Inducing mitochondriopathy-like damages by transformable nucleopeptide nanoparticles for targeted therapy of bladder cancer.

Mitochondriopathy inspired adenosine triphosphate (ATP) depletions have been recognized as a powerful way for controlling tumor growth. Nevertheless, selective sequestration or exhaustion of ATP under complex biological environments remains a prodigious challenge. Harnessing the advantages of in vivo self-assembled nanomaterials, we designed an Intracellular ATP Sequestration (IAS) system to specifically construct nanofibrous nanostructures on the surface of tumor nuclei with exposed ATP binding sites, leading to highly efficient suppression of bladder cancer by induction of mitochondriopathy-like damages. Briefly, the reported transformable nucleopeptide (NLS-FF-T) self-assembled into nuclear-targeted nanoparticles with ATP binding sites encapsulated inside under aqueous conditions. By interaction with KPNA2, the NLS-FF-T transformed into a nanofibrous-based ATP trapper on the surface of tumor nuclei, which prevented the production of intracellular energy. As a result, multiple bladder tumor cell lines (T24, EJ and RT-112) revealed that the half-maximal inhibitory concentration (IC50) of NLS-FF-T was reduced by approximately 4-fold when compared to NLS-T. Following intravenous administration, NLS-FF-T was found to be dose-dependently accumulated at the tumor site of T24 xenograft mice. More significantly, this IAS system exhibited an extremely antitumor efficacy according to the deterioration of T24 tumors and simultaneously prolonged the overall survival of T24 orthotopic xenograft mice. Together, our findings clearly demonstrated the therapeutic advantages of intracellular ATP sequestration-induced mitochondriopathy-like damages, which provides a potential treatment strategy for malignancies.

In vivo assembly enhanced binding effect augments tumor specific ferroptosis therapy.

Emerging evidence indicates that the activation of ferroptosis by glutathione peroxidase 4 (GPX4) inhibitors may be a prominent therapeutic strategy for tumor suppression. However, the wide application of GPX4 inhibitors in tumor therapy is hampered due to poor tumor delivery efficacy and the nonspecific activation of ferroptosis. Taking advantage of in vivo self-assembly, we develop a peptide-ferriporphyrin conjugate with tumor microenvironment specific activation to improve tumor penetration, endocytosis and GPX4 inhibition, ultimately enhancing its anticancer activity via ferroptosis. Briefly, a GPX4 inhibitory peptide is conjugated with an assembled peptide linker decorated with a pH-sensitive moiety and ferriporphyrin to produce the peptide-ferriporphyrin conjugate (Gi-F-CAA). Under the acidic microenvironment of the tumor, the Gi-F-CAA self-assembles into large nanoparticles (Gi-F) due to enhanced hydrophobic interaction after hydrolysis of CAA, improving tumor endocytosis efficiency. Importantly, Gi-F exhibits substantial inhibition of GPX4 activity by assembly enhanced binding (AEB) effect, augmenting the oxidative stress of ferriporphyrin-based Fenton reaction, ultimately enabling antitumor properties in multiple tumor models. Our findings suggest that this peptide-ferriporphyrin conjugate design with AEB effect can improve the therapeutic effect via induction of ferroptosis, providing an alternative strategy for overcoming chemoresistance.

Nano Proteolysis Targeting Chimeras (PROTACs) with Anti-Hook Effect for Tumor Therapy.

Proteolysis targeting chimera (PROTAC) is an emerging pharmacological modality with innovated post-translational protein degradation capabilities. However, off-target induced unintended tissue effects and intrinsic "hook effect" hinder PROTAC biotechnology to be maturely developed. Herein, an intracellular fabricated nano proteolysis targeting chimeras (Nano-PROTACs) modality with a center-spoke degradation network for achieving efficient dose-dependent protein degradation in tumor is reported. The PROTAC precursors are triggered by higher GSH concentrations inside tumor cells, which subsequently in situ self-assemble into Nano-PROTACs through intermolecular hydrogen bond interactions. The fibrous Nano-PROTACs can form effective polynary complexes and E3 ligases degradation network with multi-binding sites, achieving dose-dependent protein degradation with "anti-hook effect". The generality and efficacy of Nano-PROTACs are validated by degrading variable protein of interest (POI) such as epidermal growth factor receptor (EGFR) and androgen receptor (AR) in a wide-range dose-dependent manner with a 95 % degradation rate and long-lasting potency up to 72 h in vitro. Significantly, Nano-PROTACs achieve in vivo dose-dependent protein degradation up to 79 % and tumor growth inhibition in A549 and LNCap xenograft mice models, respectively. Taking advantages of in situ self-assembly strategy, the Nano-PROTACs provide a generalizable platform to promote precise clinical translational application of PROTAC.

