Search Results (914)

Streptococcus pneumoniae remains a major global health threat and is listed by the World Health Organization as a pathogen in urgent need of new antimicrobial strategies. While primarily considered an extracellular pathogen, S. pneumoniae can persist within splenic macrophages in severe disease, creating a protected intracellular niche that may contribute to fulminant sepsis. We recently demonstrated the concept of an mRNA-based therapeutic approach in which host cells produce the pneumococcal bacteriophage endolysin Cpl-1. Here, we investigated whether expression of Cpl-1 in macrophages can target S. pneumoniae residing within host cells. Using the human THP-1 macrophage line, we demonstrated successful translation and intracellular accumulation of bioactive Cpl-1 following IVT-mRNA transfection. Lysates from Cpl-1 mRNA-transfected cells exhibited bacteriolytic activity, and Western blotting as well as immunofluorescent staining confirmed cytosolic endolysin production. Phagocytosis assays using an encapsulated and unencapsulated pneumococcal strain showed a reduction in intracellular bacterial burden in Cpl-1 mRNA-transfected macrophages compared with control and inactive-mutant Cpl-1 mRNA groups, and a flow cytometry-based assay further corroborated a decreased intracellular bacterial signal. Together, these findings suggest that mRNA-encoded Cpl-1 enhances intracellular killing of S. pneumoniae and supports the feasibility of mRNA-based endolysin therapies to target intracellular pneumococcal reservoirs. Full article

Background: Conventional drug delivery systems often lead to fluctuating plasma concentrations (“Peak and Trough” phenomenon), causing toxicity or inefficacy. Microfluidics has emerged as a revolutionary tool to overcome, among other applications, the limitations of conventional bulk encapsulation methods, such as polydispersity and poor reproducibility. Methods: A systematic review of the literature published between 2020 and 2025 was conducted to evaluate the application of microfluidics in the synthesis of advanced nanomedicines. The review focused on Lipid Nanoparticles (LNPs), Polymeric Nanoparticles (PNPs), and Hydrogel Microspheres. Results: Microfluidics enables the production of monodisperse particles with precise control over geometry and drug loading stoichiometry. Key therapeutic applications include oncology (passive and active targeting), gene therapy (mRNA vaccines), and regenerative medicine (diabetic wound healing). Conclusions: While microfluidics offers superior quality control compared to bulk methods, industrial scalability remains the primary challenge, currently addressed through parallelization and continuous flow strategies. Full article

Background/Objectives: Intracellular delivery of RNA molecules is challenging. To solve this problem, many carrier systems are available, which are based on liposomes or polymers. Cyclodextrins are widely used excipients to increase the solubility of small molecules, but their polymer derivatives are able to deliver macromolecules. In the present study, we aimed to investigate and compare the siRNA and mRNA carrying capacity of a cationic quaternary ammonium β-cyclodextrin polymer (QABCDPS) and polyethylenimine (PEI). Methods: Cytotoxicity of the polymers was tested by the MTT method. Polyplexes were formulated with different nitrogen/phosphate ratios (NP), and their physicochemical properties were examined using dynamic light scattering and zeta potential measurements. Cellular internalization and intracellular effects of the polyplexes were investigated by confocal microscopy and flow cytometry. Results: QABCDPS exhibited lower toxicity compared to PEI, effectively binding both siRNA and mRNA and delivering them into vesicles in the cytoplasm, but showing different internalization patterns. Polyplexes formed with PEI showed stronger biological effect than those with QABCDPS, which can be attributed to the strength of interactions facilitated by the polymers. Conclusions: In summary, QABCDPS is a low-toxicity carrier that shows some promise for mRNA delivery but is ineffective for siRNA silencing under the tested conditions and requires further structural optimization. Full article

