Search Results (102)

RNA origami enables genetically encoded, single-stranded RNA nanostructures that can self-assemble through co-transcriptional folding and are increasingly deployed as scaffolds for biosensing, synthetic biology, and nanomedicine. A recurring practical bottleneck is scalable, solution-phase readout of whether a designed scaffold has reached its intended base-paired architecture, whether it undergoes slow maturation or kinetic trapping, and how its stability is distributed across motifs. Here, we adapt microfluidic modulation spectroscopy (MMS) as a label-free structural biosensor for RNA folding by exploiting the rich 1760–1600 cm⁻¹ vibrational fingerprints of RNA bases and base pairs. MMS alternates between sample and composition-matched buffer measurements in a microfluidic transmission cell to automatically subtract the solvent background, enabling high-quality spectral measurement from microliter volumes under native solution conditions. Using a six-helix-bundle-with-clasp (6HBC) RNA origami as a model, we established an analysis workflow (baselined second derivative and constrained deconvolution) to quantify paired versus unpaired populations. Thermal ramping resolves multiple unfolding events and yields an unfolding barcode that differs between young and mature ensembles. Importantly, MMS tracks post-transcriptional maturation from a kinetically trapped young conformer toward a more compact, base-paired mature state, consistent with prior cryo-EM/SAXS observations for 6HBC RNA origami. Together, these results position MMS as a rapid, automated, and scalable complement to high-resolution structure determination for engineering dynamic RNA origami biosensors. Full article

RNA-based interventions are particularly promising for next-generation therapeutic strategies and hold significant potential when integrated with diagnostic modalities. Among noncoding RNAs, microRNAs (miRNAs) regulate gene expression post-transcriptionally and represent compelling targets for cancer therapy. However, their clinical translation remains hindered by instability, off-target effects, and limited delivery efficiency. Here, we report the microfluidic synthesis of hybrid lipid–polymer nanoparticles (LiPoNs) that co-deliver an AntimiR-21 and the magnetic resonance imaging contrast agent gadolinium diethylenetriamine penta-acetic acid (Gd-DTPA). The LiPoNs were obtained using coupled Hydrodynamic Flow Focusing (cHFF), enabling precise control over lipid–polymer self-assembly and surpassing the compositional limitations reported with conventional micromixers. The resulting AntimiR-21–Gd-DTPA–LiPoNs exhibited an average hydrodynamic diameter of 124 nm, narrow polydispersity (PDI < 0.2), and encapsulation efficiency up to 60%. In MDA-MB-231 breast cancer cells, treatment with AntimiR-21–LiPoNs induced suppression of miR-21 and a corresponding decrease in migratory capacity, demonstrating effective functional delivery and gene expression modulation. These findings establish a versatile microfluidic platform for engineering multifunctional lipid–polymer nanostructures whose hybrid architecture combines the biocompatibility and membrane fusion capability of lipids with the structural robustness and controlled release properties of polymers, thereby advancing RNA-based theranostic design for precision oncology and related applications. Full article

Background/Objectives: Alveolar macrophages represent the main path of defense in the peripheral pulmonary tissue, though their role in chronic inflammatory lung diseases shows that their protective function can turn pathological. This study focused on developing a system to passively target the release of the pro-inflammatory cytokine TNF-α through the local delivery of siRNA. Methods: An inhalable aspherical microparticle made up of mesoporous silica nanoparticles, crosslinked by an electrostatic LbL-system embedding the siRNA, was developed. Results: Through testing with the NGI, adequate aerodynamic properties with an MMAD as low as 3.37 µm could be determined, with a GSD as low as 1.46, suggesting a relatively small size distribution even during inhalation. To further understand the interaction of the microrods with the lung parenchyma and the resident cells, the disintegration of the rods in different simulant body fluids, their toxicity, and the cell uptake through dTHP-1 and A549 were observed. This showed slow but continuous disintegration, no toxicity in A549 cells, and high microrod uptake by dTHP-1 cells. To demonstrate the effect of the delivered siRNA on the release of TNF-α, ELISAs were carried out, establishing an inhibitory effect of the siRNA-carrying microcarrier system compared to those without siRNA or loaded with scrambled siRNA. To increase the efficacy of the siRNA, chloroquine as an endosomal escape-enhancing compound was loaded onto the mesoporous silica nanoparticles. This resulted in a significant improvement in siRNA inhibition. Conclusions: The developed formulation is able to reach the targeted structure and inhibit the secretion of TNF-α, with CQ increasing the inhibitory effect of the siRNA. Full article

