Stepwise Size Shrinkage Cascade-Activated Supramolecular Prodrug Boosts Antitumor Immunity by Eliciting Pyroptosis.

Effective pyroptosis induction is a promising approach to potentiate cancer immunotherapy. However, the actual efficacy of the present pyroptosis inducers can be weakened by successive biological barriers. Here, a cascaded pH-activated supramolecular nanoprodrug (PDNP) with a stepwise size shrinkage property is developed as a pyroptosis inducer to boost antitumor immune response. PDNPs comprise multiple poly(ethylene glycol) (PEG) and doxorubicin (DOX) drug-polymer hybrid repeating blocks conjugated by ultra-pH-sensitive benzoic imine (bzi) and hydrazone (hyd) bonds. The PEG units endow its "stealth" property and ensure sufficient tumor accumulation. A sharp switch in particle size and detachment of PEG shielding can be triggered by the acidic extracellular pH to achieve deep intratumor penetration. Following endocytosis, second-stage size switching can be initiated by more acidic endolysosomes, and PDNPs disassociate into ultrasmall cargo to ensure accurate intracellular delivery. The cascaded pH activation of PDNPs can effectively elicit gasdermin E (GSDME)-mediated pyroptosis to enhance the immunological response. In combination with anti-PD-1 antibody, PDNPs can amplify tumor suppression and extend the survival of mice, which suggests a powerful immune adjuvant and pave the way for high-efficiency immune checkpoint blockade therapy.

Multi-chamber petaloid root-growth chip for the non-destructive study of the development and physiology of the fibrous root system of Oryza sativa.

The root system of plants is a major component of their bodies in terms of both function and bulk. The investigation of root system development is greatly assisted by microfluidic devices, which improve the spatial and temporal resolution of observations without destroying tissue. In the present study, a multi-chamber petaloid root-growth chip was developed for studying the development and physiology of root systems that have thin branching structures (i.e., fibrous root systems). The petaloid root-growth chip includes a central seed germination chamber and five root-growth chambers for observing the development of fibrous roots. The proposed device was applied for investigating the root system development of Oryza sativa. The phenotype and growth kinetics of O. sativa root systems grown in the proposed device were compared with those obtained during growth in a conventional conical flask with agar-based medium, and the results indicated that cultivation in the miniaturized device did not delay root system growth in the early stage (≤2 weeks). In addition, the transparent device enabled the non-destructive observation of the developmental and microstructural characteristics of the roots, such as the root caps, root border cells, and root hairs. Moreover, the ability to control the microenvironment in each of the five root-growth chambers individually facilitated the investigation of specific adaptations in the fibrous root growth of single O. sativa seedlings to different drought stresses. Accordingly, five polyethylene glycol (PEG)6000-induced drought stress conditions were established in the five root-growth chambers to investigate the root development of a single O. sativa seedling in the central germination chamber. In situ observations demonstrated that the different PEG6000-induced conditions affected the root growth responses and root microstructural adaptations of the single seedlings in each root-growth chamber. Therefore, the petaloid root-growth microfluidic chip can eliminate the effects of variations in different plant seeds to reveal the responses of plants to different environmental conditions more objectively while concurrently allowing for non-destructive observations at very high spatial and temporal resolution.

Spontaneous formation of tumor spheroid on a hydrophilic filter paper for cancer stem cell enrichment.

Emerging evidence has demonstrated that cancer stem cells (CSCs) play critical roles in tumor invasion, metastasis and recurrence. The specific targeting capability on CSCs is of high importance for the development of effective anti-tumor therapeutics. However, isolation, enrichment and cultivation of these special and rare groups of tumor cells for in vitro analyses is a nontrivial job and requires particular culture medium and environmental control. Herein, we established a low-cost and efficient method for CSC enrichment by culturing prostate cancer cells on a hydrophilic filter paper. We found that tumor spheroids could form spontaneously on a pristine filter paper solely with regular cell culture medium. The paper-grown cells had elevated expression of putative CSC markers, indicating increased stemness of the cancer cells. Moreover, increased resistance of the chemotherapeutic drug doxorubicin was observed on the formed CSC spheroids compared to regular culture. The properties of the filter paper were characterized to investigate the underlying mechanism behind the promoted tumor spheroid formation. The obtained results suggested that the excellent hydrophilicity of the cellulose fibers retarded the hydrophobic interaction-mediated cell anchoring on the cellulose fibers, while the limited space/niche between fibers promoted the aggregation of cells. In addition, biocompatible paper-based materials are able to realize convenient assembly of tissue-like structures for developing in vitro disease models or organs-on-paper applications. Therefore, hydrophilic filter papers could be a low-cost material for construction of various assay platforms for isolating and enriching CSCs, screening anti-tumor drugs, and constructing tumor models in vitro.

3D-Printed seed planter and well array for high-throughput seed germination screening.

Seed germination is an important means of evaluating seed quality. In the present study, a well array for a seed germination experiment was designed and fabricated by 3D printing for the first time. Each hollow cone-shaped well can hold one seed, which not only prevented the seed from falling out of the well but also ensured that part of the seed was fully exposed to the sublayer of wet filter paper, allowing it to receive water for germination. Coupled with a 3D-printed seed planter, single seeds can be quickly placed into arrayed wells. Moreover, the number of sprouts could be automatically obtained from image analysis, which greatly improved the efficiency of the entire experiment. In summary, the high-throughput, easy-to-use seed germination well array that we have developed has been shown to have potential applications in botanical seed research and agricultural production.