Chimeric antigen receptor T (CAR-T) cell therapy has transformed the treatment of hematologic malignancies, yet its broader application, particularly in solid tumors, remains constrained by high cost, labor-intensive manufacturing, limited production capacity, and variable clinical performance, as well as barriers such as poor trafficking, antigen heterogeneity, and an immunosuppressive tumor microenvironment. In vivo CAR-T cell engineering, in which CAR-T cells are generated directly within the patient, offers a paradigm shift by eliminating the need for ex vivo cell processing and complex logistical infrastructure. Among emerging approaches, messenger RNA (mRNA)-loaded lipid nanoparticles (LNPs) have emerged as a promising and clinically tractable platform for in vivo CAR-T cell generation, enabling direct reprogramming of T lymphocytes within the patient and thereby circumventing the need for leukapheresis, viral vector production, and prolonged ex vivo culture, effectively transforming the patient into their own cell therapy factory. This review synthesizes advances in mRNA-LNP-mediated in vivo CAR-T cell generation, encompassing ionizable lipid chemistry and emerging T cell-targeted delivery strategies, including surface functionalization approaches. We discuss the implications of transient CAR expression for immune activation, safety, and therapeutic durability, alongside CAR design optimization through co-stimulatory domains and safety switches. Preclinical evidence from murine tumor models and non-human primates is integrated with current regulatory considerations, and key barriers to clinical translation are highlighted. Collectively, progress in nucleic acid delivery, synthetic immunology, and precision medicine positions in vivo mRNA-CAR-T therapy as a promising modality for oncology and beyond. Full article

Lipid nanoparticles (LNPs) have emerged as popular nucleic acid delivery systems, yet the dynamic mechanisms related to their self-assembly and structural maturation remain insufficiently understood due to the limitations of traditional offline characterization tools. This study establishes a time-resolved (TR) in situ small-angle X-ray scattering (SAXS) methodology to monitor the structural evolution of LNPs during microfluidic formulation and subsequent maturation. By integrating a dual-channel microfluidic mixing system with a SAXS measurement platform, we successfully captured the real-time scattering profiles of both empty and messenger RNA-loaded nanoparticles (mRNA-LNPs). The results demonstrate distinct assembly pathways for empty-LNPs and those encapsulated with mRNA. The empty-LNPs undergo a gradual transition toward periodic nanostructures, whereas mRNA-LNPs exhibit rapid complexation into stable subunits followed by hierarchical assembly. Furthermore, the platform effectively tracked nanoscale structural rearrangements during a microfluidic dilution process, revealed by subtle shifts in scattering peaks and internal periodicity. Overall, this time-resolved approach provides a robust experimental framework for capturing transient intermediate states, offering a valuable tool to elucidate molecular assembly mechanisms and facilitate the rational design of next-generation nanomedicines. Full article

Porcine enteric coronaviruses (PECs), including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), remain major causes of neonatal diarrhea, dehydration, mortality, and economic loss in swine production. Despite substantial progress in vaccine development, durable field protection is still inconsistent. In this narrative review, this narrative review synthesizes current knowledge on PEC vaccine design from three connected perspectives: antigenic breadth, mucosal immunity, and translational evaluation. The economic and virological context of PEC vaccine development is first summarized, including the recurrent production burden of PECs, coronavirus genome organization, structural proteins, and the central role of the spike protein in receptor engagement, membrane fusion, and neutralizing antibody induction. Key issues are then discussed, including how spike diversity, conformational stability, epitope accessibility, glycan shielding, and antigen matching influence protective breadth; why intestinal secretory IgA, mucosal immune-cell trafficking, local memory responses, and lactogenic immunity should be prioritized as biologically relevant endpoints; and how delivery route, adjuvant selection, and platform design shape response quality. Current evidence on recombinant protein, viral-vectored, nanoparticle, virus-like particle, probiotic, plant-derived, and mRNA-based approaches is compared with attention to both promise and current evidentiary and translational limitations. The available literature suggests that future progress in PEC vaccinology is likely to depend less on platform novelty alone than on integrated vaccine designs that align antigen selection, mucosal delivery, maternal–neonatal protection, heterologous challenge, manufacturability, and field applicability. Full article

