oxidative stress, provoke robust ICD responses, and produce an immunogenic tumor microenvironment, successfully inhibiting CT26 tumor growth by eliciting a systemic ICD-based antitumor immunity.

Enzyme-Instructed Peptide Assembly Favored by Preorganization for Cancer Cell Membrane Engineering.

Innovative methods for engineering cancer cell membranes promise to manipulate cell-cell interactions and boost cell-based cancer therapeutics. Here, we illustrate an in situ approach to selectively modify cancer cell membranes by employing an enzyme-instructed peptide self-assembly (EISA) strategy. Using three phosphopeptides (pY1, pY2, and pY3) targeting the membrane-bound epidermal growth factor receptor (EGFR) and differing in just one phosphorylated tyrosine, we reveal that site-specific phosphorylation patterns in pY1, pY2, and pY3 can distinctly command their preorganization levels, self-assembling kinetics, and spatial distributions of the resultant peptide assemblies in cellulo. Overall, pY1 is the most capable of producing preorganized assemblies and shows the fastest dephosphorylation reaction in the presence of alkaline phosphatase (ALP), as well as the highest binding affinity for EGFR after dephosphorylation. Consequently, pY1 exhibits the greatest capacity to construct stable peptide assemblies on cancer cell membranes with the assistance of both ALP and EGFR. We further use peptide-protein and peptide-peptide co-assembly strategies to apply two types of antigens, namely ovalbumin (OVA) protein and dinitrophenyl (DNP) hapten respectively, on cancer cell membranes. This study demonstrates a very useful technique for the in situ construction of membrane-bound peptide assemblies around cancer cells and implies a versatile strategy to artificially enrich cancer cell membrane components for potential cancer immunotherapy.

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 photosensitizer-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.

Amino acid sequence determines the adjuvant potency of a D-tetra-peptide hydrogel.

The development of novel vaccine adjuvants is essential for the production of modern vaccines against infectious agents and cancer. We recently reported a supramolecular hydrogel of a self-assembling D-tetra-peptide named Nap-GDFDFDY (Gel-gffy) that can evoke potent humoral and cellular immune responses; however, the determinants of its immunostimulatory properties were not characterized. In this study, we show that the amino acid sequence of the peptide determines the adjuvant potency of Gel-gffy. We designed and synthesized five Gel-gffy variants (Sol-gfgy, Sol-ggfy, Gel-gffg, Gel-gfyf, and Gel-gyff) by substituting the phenylalanine and tyrosine to glycine or changing the position of the tyrosine in the parent D-tetra-peptide. First, we characterized their gelation properties, nanomorphology, and secondary structure using transmission electron microscopy and circular dichroism; next, we examined their immunostimulatory properties. Gel-gfyf, Gel-gyff and Gel-gffy markedly upregulated maturation marker expression on bone marrow-derived dendritic cells. Moreover, the Gel-gfyf-, Gel-gyff- or Gel-gffy-encapsulated ovalbumin (OVA) vaccine induced robust humoral and cellular immune response in vivo. Notably, Gel-gffy had the strongest immunostimulatory activity. Our findings demonstrate that both the position and number of aromatic amino acids are crucial in determining the adjuvant potency of Gel-gffy, thus providing a valuable insight into designing peptide hydrogels as vaccine adjuvants.

Enzyme-instructed self-assembly enabled fluorescence light-up for alkaline phosphatase detection.

Alkaline phosphatase (ALP) exists in both normal and pathological tissues. Spatiotemporal variations in ALP levels can reveal its potential physiological functions and changes that occur during pathological conditions. However, it is still challenging to exploit fluorescent probes that can measure ALP activity under good spatial and temporal resolutions. Herein, enzyme-instructed self-assembly (EISA) was used to construct a high-performing analytical tool (MN-pY) to probe ALP activity. MN-pY alone (free state) showed negligible fluorescence but presented an almost 13-fold increase in fluorescence intensity in the presence of ALP (assembly state). Mechanism study indicated the increase in fluorescence intensity was due to hydrogelation and formation of supramolecular fibrils, mainly consisting of dephosphorylated MN-Y. The dephosphorylation and further fibrillation of MN-pY could induce the formation of a "hydrophobic pocket", leading to a further increase in fluorescence intensity. Moreover, MN-pY could selectively illuminate HeLa cells with a higher ALP expression but not LO2 cells with lower ALP levels, promising a potential application in cancer diagnosis.

Tandem molecular self-assembly for selective lung cancer therapy with an increase in efficiency by two orders of magnitude.

In situ self-assembly of prodrug molecules into nanomedicine can elevate the therapeutic efficacy of anticancer medications by enhancing the targeting and enrichment of anticancer drugs at tumor sites. However, the disassembly and biodegradation of nanomedicine after enrichment prevents the further improvement of the efficiency, and avoiding such disassembly and biodegradation remains a challenge. Herein, we rationally designed a tandem molecular self-assembling prodrug that could selectively improve the therapeutic efficacy of HCPT against lung cancer by two orders of magnitude. The tandem molecular self-assembly utilized an elevated level of alkaline phosphatase and reductase in lung cancer cells. The prodrug first self-assembled into nanofibers by alkaline phosphatase catalysis and was internalized more efficiently by lung cancer cells than free HCPT. The resulting nanofiber was next catalyzed by intracellular reductase to form a more hydrophobic nanofiber that prevented the disassembly and biodegradation, which further significantly improved the efficacy of HCPT against lung cancer both in vitro and in vivo.