Kangfu Chen

Targeted protein degradation harnesses endogenous cellular machinery to eliminate disease-causing proteins, yet achieving phenotype-specific degradation across diverse cell types remains challenging. Here we show that antibody-enriched biomolecular condensates formed by liquid-liquid phase separation function as intracellular protein degradation tools, combining cytosolic trafficking with direct proteasome recruitment for targeted substrate clearance. These nanoscale condensates incorporate a short proteasome-targeting motif into phase-separation precursors, preserve antibody activity, enable direct proteasome recruitment, and improve delivery uniformity. When loaded with a mutation-specific antibody, these condensates selectively degrade oncogenic KRAS G12V without affecting wild-type KRAS in heterozygous cells, and suppress tumor growth in a KRAS G12V xenograft model. This strategy provides a modular platform for intracellular protein degradation that can be readily adapted by exchanging antibodies, without requiring genetic modification of cellular system.

Lipid nanoparticles (LNPs) play a critical role in the delivery of therapeutic messenger RNA (mRNA). Despite extensive efforts to optimize lipid formulations for in vivo delivery, efficacy of mRNA by LNPs remains suboptimal in many organs. Here, we demonstrate that LNP delivery efficacy is influenced by cellular metabolism, with the physiologic metabolome imposing constraints on mRNA expression from LNPs. Using an in vitro system, we found that simulated physiologic metabolic conditions led to the down-regulation of certain amino acid metabolic programs. Supplementation with an optimized formulation of methionine, arginine, and serine as an amino acid supplement (AAS) enhanced the uptake of LNPs and the expression of delivered mRNA cargo in epithelial cells in vitro. Coadministration of AAS with LNPs led to a 5- to 20-fold improvement in mRNA expression across various cell types and lipid formulations in vitro by promoting clathrin-independent carrier–mediated endocytosis. Delivery of mRNA by LNPs coadministered with AAS by multiple routes enhanced in vivo mRNA expression in preclinical models. Delivery of mRNA encoding growth hormone by LNPs with coadministration of AAS improved the liver growth hormone expression and the therapeutic outcomes in a model of inflammatory liver damage. Delivery of gene editing materials by LNP and AAS through an intratracheal route increased lung-targeted in vivo gene editing efficiency compared with LNP alone. The addition of an optimized AAS as a codelivered agent with LNPs may provide a simple strategy to broadly improve the efficacy of mRNA-based cell and gene therapies.

Microfluidics enables precise control over fluid flow at a small volume, making it highly suitable for cell analysis. Microfluidic devices, often referred to as “lab-on-a-chip”, have been extensively utilized to study individual cells with high resolution and minimal reagent use. In this chapter, we introduce the fundamental principles of microfluidics within the context of cell analysis. The behavior of fluid flow at microscale is first described, with a primary focus on laminar flow. Subsequently, basic fluidic control components, such as micropumps and microvalves, are discussed. In the third section, various advanced fluidic control techniques widely used for cell manipulations are introduced, including inertial flow, micro-magnetofluidics, droplet-based microfluidics, and acoustic flow.

Immunotherapy, which modulates the patient's immune system to treat diseases, has revolutionized cancer therapy and is now being explored for autoimmune diseases. However, cellular heterogeneity in the immune system, tumor microenvironment, and inflamed tissues poses a major challenge to the efficacy of immunotherapies. This complexity underscores the need for a deeper understanding of disease mechanisms at single‐cell resolution. In recent years, microfluidic technologies have played a key role in enabling single‐cell analysis at transcriptomic and proteomic levels, leading to the discovery of new biomarkers for immunotherapy. In this review, we summarize the state‐of‐the‐art microfluidic platforms used for single‐cell omics, focusing on their conceptual principles and practical designs. We also briefly summarize the applications of microfluidic devices in the preclinical process of immunotherapeutic target discovery.

Immunotherapies have transformed the treatment of many cancers and autoimmune diseases. However, durable therapeutic benefits are achieved in only certain subsets of patients. Due to the complexity and heterogeneity of disease, it remains challenging to design effective immunotherapies and predict their effects. Microscale systems — including microfluidics, microelectronics and microscaffolds — are now being adapted to accelerate immunotherapy discovery and development, with the potential to address efficacy, toxicity, predictability and affordability challenges. These microsystems consist of miniaturized structures, sensors and actuators that can manipulate molecules and cells of the immune system with high accuracy and throughput. Advances facilitated by microsystem technologies relevant to the discovery and translation of key types of immunotherapy (monoclonal antibodies, cytokine-based drugs, engineered immune cells and therapeutic vaccines) include the development of high-throughput devices for functional selection, miniaturized bioreactors for biomanufacturing, engineered scaffolds for therapeutic administration and sensitive biosensors for immune surveillance post-administration. Challenges facing the clinical translation of microsystem-based immunotherapies include issues related to standardization and integration as well as the need for new regulatory guidance.