Segmental bone defects and age-related osteoarthritis (OA) are clinically challenging in terms of treatment. Although preclinical studies have demonstrated efficacy for bone defect healing and OA using ex vivo gene therapy or biomaterial sustained-release delivery, few such treatments have translated into clinical therapies due to safety concerns. Bone morphogenetic proteins belong to the transforming growth factor β (TGFβ) superfamily and are effective in bone and cartilage regeneration/repair. Among BMPs, BMP4 is not only effective in promoting bone and cartilage repair but also promotes stem cell renewal potential and exhibits anti-aging effects. Therefore, the aim of this study is to investigate whether human BMP4 mRNA encapsulated in lipid nanoparticles (hBMP4 mRNA/LNP) can promote bone and cartilage repair. In vitro data demonstrated that hBMP4 mRNA/LNP-treated human MSCs secreted BMP4 protein, as detected by ELISA, and enhanced osteogenic differentiation. In vivo results demonstrated that hBMP4 mRNA/LNP at a 50 µg dose promoted limited new bone formation only at 2 weeks after creation of defect in critical-sized calvarial bone defects in aged mice when delivered using fibrin sealant hydrogel, as revealed by micro-CT and histology. However, intra-articular injection (IA) of lower doses (2.5 and 5 µg) in aged mice knee joints prevented cartilage loss, as demonstrated by micro-CT; decreased OARSI histology scores; and improved cartilage-specific matrix COL2. hBMP4 mRNA/LNP at a 5 μg dose significantly increased SOX9⁺ cells per normalized cartilage area as well as the percentage of SOX9⁺ cells in the cartilage area. hBMP4 mRNA/LNP treatment showed a trend of pain alleviation and did not change serum hyaluronic acid levels. In conclusion, human BMP4 mRNA encapsulated in lipid nanoparticles improved cartilage repair and delayed cartilage degeneration in aged mice, while having a limited effect on bone healing, even at a higher dosage. These results suggest that hBMP4 mRNA encapsulated with lipid nanoparticles represents a promising treatment for age-related OA. Full article

Local pulmonary delivery offers a non-invasive application route for mRNA therapeutics with the potential for high bioavailability at the target-site of applications such as mucosal vaccination or the treatment of lung diseases. However, efficient delivery remains challenging due to major lung-specific barriers, particularly mucus. Herein, pH-responsive, amphiphilic xenopeptides comprising lipoamino fatty acids and oligoamino acids (OAAs) connected in distinct branched U-shape or bundle topologies were evaluated as mRNA polyplexes for delivery to A549 and Calu-3 lung cells under standard submerged or air–liquid interface (ALI) transfection conditions, and upon intratracheal application in BALB/c mice. Optionally, polyplexes were coated with negatively charged hyaluronic acid (HA) or colloidally stabilized with poly(ethylene glycol) (PEG). For U-shapes, hydrophobic modification of the OAA domain boosted their efficiency. Interestingly, best-performing formulations varied across transfection conditions. While the bundle topology showed the highest potential in submerged cell culture, U-shaped carriers were more efficient under ALI conditions. Polyplex surface modification with HA or PEG did not strongly alter in vitro transfections, whereas hydrophobized U-shape core polyplexes combined with surface modification enhanced their efficiency in vivo. Thus, the cationizable core and surface properties of mRNA nanoparticles require specific balancing in various lung cell models and lung. Full article

