The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein plays an essential role in the cisplatin (CDDP)-induced generation of reactive oxygen species (ROS). In this study, we evaluated the suitability of ultrasound-mediated lysozyme microbubble (USMB) cavitation to enhance NOX4 siRNA transfection in vitro and ex vivo. Lysozyme-shelled microbubbles (LyzMBs) were constructed and designed for siNOX4 loading as siNOX4/LyzMBs. We investigated different siNOX4-based cell transfection approaches, including naked siNOX4, LyzMB-mixed siNOX4, and siNOX4-loaded LyzMBs, and compared their silencing effects in CDDP-treated HEI-OC1 cells and mouse organ of Corti explants. Transfection efficiencies were evaluated by quantifying the cellular uptake of cyanine 3 (Cy3) fluorescein-labeled siRNA. In vitro experiments showed that the high transfection efficacy (48.18%) of siNOX4 to HEI-OC1 cells mediated by US and siNOX4-loaded LyzMBs significantly inhibited CDDP-induced ROS generation to almost the basal level. The ex vivo CDDP-treated organ of Corti explants of mice showed an even more robust silencing effect of the NOX4 gene in the siNOX4/LyzMB groups treated with US sonication than without US sonication, with a marked abolition of CDDP-induced ROS generation and cytotoxicity. Loading of siNOX4 on LyzMBs can stabilize siNOX4 and prevent its degradation, thereby enhancing the transfection and silencing effects when combined with US sonication. This USMB-derived therapy modality for alleviating CDDP-induced ototoxicity may be suitable for future clinical applications.

Targeting genes using siRNA shows promise as an approach to alleviate symptoms of diabetic neuropathy. It focuses on neuropathies and distal symmetric polyneuropathy (DSPN) to explore the potential use of small interfering RNA (siRNA) as a treatment for diabetic neuropathy. Timely identification and management of neuropathy play a critical role in mitigating potential complications. RNAi success depends on understanding factors affecting small interfering RNA (siRNA) functionality and specificity. These include sequence space restrictions, structural and sequence features, mechanisms for nonspecific gene modulation, and chemical modifications. Addressing these factors enhances siRNA performance for efficient gene silencing and confidence in RNAi-mediated genomic studies. Diabetic retinopathy, particularly in South Asian, African, Latin American, and indigenous populations, is a significant concern due to its association with diabetes. Ethnicity plays a crucial role in its development and progression. Despite declining rates in the US, global trends remain concerning, and further research is needed to understand regional differences and reinforce ethnicity-based screening and treatment protocols. In this regard, siRNA emerges as a valuable instrument for early intervention strategies. While presenting promising therapeutic applications, siRNA utilization encounters challenges within insect pest control contexts, thereby providing insights into enhancing its delivery mechanisms for neuropathy treatment purposes. Recent advancements in delivery modalities, such as nanoparticles, allow for the controlled release of siRNA. More investigation is necessary to grasp the safety and efficacy of siRNA technology fully. It holds promise in transforming the treatment of diabetic neuropathy by honing in on particular genes and tackling issues such as inflammation and oxidative stress. Continuous advancements in delivery techniques have the potential to enhance patient results significantly. SiRNA targets genes in diabetic neuropathy, curbing nerve damage and pain and potentially preventing or delaying the condition. Customized treatments based on genetic variations hold promise for symptom management and enhancing quality of life.

Epithelial ovarian cancer (EOC) is associated with a dismal prognosis due to development of resistance to chemotherapy and metastasis in the peritoneal cavity and distant organs. In order to identify new targets and treatment modalities we searched the literature for up- and and down-regulated circRNAs with efficacy in preclinical EOC-related in vivo systems. Our search yielded circRNAs falling into the following categories: cisplatin and paclitaxel resistance, transmembrane receptors, secreted factors, transcription factors, RNA splicing and processing factors, RAS pathway-related components, proteolysis and cell-cycle regulation, signaling-related proteins, and circRNAs regulating proteins in additional categories. These findings can be potentially translated by validation and manipulation of the corresponding targets, inhibition of circRNAs with antisense oligonucleotides (ASO), small interfering RNAs (siRNA) or small hairpin RNA (shRNA) or by reconstituting their activity.

