Mitochondria are essential organelles that govern energy metabolism, redox balance, and cell survival; their dysfunction is implicated in a wide range of pathologies, including neurodegenerative disorders, cardiovascular diseases, metabolic syndromes, and cancer. Despite their significance as therapeutic targets, the unique structural and electrochemical properties of mitochondria, particularly the impermeable inner mitochondrial membrane and high membrane potential pose major challenges for the targeted delivery of therapeutic agents. Recent advances in biomaterials have spotlighted peptide-polymer conjugates as versatile platforms, capable of navigating intracellular barriers and achieving precise mitochondrial localization. These hybrid systems combine the physicochemical tunability of polymers with the biofunctionality of peptides, enhancing cellular uptake, endosomal escape, and suborganelle trafficking. The incorporation of stimuli-responsive elements further enables spatiotemporal control of cargo release in response to intracellular cues such as pH shifts, thermal fluctuations, redox gradients, or enzymatic activity. Such systems are especially promising for mitochondrial gene and protein delivery, offering improved selectivity, reduced systemic toxicity, and the potential to restore mitochondrial function under pathological conditions. This review showcases advanced strategies in stimuli-responsive peptide-polymer systems for mitochondria-targeted delivery, highlighting how their smart, responsive functions enable precise, controllable therapeutic interventions and drive the development of next-generation, transformative biomaterials in precision nanomedicine.

Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for hematologic diseases, but its broader application remains constrained by challenges in sourcing, manipulating, and reliably expanding functional HSCs. In this review, we discuss strategies to expand and engineer HSCs by recreating essential aspects of the bone marrow niche. These include defined cytokine cocktails, small molecule modulators, stromal co-culture systems, and biomaterials that promote self-renewal while limiting differentiation. We highlight advances in three-dimensional organoid models and microfluidic platforms that better support long-term repopulating cells and reflect native microenvironments. In parallel, progress in gene delivery platforms, including both viral and nonviral approaches, is enabling more efficient and targeted modification of HSCs for therapeutic use in genetic disorders such as sickle cell disease and β-thalassemia. While these tools have advanced significantly, significant hurdles remain in scaling, preserving stem cell identity, and reducing culture-induced stress. Continued refinement of biomimetic systems and genome engineering technologies will be central to expanding the clinical utility of HSC-based therapies.

The clinical translation of adeno-associated virus (AAV)-based gene therapies is often hindered by nonselective tissue transduction, off-target uptake by nontarget cells, and unintended toxicity to healthy tissues. To overcome these challenges, we previously developed a site-specific AAV capsid engineering strategy involving the incorporation of an azide-bearing unnatural amino acid (NAEK) into defined capsid positions, enabling precise, bioorthogonal conjugation of targeting ligands. In this study, we applied this approach to generate a series of folate receptor α (FRα)-targeted AAV2 vectors through covalent tethering of folic acid (FA) at specific capsid residues. FA conjugation at residues S264 + 1 and Q325 significantly enhanced FRα-mediated transduction, yielding a 3-5-fold increase in gene transfer efficiency in FRα-positive tumor cells. Structure-activity relationship analysis revealed that transduction selectivity is governed not only by ligand-receptor binding affinity but also by the spatial location of the conjugation site, which influences competition with the native AAV receptor (AAVR). Importantly, this modular conjugation platform allows for facile replacement of ligands, enabling the rational design of receptor-directed AAV vectors for targeted and cell-specific gene therapy. These findings provide mechanistic insights into capsid-receptor interactions and establish a flexible strategy for precision engineering of AAV-based delivery systems.