Messenger RNA (mRNA) therapeutics are redefining treatment approaches in vaccines, cancer immunotherapy, protein replacement, and gene editing. Lipid nanoparticles have enabled early clinical successes, but they can be limited by liver-dominant biodistribution, long-term storage stability, and systemic tolerability. Polymeric delivery systems offer a versatile alternative, with tunable physicochemical properties enabling precise control over mRNA complexation, protection, release, and targeting. This review examines recent progress across polyethyleneimine derivatives, poly(β-amino ester)s, poly(amino acid)s, polyesters, dendrimers, charge-altering releasable transporters, and lipid-polymer hybrids. We highlight strategies such as structural modification, stimuli-responsive designs, and high-throughput polymer screening that enhance stability, reduce cytotoxicity, and enable organ- or cell-specific delivery. Addressing challenges in immunogenicity, biodistribution, and manufacturing scalability will be pivotal to translating these innovations into safe and effective mRNA therapeutics. Full article

Residual cardiovascular risk arises from dysregulated expression of genes encoding apolipoprotein(a) (LPA), apolipoprotein C-III (APOC3), angiopoietin-like gene 3 (ANGPTL3), and proprotein convertase subtilisin/kexin type 9 (PCSK9). RNA-based therapies, small interfering RNAs (siRNAs), and antisense oligonucleotides (ASOs) modulate these targets at the post-transcriptional level through RNA interference and RNase H-mediated degradation, respectively. This scoping review maps the molecular mechanisms, target involvement, and pharmacodynamic outcomes of RNA therapies for managing residual cardiovascular risk, with contextual comparison to traditional lipid-lowering agents. A systematic search of PubMed, Embase, Web of Science, and Scopus was performed from 2020 to February 2026. Of the 1088 records identified, 30 studies met the inclusion criteria. RNA therapies have demonstrated potential for engagement, with 80–98% reductions in Lp(a) (pelacarsen, olpasiran, zerlasiran, lepodisiran), 50–80% reductions in triglycerides (olezarsen, plozasiran, volanesorsen), and 36–44% reductions in low-density lipoprotein cholesterol (LDL-C). Mechanistically, siRNAs achieve gene silencing through RISC-mediated mRNA cleavage, with sustained pharmacodynamic effects (3–6 months) because of Argonaute-2 stability, while gapmer ASOs recruit RNase H1 for mRNA degradation. Conjugation with GalNAc allows for hepatocyte-specific delivery with a subcutaneous bioavailability of 70–85%. Safety profiles were favorable, with injection site reactions (4–12%) being the most common adverse event. This analysis maps the emerging molecular landscape of RNA therapies, highlighting their substantial precision for targeting residual cardiovascular risk pathways that cannot be addressed by traditional agents. Full article

RNA-based therapeutics are becoming increasingly important in oncology, particularly following the rapid development of mRNA technologies during the COVID-19 pandemic, but their success strongly depends on how efficiently they can be delivered to target cells. Microfluidic technologies have redefined the design and manufacturing of RNA-based nanocomplexes, as they enable precise control over physicochemical features that are critical for clinical translation in oncology. This review examines recent developments in microfluidic-assisted synthesis of RNA nanocarriers, with a focus on cancer applications. Through a detailed analysis of material systems, device architectures, and formulation strategies, we explore how laminar flow environments enable reproducible encapsulation, tunable particle size, and improved payload stability. We examine the microfluidic assembly of lipid nanoparticles and polymeric carriers for RNA delivery, highlighting strategies to enhance durability, bioavailability, and cellular uptake. Advancements in process optimization, including flow parameter refinement and inline monitoring, are discussed alongside the influence of device geometries on mixing dynamics and nucleation. Beyond formulation, we explore the integration of microfluidics with tumor-on-chip platforms to evaluate transport, penetration, and therapeutic response in physiologically relevant cancer models. By connecting technological innovation with preclinical application, this work outlines the trajectory toward next-generation, personalized RNA nanomedicines enabled by microfluidic precision. Full article