Background: Diabetic nephropathy (DN) is largely driven by transforming growth factor-β1 (TGF-β1)-mediated fibrosis. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes offer precise gene disruption, yet effective non-viral delivery remains a challenge. This study developed cationic lipid-based hybrid nanostructured lipid carriers (NLCs) for intracellular delivery of TGFB1-targeting RNP as an early-stage platform for DN gene modulation. Methods: A single-guide RNA (sgRNA) targeting human TGFB1 was assembled with Cas9 protein (1:1 and 1:2 molar ratios). Hybrid NLCs comprising squalene, glyceryl trimyristate, and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were formulated via optimized emulsification–sonication to achieve sub-100 nm particles. Physicochemical properties, including polydispersity index (PDI), were assessed via dynamic light scattering (DLS), while silencing efficacy in HEK293T cells was quantified using quantitative reverse transcription PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Results: Optimized NLCs achieved hydrodynamic diameters of 65–99 nm (PDI < 0.5) with successful RNP complexation. The 1:2 Cas9:sgRNA formulation produced the strongest gene-editing response, reducing TGFB1 mRNA by 67% (p < 0.01) compared with 39% for the 1:1 ratio. This translated to a significant reduction in TGF-β1 protein (p < 0.05) within 24 h. Conclusions: DOTAP-based hybrid NLCs enable efficient delivery of CRISPR–Cas9 RNP and achieve significant suppression of TGFB1 expression at both transcriptional and protein levels. These findings establish a promising non-viral platform for upstream modulation of profibrotic signaling in DN and support further evaluation in kidney-derived cells and in vivo renal models. Full article

Lipid nanoparticles (LNPs) are now the go-to method for delivering genetic medicines, backed by real-world use in patients. Things like which fats they are made of, their shape at the molecular level, how ingredients mix, and how they are built, matter a lot. This review attempts to take a close look at how different components, such as ionizable lipids, auxiliary lipids (DSPC, DOPE), cholesterol, and PEG-based lipids, affect the bioavailability of LNPs. It also focuses on key functions of LNPs, including packaging genetic material, escaping cellular traps, spreading in the body, and remaining active in the blood. New data show that lipids with the right handedness and highly sensitive chiroptical quality control can sharpen delivery accuracy and boost transport rates, turning stereochemistry into a practical design knob. Rather than simply listing results, we examine real-world examples that are already used to regulate gene expression, enhance mRNA expression, splenic targeting, and show great potential for gene repair, protein replacement, and DNA base-editing applications. Also, recent advances in AI-based designs for LNPs that take molecular shape into account and help speed up modifications to lipid arrangements and mixture configurations are highlighted. In summary, this paper presents a practical and scientific blueprint to support smarter production of advanced LNPs used in genetic medicine, addressing existing obstacles, balanced with future opportunities. Full article

Background: Hepatocellular carcinoma (HCC) remains a major global health challenge with limited therapeutic options. Although RNA interference (RNAi) enables precise gene silencing, its clinical application is restricted by siRNA instability, inefficient cellular uptake, and the requirement for potentially toxic delivery carriers. To address these limitations, a dual-targeted branched siRNA nanostructure (GT-multi-siRNA) was developed to simultaneously silence two HCC-related oncogenes, GP73 and hTERT. Methods: GT-multi-siRNA was synthesized in Escherichia coli and characterized for particle size, stability, Dicer processing efficiency, intracellular retention, and cytotoxicity. Its therapeutic effects were evaluated through gene-silencing assays, proliferation and migration assays in Hep3B cells, and intratumoral administration in a xenograft mouse model. Histopathology and cytokine profiling were conducted to assess biosafety. Results: GT-multi-siRNA formed uniform nanoparticles (50–100 nm) with moderate physicochemical stability and minimal cytotoxicity at concentrations ≤ 200 ng/μL. The nanostructure was efficiently processed by Dicer into functional siRNAs and remained detectable intracellularly for at least 36 h. In Hep3B cells, GT-multi-siRNA reduced GP73 and hTERT mRNA and protein levels by approximately 50%, accompanied by significant inhibition of cell proliferation and migration. In vivo, a single intratumoral dose suppressed tumor growth, while a two-dose regimen markedly limited tumor progression. No liver toxicity was observed, and cytokine analysis showed selective IL-4 upregulation without influencing IL-6 levels. Conclusions: GT-multi-siRNA demonstrates potent dual-gene silencing activity and favorable biosafety, providing a promising RNAi-based therapeutic strategy for targeted HCC treatment. Full article