Polymeric nanogels hold strong promise for gene delivery, but their production is often limited by poor scalability and inconsistent control over physicochemical properties. To address this challenge, we present a scalable microfluidic strategy for engineering carboxymethyl chitosan-grafted branched polyethyleneimine plasmid DNA nanogels (CMC-bPEI-pDNA NGs) using a coaxial flow reactor. This continuous flow platform enables precise control over nanogel formation, offering tunability in particle size, surface charge, and encapsulation efficiency. Through systematic process development and parametric optimisation - including investigations into hydrodynamics, mixing, reactor geometry, and effect of reagent concentrations - we designed a novel process achieving high-throughput, reproducible nanogel production suitable for in vitro gene delivery. Optimised formulations, produced in as little as 3 s residence time, exhibited excellent monodispersity (polydispersity index, PDI < 0.2), sub-200 nm particle size, and pDNA encapsulation efficiency exceeding 90%. Fluorescence microscopy-based transfection assays confirmed effective intracellular delivery with high green fluorescent protein (GFP) expression in HEK293T cells 72 h post-transfection. We successfully scaled the process 100-fold by extending the reactor length, while maintaining similar physicochemical properties and biological performance. Nanogels produced at high throughput (1.14 L h-1) maintained a high GFP expression, confirming functional gene delivery and process scalability. We identified critical process parameters governing nanogel properties and scalability, including minimum residence time for nanogel formation, optimal flow rate ratios, reagent feeds configuration and reactor design for large-scale implementation. This work establishes a robust and scalable microfluidic process for producing functional polymeric nanogel gene delivery vectors, demonstrating its feasibility for translation from laboratory to larger-scale manufacturing, thereby serving as a proof of concept for future industrial-scale gene therapy applications.

Dendrimer-based nucleic acid (NA) delivery systems have attracted significant attention due to their synthetic versatility, monodispersity, nuclease resistance, high payload release, and transfection efficiency. The conventional dendrimers are non-fluorescent, limiting their utility in real-time tracking and monitoring of drug delivery. Although terminal functionalization with fluorophores can partially address this issue, it often alters critical physicochemical properties and transfection efficiency. In this study, we report the design and development of far-red fluorescent dendrimers with a naphthalene diimide (NDI)-core for efficient and traceable gene and RNA delivery. These intrinsically fluorescent dendrimers enable real-time monitoring of cellular uptake and delivery. The NDI G3 formulation effectively condenses DNA, protects it from DNase-mediated degradation, and facilitates efficient transfection in cells. Therapeutically, NDI G3 demonstrated efficient glutathione peroxidase 4 (GPX4) siRNA delivery, comparable to PAMAM G3 and Lipofectamine 3000. Notably, the NDI G3-Ca2+-GPX4 siRNA-FINO2 formulation sensitizes human colon cancer-derived cells to ferroptosis, synergistically annihilating cancer cells compared to treatment with FINO2 alone. The intrinsically far-red fluorescent NDI G3 dendrimer with a dynamic fluorescence response developed for the combinatorial delivery of siRNA and drug molecules offers a generalized framework for designing next-generation far-red fluorescent dendrimers for nucleic acid therapeutics and theranostic applications.

RNAi therapeutics targeting Japanese encephalitis virus: Gene targets, delivery platforms, and translational barriers.

Triggered ROS cyclic responsive silicon nanowire arrays for gene transfection of stem cells.

Lipid nanoparticles (LNPs) have gained significant attention because of the clinical success of the Onpattro drug and mRNA vaccines. Two major challenges remain: (i) designing LNPs for gene therapy targeting non-liver tissues and (ii) overcoming inefficient endosomal escape of conventional LNPs. Cationic LNPs have been reported to shift the organ tropism, but their endosomal escape is yet to be evaluated. Here, we investigated the fusion dynamics of cationic LNPs with model membranes at the single-particle level. We found that the membrane fusion occurs through a unique mass transfer pathway, involving a one-step transition that forms a metastable intermediate that fully coalesces with the target membrane. A moderately high concentration (31 mol%) of the cationic lipid (DOTAP), combined with either DOPE or DSPC + cholesterol helper lipids, accelerates the fusion kinetics by reducing the lag time. The enhanced fusogenicity of these compositions aligns with the bulk-phase lipid mixing results. Endosomal localization and eGFP expression upon gene delivery in a range of mammalian cell lines confirm effective endosomal escape of DOPE- or DSPC + cholesterol-rich cationic LNPs. Overall, these findings represent a step toward designing optimal cationic LNP candidates for efficient gene delivery to organs beyond the liver.