Background: Yes-associated protein 1 (YAP1), a key effector of the Hippo signaling pathway and mechanosensitive transcriptional coactivator, plays a complex role in osteoarthritis (OA) and cartilage regeneration. While YAP1 is essential for tissue homeostasis, its dysregulation has been implicated in both inflammatory and degenerative joint pathologies. However, its precise function remains ambiguous. Methods: We silenced YAP1 with small interfering RNA (siYAP1) in two human-cell-based models relevant to OA pathogenesis and cartilage repair: (1) IL-1β (10 ng/mL)-stimulated articular chondrocytes in monolayer and pellet cultures, and (2) TGF-β1 (10 ng/mL)-induced chondrogenesis in MSC pellet cultures. Outcome measures comprised YAP1 nuclear localization; inflammatory/catabolic markers in chondrocytes (IL6, IL8, ADAMTS5, MMP13); and, in MSC pellets, chondrogenic or hypertrophic markers (COL2A1, ACAN, RUNX2, MMP13, COL10A1) together with glycosaminoglycan (GAG) deposition. Statistical significance was assessed using an ANOVA or Friedman test with post hoc correction (Tukey or Dunn’s test, respectively); p < 0.05 was considered significant. Results: In human chondrocytes, siYAP1 reduced IL-1β-induced nuclear YAP1 localization and suppressed pro-inflammatory mediators IL6 and IL8, indicating an anti-inflammatory effect. YAP1 silencing also downregulated ADAMTS5 expression in 2D monolayers but not in 3D pellet cultures, suggesting reduced regulatory influence in the three-dimensional environment. Notably, MMP13 expression was paradoxically increased following YAP1 knockdown, underscoring the complexity of YAP1’s role in catabolic regulation. In MSC chondrogenesis, siYAP1 enhanced TGF-β1-induced chondrogenesis by increasing COL2A1 and ACAN expression and promoting GAG deposition on day 21. Additionally, it reduced hypertrophic markers RUNX2 and MMP13 on day 7, though COL10A1 remained elevated compared to negative siRNA, indicating only partial suppression of hypertrophic differentiation. Nuclear YAP1 levels were increased by day 21 despite reduced mRNA, suggesting post-transcriptional regulation or enhanced nuclear translocation. Conclusions: These findings demonstrate that YAP1 knockdown exerts context-specific anti-inflammatory and pro-chondrogenic effects while partially mitigating hypertrophy. However, divergent outcomes, namely elevated MMP13 in chondrocytes and upregulated COL10A1 in MSCs, indicate that YAP1 silencing does not uniformly suppress inflammation or hypertrophy. YAP1 represents a potential therapeutic target for OA, but its modulation requires careful consideration of cellular context, siRNA delivery method, and timing to optimize outcomes for cartilage repair and joint preservation. Full article

Messenger RNA (mRNA) vaccines are emerging as promising tools capable of reshaping how cancer interacts with the immune system and responds to immunotherapy. These vaccines not only act as platforms for antigen delivery but can also influence the tumor microenvironment (TME), fostering a shift from immunologically “cold’’ conditions toward “hotter’’ and treatment-responsive states. In melanoma, this capability has been found to enhance the efficacy of the immune checkpoint inhibitors (ICIs), as mRNA-based priming can provide the robust antitumor activation needed for more effective checkpoint blockade. Early clinical studies with personalized or off-the-shelf vaccines showed benefits in patients with high-risk resected melanoma or refractory to PD-1 inhibition. Combining mRNA vaccines with ICIs, along with other immunomodulatory strategies, may be helpful to overcome resistance arising from the TME and achieve more durable clinical benefits. Besides these advances, computational and in silico modeling are providing new insights into how mRNA vaccines modulate the TME, helping to identify factors such as antigen-presenting cell (APC) density, CD8⁺ T-cell infiltration, and macrophage polarization that may predict treatment success and guide personalized strategies. Together, these developments indicate that combining mRNA vaccination with ICIs, supported by computational tools, may improve clinical outcomes in melanoma and, potentially, in selected tumor types with favorable immunological features, although important biological constraints limit direct extrapolation to less immunogenic malignancies. Full article