Background: Rabies virus (RABV) causes approximately 59,000 human deaths annually. Current pre- and post-exposure vaccination relies on inactivated vaccines (INVs) with limited yield and immunogenicity. We engineered a dual-cationic LNP-based nucleocapsid-like nanostructure (NLS) that co-encapsulates RABV G-mRNA and recombinant RABV-N to engage MHC-I/II pathways and enhance protection. Methods: A pVAX-RABV-G plasmid containing 5′/3′UTRs, Kozak, and poly(A) was transcribed in vitro. RABV-N with an N-terminal 6× His tag was expressed in E. coli BL21(DE3) and purified by Ni-Sepharose affinity chromatography. Dual-cationic LNPs (DHA, DOTAP Cl, mPEG-DTA2K, DOPC) were formulated by microfluidics at a 4:1 (G-mRNA:RABV-N) mass ratio. Vaccine quality was assessed by encapsulation efficiency, DLS, PDI, zeta potential, and TEM. Mice received empty LNPs, INV, G-mRNA, or NLS under varied schedules and doses. ELISA measured RABV-G/N-IgG; RFFIT determined neutralizing antibody (nAb) titers; ELISPOT quantified CTL response; qPCR assessed T-cell activation genes. On day 35 after the first immunization of vaccines, mice were challenged intramuscularly with 25 LD₅₀ of CVS-24. Results: G-mRNA purity was >95% and drove strong RABV-G expression in 293T cells. Purified RABV-N was approximately 52 kDa, >90% pure, and reactive to anti-His and anti-N antibodies. NLS achieved >95% encapsulation, a diameter of 136.9 nm, PDI 0.09, and a +18.7 mV zeta potential. A single dose yielded approximately 10 IU mL⁻¹ nAb by day 7; two doses peaked at approximately 1000 IU mL⁻¹. Mice showed 100% survival and no viral rebound in brain, spinal cord, and sciatic nerve. NLS induced stronger MHC-I/II-linked cellular immunity and higher RABV G/N-specific IFN-γ spot frequencies than G-mRNA or INV. Conclusions: The dual-antigen NLS vaccine co-delivering G-mRNA and RABV-N via dual-cationic LNPs robustly activates MHC-I/II, rapidly generates high-titer nAb (≥10 IU mL⁻¹ within 1 week), and sustains potent CD8⁺ CTL and CD4⁺ Th responses. A two-dose regimen (days 0 and 21) conferred complete protection, supporting the NLS platform as a next-generation rabies vaccine candidate. Full article

Chimeric antigen receptor (CAR) therapy represents a promising modality for treating cancer and autoimmune diseases, employing genetically engineered immune cells. Despite remarkable clinical outcomes, its broad implementation is constrained by significant challenges, including toxicity, limited specificity, and complexities associated with genetic material delivery. Biological nanoparticles, such as exosomes, virus-like particles, and biomimetic nanostructures, possess unique properties that can address these limitations. These nanoplatforms enable targeted delivery of genetic constructs, mitigate the risk of cytokine release syndrome, modulate CAR cell activity, and can function as biosensors. Furthermore, they facilitate non-viral, in vivo CAR cell engineering, streamlining the process compared to conventional ex vivo methods. The advancement of in vivo strategies underscores the critical need to overcome toxicity hurdles inherent to current CAR-T platforms. In this context, exosomes and biomimetic nanoparticles offer considerable potential due to their innate biocompatibility, programmability, and versatile cargo capacity for payloads like mRNA and circular RNA. This review comprehensively outlines contemporary genetic platforms for CAR expression and examines the opportunities presented by biological delivery vehicles. It focuses on recent achievements and revisits fundamental CAR principles through the lens of emerging technologies aimed at confronting persistent challenges in the field. Full article