Fully wearable devices are crucial for real-time health monitoring, but existing devices often lack stable power, on-site signal processing, and multimodal sensing. To overcome these limitations, we introduce the first self-powered and fully wearable sensor (MESFW) based on multiplexed green biofuels. The MESFW integrates a microfluidic module, sensing module, laser-induced graphene (LIG) electrodes, and customized electronics, enabling highly sensitive detection of glucose and alcohol in noninvasive biofluids (sweat, breath, saliva, tears) while monitoring daily activities (temperature, pressure, touch). Experiments show the MESFW can track glucose and alcohol levels before and after meals/drinking, powered continuously for 24 h. With machine learning, it accurately identifies wearing types and enables real-time health prediction. This innovation offers a novel approach to real-time physiological monitoring.

Nanomedicine is revolutionizing precision medicine, providing targeted, personalized treatment options. Lipid-based nanomedicines offer distinct benefits including high potency, targeted delivery, extended retention in the body, reduced toxicity, and lower required doses. These characteristics make lipid-based nanoparticles ideal for drug delivery in areas such as gene therapy, cancer treatment, and mRNA vaccines. However, traditional bulk synthesis methods for LNPs often produce larger particle sizes, significant polydispersity, and low encapsulation efficiency, which can reduce the therapeutic effectiveness. These issues primarily result from uneven mixing and limited control over particle formation during the synthesis. Microfluidic technology has emerged as a solution, providing precise control over particle size, uniformity, and encapsulation efficiency. In this mini review, we introduce the state-of-the-art microfluidic systems for lipid-based nanoparticle synthesis and functionalization. We include the working principles of different types of microfluidic systems, the use of microfluidic systems for LNP synthesis, cargo encapsulation, and nanomedicine delivery. In the end, we briefly discuss the clinical use of LNPs enabled by microfluidic devices.

Numerous human cancers have exhibited the ability to elude immune checkpoint blockade (ICB) therapies. This type of resistance can be mediated by immune-suppressive macrophages that limit antitumor immunity in the tumor microenvironment (TME). Here, we elucidate a strategy to shift macrophages into a proinflammatory state that down-regulates V domain immunoglobulin suppressor of T cell activation (VISTA) via inhibiting AhR and IRAK1. We used a high-throughput microfluidic platform combined with a genome-wide CRISPR knockout screen to identify regulators of VISTA levels. Functional characterization showed that the knockdown of these hits diminished VISTA surface levels on macrophages and sustained an antitumor phenotype. Furthermore, targeting of both AhR and IRAK1 in mouse models overcame resistance to ICB treatment. Tumor immunophenotyping indicated that infiltration of cytotoxic CD8 ⁺ cells, natural killer cells, and antitumor macrophages was substantially increased in treated mice. Collectively, AhR and IRAK1 are implicated as regulators of VISTA that coordinate a multifaceted barrier to antitumor immune responses.

A triple signal amplification strategy was integrated with a built-in double electrode and external energy storage device to fabricate a novel self-powered biosensor for ultrasensitive detection of miRNA-21. Specifically, DNA tetrahedra and haripin2-glucose oxidase are modified on the surface of the biocathode and bioanode by catalytic hairpin assembly (CHA) to achieve dual signal amplification. Moreover, triple signal amplification is realized by including an external capacitor. Consequently, the as-constructed self-powered biosensor demonstrates a low detection limit of 0.06 fM toward the miRNA-21 assay within the range of 0.1 fM to 10 pM. This study presents a practical and sensitive approach to timely cancer detection.

Maintenance of the mitochondrial thiol redox state is essential for cell survival. However, we lack a comprehensive understanding of the redox response to mitochondrial glutathione depletion. We developed a mitochondria-penetrating peptide, mtCDNB, to specifically deplete mitochondrial glutathione. A genome-wide CRISPR/Cas9 screen in tandem with mtCDNB treatment was employed to uncover regulators of the redox response to mitochondrial glutathione depletion. We identified nucleoside diphosphate kinase 3 (NME3) as a regulator of mitochondrial dynamics. We show that NME3 is recruited to the mitochondrial outer membrane when under redox stress. In the absence of NME3, there is impaired mitophagy, which leads to the accumulation of dysfunctional mitochondria. NME3 knockouts depleted of mitochondrial glutathione have increased mitochondrial ROS production, accumulate mtDNA lesions, and present a senescence-associated secretory phenotype. Our findings suggest a novel role for NME3 in selecting mitochondria for degradation through mitophagy under conditions of mitochondrial redox stress.