Cancer is a complex and evolutionary disease, with the development of different types of cancers leading to various different defective gene mutations. Synthetic lethality is a genetic-level precision medical strategy. Currently, treating BRCA (BReast CAncer)-mutated breast or ovarian cancer cells with a chemical inhibitor (Poly(ADP-ribose) polymerase, PARPi) is a typical synthetic lethal application in clinical practice. However, PARPi therapy has been found to cause off-target effects and therapy-induced immune escape driven by PD-L1 upregulation, allowing for cancer cells to escape attack from the immune response. To overcome these challenges, we developed a core–shell structure comprising a hydrophobic core of quercetin (Q)-mediated PARP inhibition and iron oxide nanoparticles (IONPs), enveloped by a hydrophilic fucoidan (Fu) shell to encapsulate short hairpin RNA targeting Programmed Death Ligand 1 (shPD-L1) for efficient gene transfection (shPD-L1@QIO@Fu). Structurally, the incorporation of quercetin into the intermediate hydrophobic layer enables modulate of the PARP effect, while the inner aqueous core with shPD-L1 gene silencing can inhibit the expression of PD-L1 protein. In this study, we proved that shPD-L1@QIO@Fu demonstrated a dual therapeutic mechanism against BRCA-mutant cancer cells by inducing extensive DNA double-strand breaks and promoting apoptosis. Furthermore, the combined action of quercetin-mediated DNA damage and shPD-L1-driven PD-L1 suppression led to a significant reduction in PD-L1 mRNA to approximately 5% at 72 h and decreased surface PD-L1 below baseline by 96 h. This effectively suppresses PARPi-induced PD-L1 upregulation and enhances antitumor immunity. These findings demonstrate the therapeutic efficacy of shPD-L1@QIO@Fu nanomedicine, providing a promising foundation for advanced co-delivery strategies to synergize PARP inhibition mediated synthetic lethality with immune checkpoint blockade in next-generation precision medicine. Full article

Therapeutic cancer vaccines represent a rational immunotherapeutic strategy aimed at inducing tumor-specific adaptive immune responses in patients with established malignancies. In contrast to prophylactic vaccines, these approaches must function within immunosuppressive tumor microenvironments characterized by antigenic heterogeneity, immune dysfunction, and dynamic tumor evolution. Effective vaccine design requires the integration of three essential components: the selection of appropriate tumor-associated or tumor-specific antigens, efficient delivery platforms that enable antigen presentation, and adjuvant systems that promote robust T-cell priming and expansion. Initial clinical investigations in B-cell malignancies and multiple myeloma demonstrated that idiotype-based vaccines can elicit tumor-specific immune responses. However, durable clinical benefit has been inconsistent, reflecting limitations in antigen selection, suboptimal immunogenicity, and tumor-mediated immune evasion. Over the past decade, advances in tumor genomics, next-generation sequencing, and immune monitoring have enabled the development of next-generation vaccine platforms, including dendritic cell-based approaches, personalized neoantigen vaccines, and mRNA-based technologies. Emerging evidence suggests that vaccine efficacy is highly dependent on disease context. Biologically favorable settings such as minimal residual disease (MRD) and post-transplant immune reconstitution provide reduced tumor burden and improved immune competence, thereby enhancing the likelihood of effective immune priming. In parallel, combination strategies incorporating immune checkpoint inhibitors, immunomodulatory agents, and cellular therapies are increasingly being explored to overcome tumor-induced immunosuppression. This review synthesizes current knowledge of therapeutic cancer vaccines in B-cell malignancies and multiple myeloma, with emphasis on immunologic mechanisms, antigen selection, vaccine platforms, and clinical evidence. We further propose a conceptual framework integrating tumor biology, immune context, and combination strategies to guide the rational development of next-generation vaccine therapies. Full article