Highly efficient gene delivery using lentiviral vectors is beneficial not only for basic cell science research but also for human gene therapy applications. Lentiviral pseudotyping with vesicular stomatitis virus G (VSV-G) is a commonly used method that enables the transduction of various cell types. However, the low expression of low-density lipoprotein-receptor, the primary receptor of VSV-G, limits the efficiency of lentiviral vector transduction. Sendai virus hemagglutinin-neuraminidase glycoproteins recognize terminal sialic acids on the host cell plasma membrane, facilitating viral entry. Here, we describe methods for lentiviral dual-pseudotyping with VSV-G and Sendai virus hemagglutinin-neuraminidase to broaden viral tropism.

The controlled synthesis of telechelic polymers with precisely functionalized chain-ends and predictable main-chain structures is highly desirable. Herein, a series of air-stable trans-bis(phenylethynyl)palladium catalysts were designed and efficiently synthesized, which initiate the polymerization of phenyl isocyanides via a living chain-growth process, resulting in polyisocyanides with precise control of molecular weights (M ns) and narrow molecular weight distributions (Đ). The substituents of the catalyst can regulate the polymerization rate while serving as entire chain-end functional groups of the resulting polymers. Given the precise control over the length of rigid polyisocyanides, these polyisocyanides are an ideal building block for constructing covalent polymer frameworks (CPF m s) with tuneable pore apertures and functionalities. As a proof of concept, water-soluble CPFs with tuneable pore-apertures were prepared and the ordering of the resulting CPFs was systematically verified by dynamic light scattering (DLS), high-resolution transmission electron microscopy (HR-TEM), and small-angle X-ray scattering (synchrotron radiation facility). Moreover, the pore aperture can be directly controlled by tuning the length of the polyisocyanide link. Owing to the tuneable pore size and charge attraction effects, the CPFs with pore apertures matching the target single-stranded deoxyribonucleic acid (ssDNA) exhibit good performance on gene delivery. The percentage of delivered ssDNA into cells is up to ca. 98% (21 and 35 units).

Recent advancements in nanocarriers, particularly liposomes, have shown promising prospects for enhancing the pharmacokinetics, biodistribution, and therapeutic efficacy of chemotherapeutic drugs. However, liposome-based drug delivery systems are often constrained by high immunogenicity, poor targeting efficiency, and limited functional capabilities. In this context, the exploration of biomimetic liposomes has revealed their potential in targeted therapy, immune camouflage, immune modulation, gene delivery and vaccine development. By integrating the beneficial features of functional molecules and natural cell membrane components with the unique properties of liposomes, biomimetic liposomes have demonstrated considerable promise in drug delivery. This review aims to emphasize recent progress in biomimetic liposomes and systematically elucidate their design mechanisms and preparation methods. Additionally, it provides a comprehensive overview of the current applications of biomimetic liposomes as an innovative drug delivery platform, with the goal of advancing knowledge for their effective utilization.

Chronic kidney disease (CKD) is a progressive disorder that leads to significant structural and functional changes in the kidneys, posing a major global health concern and contributing to high mortality rates. The urgent need for innovative treatments is evident. Mesenchymal stem cells (MSCs) are well-regarded in regenerative medicine for their ability to repair tissue and modulate immune responses. Emerging research indicates that the therapeutic benefits of MSCs are largely mediated by the secretion of extracellular vesicles (EVs), particularly exosomes (MSC-Exos), which replicate the effects of MSCs by delivering genetic materials and proteins to target cells. MSC-Exos are novel natural carriers for targeted gene or drug delivery, offering biocompatibility, intrinsic targeting capabilities, and bioactive cargo to modulate recipient cells. They represent a groundbreaking platform for precision medicine, enhancing therapeutic efficacy with minimal immunogenicity and off-target effects. Moreover, embedding exosomes within hydrogels has emerged as a promising strategy to maintain their biological activity and enable a controlled release. This review explores the roles of MSC-Exos in CKD pathophysiology, highlights the renoprotective effects of MSC-Exos for various sources, and provides a comprehensive overview of how hydrogel biomaterials present a promising approach for integrating exosomes to enhance therapeutic outcomes. The use of hydrogels to encapsulate exosomes improves their sustained release and stability in diseased kidney tissues, providing an innovative strategy to enhance precision therapies.