Programmable Peptides Activated Macropinocytosis for Direct Cytosolic Delivery.

Bioactive macromolecules show great promise for the treatment of various diseases. However, the cytosolic delivery of peptide-based drugs remains a challenging task owing to the existence of multiple intracellular barriers and ineffective endosomal escape. To address these issues, herein, programmable self-assembling peptide vectors are reported to amplify cargo internalization into the cytoplasm through receptor-activated macropinocytosis. Programmable self-assembling peptide vector-active human epidermal growth factor receptor-2 (HER2) signaling induces the receptor-activated macropinocytosis pathway, achieving efficient uptake in tumor cells. Shrinking macropinosomes accelerate the process of assembly dynamics and form nanostructures in the cytoplasm to increase peptide-based cargo accumulation and retention. Inductively coupled plasma mass (ICP-MS) spectrometry quantitative analysis indicates that the Gd delivery efficiency in tumor tissue through the macropinocytosis pathway is improved 2.5-fold compared with that through the use of active targeting molecular delivery. Finally, compared with nanoparticles and active targeting delivery, the delivery of bioactive peptide drugs through the self-assembly of peptide vectors maintains high drug activity (the IC50 decreased twofold) in the cytoplasm and achieves effective inhibition of tumor cell growth. Programmable self-assembling peptide vectors represent a promising platform for the intracellular delivery of diverse bioactive drugs, including molecular drugs, peptides, and biologics.

An In Vivo Self-Assembled Bispecific Nanoblocker for Enhancing Tumor Immunotherapy.

Anti-PD-L1 monoclonal antibody has achieved substantial success in tumor immunotherapy by T-cells activation. However, the excessive accumulation of extracellular matrix components induced by unsatisfactory T-cells infiltration and poor tumor penetration of antibodies make it challenging to realize efficient tumor immunotherapy. Herein, a peptide-based bispecific nanoblocker (BNB) strategy is reported for in situ construction of CXCR4/PD-L1 targeted nanoclusters on the surface of tumor cells that are capable of boosting T-cells infiltration through CXCR4 blockage and enhancing T-cells activation by PD-L1 occupancy, ultimately realizing high-performance tumor immunotherapy. Briefly, the BNB strategy selectively recognizes and bonds CXCR4/PD-L1 with deep tumor penetration, which rapidly self-assembles into nanoclusters on the surface of tumor cells. Compared to the traditional bispecific antibody, BNB exhibits an intriguing metabolic behavior, that is, the elimination half-life (t1/2 ) of BNB in the tumor is 69.3 h which is ≈50 times longer than that in the plasma (1.4 h). The higher tumor accumulation and rapid systemic clearance overcome potential systemic side effects. Moreover, the solid tumor stress generated by excessive extracellular matrix components is substantially reduced to 44%, which promotes T-cells infiltration and activation for immunotherapy efficacy. Finally, these findings substantially strengthen and extend clinical applications of PD-1/PD-L1 immunotherapy.

A bispecific glycopeptide spatiotemporally regulates tumor microenvironment for inhibiting bladder cancer recurrence.