Surface modification of nucleic acid-based electrochemical biosensors has been at the forefront of research since their inception. Effective modification ensures the optimization of the sensitivity, specificity, and stability of modern biosensors. Recent advances in DNA nanotechnology have enabled the development of novel electrochemical biosensor interfaces with precise assembly and high biocompatibility. In this review, we explore three strategies for enhancing biosensor performance: the integration of tetrahedral DNA nanostructures (TDNs), self-assembled monolayers (SAMs), and DNA-based hydrogels. TDNs offer well-defined geometry and controlled spatial presentation of capture probes, significantly reducing background noise and improving target accessibility. SAMs provide a robust and tunable platform for anchoring these nanostructures, enabling reproducible and chemically stable interfaces. DNA hydrogels serve as a responsive and flexible scaffold capable of signal amplification and analyte retention. These surface architectures enhance sensitivity and minimize non-specific adsorption (NSA). We discuss recent applications and experimental outcomes, highlighting how each component is driving the next generation of nucleic acid-based biosensors. Full article

Botrytis cinerea, a necrotrophic fungal pathogen responsible for gray mold, poses a severe threat to over 1400 plant species, causing significant pre- and postharvest losses worldwide. RNA interference (RNAi)-based strategies, particularly spray-induced gene silencing (SIGS), have emerged as environmentally friendly alternatives to chemical fungicides. However, the application of naked double-stranded RNA (dsRNA) suffers from poor stability and low cellular uptake. In this study, we engineered a self-assembling triangular RNA nanoparticle, termed Bc-triangle, targeting four virulence genes of B. cinerea—BcDCL1, BcPPI10, BcNMT1 and BcBAC. The nanostructure was designed using RNA origami principles and produced in Escherichia coli. Functional assays demonstrated that Bc-triangle significantly inhibited conidial germination and mycelial growth in vitro, and markedly reduced disease severity in plants. Compared with linear dsRNA, Bc-triangle showed superior persistence and efficacy, with lesion area reduction sustained up to 10 days post-spraying. qRT-PCR analysis revealed substantial downregulation of the target genes, especially BcNMT1, indicating enhanced RNAi activation. These findings establish RNA nanotechnology as a powerful platform for transgene-free, programmable, and sustainable control of fungal pathogens in crop production. Full article

Liquid biopsy offers dynamic and noninvasive analysis of cellular biomarkers, thereby presenting enormous potential for early detection of cancer, cancer staging, prediction of relapse, real-time examination of therapeutic efficacy, perception of therapeutic targets, and understanding the resistance mechanisms. Nanotechnology has emerged as a novel tool to widen the application horizon of liquid biopsy. Several nanomaterials, nanodevices, nanostructures, and nanosensors have been explored for improved application of liquid biopsy for biomarker detection. The circulating tumor cells (CTCs), circulating tumor proteins (CTP), miRNA and extracellular vesicles (EVs) are some of the important biomarkers for detection by liquid biopsy in bodily fluids. Herein, we have discussed the state of the art and beyond in advances in nanotechnology and in increasing the specificity, sensitivity, and purity with which we detect liquid biopsy biomarkers. The opportunities and prospects of these advanced innovative nanomaterials and technologies in clinical applications are explored. Furthermore, various isolation and biosensing strategies for visualization and signal amplification using nanomaterials are summarized. The utilization of nanotechnology-based liquid biopsy may provide greater insights for improved treatment, diagnosis, and prognosis of cancer. Full article

Exosomes are nanoscale extracellular vesicles (EVs) that carry biomolecular signatures reflective of their parent cells, making them powerful tools for non-invasive diagnostics and therapeutic monitoring. Despite their potential, clinical application is hindered by challenges such as low abundance, heterogeneity, and the complexity of biological samples. To address these limitations, plasmonic biosensing technologies—particularly propagating surface plasmon resonance (PSPR), localized surface plasmon resonance (LSPR), and surface-enhanced Raman scattering (SERS)—have been developed to enable label-free, highly sensitive, and multiplexed detection at the single-vesicle level. This review outlines recent advancements in nanoplasmonic platforms for exosome detection and profiling, emphasizing innovations in nanostructure engineering, microfluidic integration, and signal enhancement. Representative applications in oncology, neurology, and immunology are discussed, along with the increasingly critical role of artificial intelligence (AI) in spectral interpretation and diagnostic classification. Key technical and translational challenges—such as assay standardization, substrate reproducibility, and clinical validation—are also addressed. Overall, this review highlights the synergy between exosome biology and plasmonic nanotechnology, offering a path toward real-time, precision diagnostics via sub-femtomolar detection of exosomal miRNAs through next-generation biosensing strategies. Full article