CD200, an immunoregulatory glycoprotein of the immunoglobulin superfamily, suppresses inflammatory signaling by engaging its receptor CD200R, which is predominantly expressed on myeloid cells. To enhance the immune-evading properties of viral vectors, we engineered lentiviral particles displaying the CD200 ectodomain (CD200ED) to exploit anti-inflammatory response and phagocytosis resistance. A fusion gene encoding the mouse CD200 ectodomain and core streptavidin (CD200ED-coreSA) was cloned into the pET-30a(+) plasmid, expressed in E. coli Lemo21(DE3), and purified via immobilized metal affinity chromatography (IMAC). Successful protein assembly was confirmed by SDS-PAGE and western blot. Biotinylated VSV-G pseudotyped lentiviral vectors, encoding a green fluorescent protein reporter, were functionalized with CD200ED-coreSA. When exposed to murine J774A.1 macrophages, CD200ED-modified lentiviruses significantly reduced pro-inflammatory cytokine production - evidenced by 47.1% decrease in TNF-α and 55% decrease in IL-6 - compared to unmodified controls. Additionally, CD200ED anchoring reduced macrophage phagocytosis of lentiviral particles by 25%. These findings demonstrate that CD200-tethering confers dual anti-inflammatory and phagocytosis resistance capabilities to viral vectors, offering a promising strategy to improve gene delivery efficiency in inflammatory environments.

Lentiviral (LV) vectors derived from human immunodeficiency virus type 1 (HIV-1) mediate efficient gene transfer into various cell types, including neuronal, glial, and neural stem cells in the central nervous system. The HIV-1-based LV vectors commonly used are self-inactivating and pseudotyped with a variety of viral envelope glycoproteins. Pseudotyping with vesicular stomatitis virus glycoprotein (VSV-G) confers broad tropism to wide variety of cells and enhances vector stability of the vectors. Pseudotyping with other glycoproteins alters the tropism. In particular, the use of rabies virus glycoprotein (RV-G) or fusion glycoproteins (FuGs) consisting of VSV-G and RV-G segments increases the efficiency of retrograde gene delivery into neuronal cells. The LV vector strategy provides useful approaches to genetic manipulation of specific neural pathways for elucidating the structure and function of neural circuits, and also to genome-wide screening of key molecules and gene regulatory elements in development and survival of the nervous system. Such a strategy also provides a powerful tool for clinical applications aimed at gene therapy for neurological and neurodegenerative diseases as well as central nervous system tumors.

Developmental regulators and additives in promoting genetic transformation and genome editing efficiency.

This chapter discusses the development and application of constitutive and brain cell-type-specific promoters and enhancers in lentiviral vectors to enable precise and efficient gene delivery. The fundamental mechanisms of transcriptional regulation by promoters, distinguished between constitutive and cell-type-specific promoters, are outlined and the role of enhancers in modulating transcriptional activity is emphasized. Recent breakthroughs in single-cell transcriptomics and assay for transposase-accessible chromatin (ATAC) sequencing have led to the identification of novel enhancer regions, thus paving the way for the development of more refined and targeted lentiviral vectors. These advances have the potential to significantly improve the specificity and efficacy of transgene expression in distinct brain cell populations, including excitatory and inhibitory neurons, astrocytes, and microglia.

Oral gene delivery platform based on glycol chitosan-PEG-lactoferrin conjugate.

ASGPR-targeted micelles co-delivering lenvatinib and COP1 siRNA for hepatocellular carcinoma via dual-targeting.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising immunotherapy for central nervous system (CNS) tumors, despite the unique biological barriers posed by the blood-brain barrier (BBB) and immunosuppressive tumor microenvironment (TME). This chapter comprehensively reviews the evolution of CAR designs, gene delivery methods using lentiviral vectors (LVVs), and clinical applications for CNS tumors. We discuss current issues in CAR-T cell therapy for CNS tumors, while highlighting the strategies about new CAR designs and novel delivery methods to overcome these issues.

Muscle satellite cell editing by LNP-CRISPR-Cas9 to resist muscle injury.