Up to 75% of bladder cancer patients suffer from recurrence due to postoperative tumor implantation. However, clinically used Bacillus Calmette-Guerin (BCG) treatment failed to inhibit the recurrence. Here, we report a bispecific glycopeptide (bsGP) that simultaneously targets CD206 on tumor-associated macrophages (TAMs) and CXCR4 on tumor cells. bsGP repolarizes protumoral M2-like TAMs to antitumor M1-like that mediated cytotoxicity and T cell recruitment. Meanwhile, bsGP is cleaved by the MMP-2 enzyme to form nanostructure for the long-term inhibition of CXCR4 downstream signaling, resulting in reduced tumor metastasis and promoted T cell infiltration. In orthotopic bladder tumor models, bsGP reduced the postoperative recurrence rate to 22%. In parallel, the recurrence rates of 89 and 78% were treated by doxycycline and BCG used in clinic, respectively. Mechanistic studies reveal that bsGP reduces the matrix microenvironment barrier, increasing the spatially redirected CD8+ T cells to tumor cells. We envision that bis-targeting CD206 and CXCR4 may pave the way to inhibit tumor metastasis and recurrence.

Pro-oxidant response and accelerated ferroptosis caused by synergetic Au(I) release in hypercarbon-centered gold(I) cluster prodrugs.

Medicinal applications of gold complexes have recently attracted attention due to their innovative antitumor mechanisms. In this work, two hypercoordinated carbon-centered gold clusters PAA4 and PAA5 are quantitatively synthesized by an intramolecular 6-exo-dig cyclization of polymetalated precursors. The on-bench and in vitro experimental studies demonstrate that the characteristic hypercarbon-tetragold(I) multi-center bonding in PAA4 and PAA5 not only guarantees their stability under common physiological conditions, but also facilitates a glutathione (GSH)-triggered prompt and synergetic release of active Au(I) ions in the GSH-overexpressed and acidic microenvironment of human bladder cancer EJ cells. The instantly massive release of coordination unsaturated Au(I) ions causes the efficient inhibition of thioredoxin reductases and then induces a rapid pro-oxidant response, consequently causing the occurrence of accelerated ferroptosis of EJ cells. As a result, these hypercarbon-centered gold(I) cluster prodrugs show high cytotoxicity to bladder cancer cell lines and thus exhibit a significant inhibition effect towards bladder tumors in vivo. Correlation of the synergetic domino dissociation of carbon-polymetal multi-center bonding in metal clusters with the accelerated ferroptosis of cancer cells provides a strategy for metallo-prodrugs and opens a broader prospect for the biological application of metal cluster compounds.

Programmable design and self assembly of peptide conjugated AIEgens for biomedical applications.

Aggregation-induced emission luminogens (AIEgens) possess enhanced fluorescence in highly aggregated states, thus enabling AIEgens as a promising module for highly emissive fluorescence biomaterials. So far, AIEgens-based nanomaterials and their hybrids have been reported for biomedical applications. Benefiting from the intrinsic biocompatibility and biofunction-editing properties of peptides, peptide-AIEgens hybrid biomaterials reveal unlimited possibilities including target capacity, specificity, stimuli-responsiveness, self-assembly, controllable structural transformation, etc.. In the last two decades, peptide-AIEgens hybrid nanomaterials with a unique design concept in aggregated states have achieved various biomedical applications such as biosensing, bioimaging, imaging-guided surgery, drug delivery and therapy. More recently, programmable design of peptide-AIEgens for in situ self-assembly provides a unique strategy for constructing intelligent entities with defined biological functions. In this review, we summarize the basic design principle of programmable peptide-AIEgens, structure-effect relationship and their unusual biomedical effects. Finally, an outlook and perspective toward future challenges and developments of peptide-AIEgens nanomaterials are concluded.

A Lysosome-Targeting Self-Condensation Prodrug-Nanoplatform System for Addressing Drug Resistance of Cancer.

Lysosome-targeting self-assembling prodrugs had emerged as an attractive approach to overcome the acquisition of resistance to chemotherapeutics by inhibiting lysosomal sequestration. Taking advantage of lysosomal acidification induced intracellular hydrolytic condensation, we developed a lysosomal-targeting self-condensation prodrug-nanoplatform (LTSPN) system for overcoming lysosome-mediated drug resistance. Briefly, the designed hydroxycamptothecine (HCPT)-silane conjugates self-assembled into silane-based nanoparticles, which were taken up into lysosomes by tumor cells. Subsequently, the integrity of the lysosomal membrane was destructed because of the acid-triggered release of alcohol, wherein the nanoparticles self-condensed into silicon particles outside the lysosome through intracellular hydrolytic condensation. Significantly, the LTSPN system reduced the half-maximal inhibitory concentration (IC50) of HCPT by approximately 4 times. Furthermore, the LTSPN system realized improved control of large established tumors and reduced regrowth of residual tumors in several drug-resistant tumor models. Our findings suggested that target destructing the integrity of the lysosomal membrane may improve the therapeutic effects of chemotherapeutics, providing a potent treatment strategy for malignancies.