Magnesium ions (Mg²⁺) play an important role in animal health, with their concentration in the bloodstream serving as a key indicator for hypomagnesemia diagnosis. In this study, a flexible hydrophobic paper-based microfluidic field-effect biosensor was developed for point-of-care Mg²⁺ detection, which integrated flexible hydrophobic paper, semiconducting single-walled carbon nanotubes (SWNTs) and a Mg²⁺-specific RNA-cleaving DNAzyme(RCD)-based DNA nanostructure. Flexible hydrophobic paper was synthesized by using cellulose paper and octadecyltrichlorosilane, improving mechanical strength and decreasing biological interference. To achieve high sensitivity, the Mg²⁺-specific RCD was functionalized with SWNTs, and then repeatedly self-assembled two different Y-shaped DNAs to construct a DNA nanostructure based on a similar DNA origami technique. This proposed biosensor exhibited a linear detection range from 1 μM to 1000 μM, with a detection limit of 0.57 μM, demonstrating its great stability, selectivity, and anti-interference performance. This innovative design offers promising potential for Mg²⁺ monitoring in real applications. Full article

Background/Objectives: Cancer peptide vaccines represent a promising strategy to develop targeted and personalized treatments for cancer patients. While tumor peptides alone are insufficient in mounting effective immune responses, the addition of adjuvants can enhance their immunogenicity. Nanoparticle delivery systems have been explored as vaccine carriers to incorporate both adjuvants and peptides. One such nanoparticle is RNA origami (RNA-OG), a nucleic acid nanostructure that is programmed to form different sizes and shapes. Our designed RNA-OG can incorporate various biomolecules and has intrinsic adjuvant activity by acting as a toll-like receptor 3 agonist. We previously showed that the RNA-OG functions as an adjuvanted, carrier-free vaccine platform to assemble peptides. Although effective, only a fixed number of peptides (13) could be covalently linked to each RNA-OG. Methods: Here, we developed a simple physical assembly strategy to attach polylysine-linked neopeptides onto RNA-OG so that the number of peptides per RNA-OG could be readily tuned and tested for their immunogenicity. Results: Although the vaccines with a high number of peptides, i.e., 100–200 peptides/RNA-OG, led to greater peptide presentation by bone marrow-derived dendritic cells, they failed to mount effective CD8⁺ T cell responses against engrafted tumor cells, probably owing to an induction of early T cell exhaustion. Interestingly, the same vaccine format with a low number of peptides, i.e., 10–15 peptides/RNA-OG, enhanced CD8⁺ T cell responses without provoking T cell exhaustion in tumor-bearing mice, leading to strong protective anti-tumor immunity. In comparison, the covalently assembled RNA-OG-peptide vaccine, having a similarly low peptide dosage, offered the highest therapeutic efficacy. Thus, our RNA-OG nanostructure provides a simple and tunable platform for peptide loading to optimize vaccine efficacy. Conclusions: Our findings have significant implications for peptide vaccine design regarding peptide dosages and structural stability of RNA-OG complexed with peptides, which could guide the development of more effective peptide vaccines for cancer immunotherapy. Full article

GBM is the most common and aggressive primary brain tumor in adults, characterized by low survival rates, high recurrence, and resistance to conventional therapies. Traditional diagnostic and therapeutic methods remain limited due to the difficulty in permeating the blood–brain barrier (BBB), diffuse tumor cell infiltration, and tumor heterogeneity. In recent years, nano-based technologies have emerged as innovative approaches for the detection and treatment of GBM. A wide variety of nanocarriers, including dendrimers, liposomes, metallic nanoparticles, carbon nanotubes, carbon dots, extracellular vesicles, and many more demonstrate the ability to cross the BBB, precisely deliver therapeutic agents, and enhance the effects of radiotherapy and immunotherapy. Surface functionalization, peptide modification, and cell membrane coating improve the targeting capabilities of nanostructures toward GBM cells and enable the exploitation of their photothermal, magnetic, and optical properties. Furthermore, the development of miRNA nanosponge systems offers the simultaneous inhibition of multiple tumor growth mechanisms and the modulation of the immunosuppressive tumor microenvironment. This article presents current advancements in nanotechnology for GBM, with a particular focus on the characteristics and advantages of specific groups of nanoparticles, including their role in radiosensitization. Full article