An activated excretion-retarded tumor imaging strategy towards metabolic organs.

Intraoperative fluorescence-based tumor imaging plays a crucial role in performing the oncological safe tumor resection with the advantage of differentiating tumor from normal tissues. However, the application of these fluorescence contrast agents in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC) was dramatically hammered as a result of lacking active targeting and poor retention time in tumor, which limited the Signal to Noise Ratio (SNR) and narrowed the imaging window for complicated surgery. Herein, we reported an activated excretion-retarded tumor imaging (AERTI) strategy, which could be in situ activated with MMP-2 and self-assembled on the surface of tumor cells, thereby resulting in a promoted excretion-retarded effect with an extended tumor retention time and enhanced SNR. Briefly, the AERTI strategy could selectively recognize the Integrin αvß3. Afterwards, the AERTI strategy would be activated and in situ assembled into nanofibrillar structure after specifically cleaved by MMP-2 upregulated in a variety of human tumors. We demonstrated that the AERTI strategy was successfully accumulated at the tumor sites in the 786-O and HepG2 xenograft models. More importantly, the modified modular design strategy obviously enhanced the SNR of AERTI strategy in the imaging of orthotopic RCC and HCC. Taken together, the results presented here undoubtedly confirmed the design and advantage of this AERTI strategy for the imaging of tumors in metabolic organs.

In Situ Constructed Nano-Drug Depots through Intracellular Hydrolytic Condensation for Chemotherapy of Bladder Cancer.

Intravesical administration of first-line drugs has shown failure in the treatment of bladder cancer owing to the poor tumor retention time of chemotherapeutics. Herein, we report an intracellular hydrolytic condensation (IHC) system to construct long-term retentive nano-drug depots in situ, wherein sustained drug release results in highly efficient suppression of bladder cancer. Briefly, the designed doxorubicin (Dox)-silane conjugates self-assemble into silane-based prodrug nanoparticles, which condense into silicon particle-based nano-drug depots inside tumor cells. Significantly, we demonstrate that the IHC system possesses highly potent antitumor efficacy, which leads to the regression and eradication of large established tumors and simultaneously extends the overall survival of air pouch bladder cancer mice compared with that of mice treated with Dox. The concept of intracellular hydrolytic condensation can be extended via conjugating other chemotherapeutic drugs, which may facilitate rational design of novel nanomedicines for augmentation of chemotherapy.

In Situ Self-Assembly of Bispecific Peptide for Cancer Immunotherapy.

Precise and effective manipulation of protein functions still faces tremendous challenges. Herein we report a programmable peptide molecule, consisted of targeting and self-assembly modules, that enables specific and highly efficient assembly governed by targeting receptor proteins. Upon binding to the cell membrane receptor, peptide conformation is somewhat stabilized along with decreased self-assembly activation energy, promoting peptide-protein complex oligomerization. We first design a GNNQQNY-RGD peptide (G7-RGD) to recognize integrin αV ß3 receptor for proof-of-concept study. In the presence of αV ß3 protein, the critical assembly concentration of free G7-RGD decreases from 525 to 33 µM and the resultant G7-RGD cluster drives integrin receptor oligomerization. Finally, a bispecific assembling peptide antiCD3-G7-RGD is rationally designed for cancer immunotherapy, which validates CD3 oligomerization and concomitant T cell activation, leading to T cell-mediated cancer cell cytolysis.

Targeted in situ self-assembly augments peptide drug conjugate cell-entry efficiency.

Peptide drug conjugate (PDC) has emerged as one of the new generations of targeted therapeutics for cancer, which owns the advantages of improved drug targetability and reduced adverse effects compared with traditional chemotherapy. However, the poor permeability of PDC drugs regarding tumor cells is an urgent problem to be solved. Herein, we design a PDC drug molecule, which is composed of three modules: targeting motif (RGD target), assembly motif (GNNNQNY) and cytotoxic payload (CPT molecule). This PDC in situ forms nanoclusters upon binding cellular receptor, resulting in improved PDC cell-entry efficiency and treatment efficacy. In addition, the PDC shows increased therapeutic efficacy and raises the maximum tolerance dose of the drug in breast and bladder xenografted mice models. This strategy leverages the assembly principle to promote penetration of peptide molecules into cells and increase intracellular drug bioavailability, which is of great significance for the development of PDC drugs in the future.

Biodegradable ß-Cyclodextrin Conjugated Gelatin Methacryloyl Microneedle for Delivery of Water-Insoluble Drug.

Transdermal delivery of water-insoluble drugs via hydrogel-based microneedle (MN) arrays is crucial for improving their therapeutic efficacies. However, direct loading of water-insoluble drug into hydrophilic matrices remains challenging. Here, a biodegradable MN array patch that is fabricated from naturally derived polymer conjugates of gelatin methacryloyl and ß-cyclodextrin (GelMA-ß-CD) is reported. When curcumin, an unstable and water-insoluble anticancer drug, is loaded as a model drug, its stability and solubility are improved due to the formation of an inclusion complex. The polymer-drug complex GelMA-ß-CD/CUR can be formulated into MN arrays with sufficient mechanical strength for skin penetration and tunable drug release profile. Anticancer efficacy of released curcumin is observed in three-dimensional B16F10 melanoma models. The GelMA-ß-CD/CUR MN exhibits relatively higher therapeutic efficacy through more localized and deeper penetrated manner compared with a control nontransdermal patch. In vivo studies also verify biocompatibility and degradability of the GelMA-ß-CD MN arrays patch.

A Human Liver-on-a-Chip Platform for Modeling Nonalcoholic Fatty Liver Disease.

The liver possesses a unique microenvironment with a complex internal vascular system and cell-cell interactions. Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease, and although much effort has been dedicated to building models to target NAFLD, most in vitro systems rely on simple models failing to recapitulate complex liver functions. Here, an in vitro system is presented to study NAFLD (steatosis) by coculturing human hepatocellular carcinoma (HepG2) cells and umbilical vein endothelial cells (HUVECs) into spheroids. Analysis of colocalization of HepG2-HUVECs along with the level of steatosis reveals that the NAFLD pathogenesis could be better modeled when 20% of HUVECs are presented in HepG2 spheroids. Spheroids with fat supplements progressed to the steatosis stage on day 2, which could be maintained for more than a week without being harmful for cells. Transferring spheroids onto a chip system with an array of interconnected hexagonal microwells proves helpful for monitoring functionality through increased albumin secretions with HepG2-HUVEC interactions and elevated production of reactive oxygen species for steatotic spheroids. The reversibility of steatosis is demonstrated by simply stopping fat-based diet or by antisteatotic drug administration, the latter showing a faster return of intracellular lipid levels to the basal level.

Photo-Controlled Reversible Microtubule Assembly Mediated by Paclitaxel-Modified Cyclodextrin.

The design and construction of multi-stimuli-responsive supramolecular nanoassemblies that can mimic and regulate the fundamental biological processes have become a focus of interest in supramolecular chemistry. In this work, a perfect combination has been achieved between naturally occurring microtubules and artificially macrocyclic receptors. The self-assembling morphology of microtubules can be photo-tuned by the host-guest interaction of paclitaxel-modified ß-cyclodextrin (PTX-CD) and photochromic arylazopyrazole (PTX-AAP). Moreover, the supramolecularly aggregated microtubules in a cellular environment can induce a pronounced cell morphological change and cell death. This supramolecular approach based on the secondary PTX-AAP⊂PTX-CD complexation provides us a facile method to reversibly control the intertubular aggregation behaviors of microtubules, which may bring new perspectives in the treatment of diseases related to improper protein aggregation.