RNA interference (RNAi) is a biological process that evolved to protect eukaryotic organisms from foreign genes delivered by viruses. This process has been adapted as a powerful tool to treat numerous diseases through the delivery of small-interfering RNAs (siRNAs) to target cells to alter aberrant gene expression.Antibody-oligonucleotide conjugates (AOCs) are monoclonal antibodies with complexed siRNA or antisense oligonucleotides (ASOs) that have emerged to address some of the challenges faced by naked or chemically conjugated siRNA, which include rapid clearance from systemic circulation and lack of selective delivery of siRNA to target cells.It is essential to characterize the ADME properties of an AOC during development to optimize distribution to target tissues, to minimize the impact of biotransformation on exposure, and to characterize the PK/PD relationship to guide translation. However, owing to the complexity of AOC structure, this presents significant bioanalytical challenges, and multiple bioanalytical measurements are required to investigate the pharmacokinetics and biotransformation of the antibody, linker, and siRNA payload.In this paper, we describe an analytical workflow that details in vivo characterization of AOCs through measurement of their distinct molecular components to provide the basis for greater understanding of their ADME properties. Although the approaches herein can be applied to in vitro characterization of AOCs, this paper will focus on in vivo applications. This workflow relies on high-resolution mass spectrometry as the principal means of detection and leverages chromatographic, affinity-based, and enzymatic sample preparation steps.

Polyethylene glycol (PEG)-modified (PEGylated) cationic liposomes are frequently used as delivery vehicles for small interfering RNA (siRNA)-based drugs because of their ability to encapsulate/complex with siRNA and prolong the circulation half-life in vivo. Nevertheless, we have reported that subsequent intravenous (IV) injections of siRNA complexed with PEGylated cationic liposomes (PLpx) induces the production of anti-PEG immunoglobulin M (IgM), which accelerates the blood clearance of subsequent doses of PLpx and other PEGylated products. In this study, it is interesting that splenectomy (removal of spleen) did not prevent anti-PEG IgM induction by IV injection of PLpx. This indicates that B cells other than the splenic version are involved in anti-PEG IgM production under these conditions. In vitro and in vivo studies have shown that peritoneal cells also secrete anti-PEG IgM in response to the administration of PLpx. Interleukin-6 (IL-6) is a glycoprotein that is secreted by peritoneal immune cells and has been detected in response to the in vivo administration of PLpx. These observations indicate that IV injection of PLpx stimulates the proliferation/differentiation of peritoneal PEG-specific B cells into plasma cells via IL-6 induction, which results in the production of anti-PEG IgM from the peritoneal cavity of mice. Our results suggest the mutual contribution of peritoneal B cells as a potent anti-PEG immune response against PLpx.

The prevalence of metabolic disorders is increasing exponentially and has recently reached epidemic levels. Over the decades, a large number of therapeutic options have been proposed to manage these diseases but still show several limitations. In this circumstance, RNA therapeutics have rapidly emerged as a new hope for patients with metabolic diseases. 57 years have elapsed from the discovery of mRNA, a large number of RNA-based drug candidates have been evaluated for their therapeutic effectiveness and clinical safety under clinical studies. To date, there are seven RNA drugs for treating metabolic disorders receiving official approval and entering the global market. Their targets include hereditary transthyretin-mediated amyloidosis (hATTR), familial chylomicronemia syndrome, acute hepatic porphyria, primary hyperoxaluria type 1 and hypercholesterolemia, which are all related to liver proteins. All of these seven RNA drugs are antisense oligonucleotides (ASO) and small interfering RNA (siRNA). These two types of treatment are both based on oligonucleotides complementary to target RNA through Watson-Crick base-pairing, but their mechanisms of action include different nucleases. Such treatments show greatest potential among all types of RNA therapeutics due to consecutive achievements in chemical modifications. Another method, mRNA therapeutics also promise a brighter future for patients with a handful of drug candidates currently under development.

In recent years, nucleic acids have emerged as powerful biomaterials, revolutionizing the field of biomedicine. This review explores the multifaceted applications of nucleic acids, focusing on their pivotal role in various biomedical applications. Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), possess unique properties such as molecular recognition ability, programmability, and ease of synthesis, making them versatile tools in biosensing and for gene regulation, drug delivery, and targeted therapy. Their compatibility with chemical modifications enhances their binding affinity and resistance to degradation, elevating their effectiveness in targeted applications. Additionally, nucleic acids have found utility as self-assembling building blocks, leading to the creation of nanostructures whose high order underpins their enhanced biological stability and affects the cellular uptake efficiency. Furthermore, this review delves into the significant role of oligonucleotides (ODNs) as indispensable tools for biological studies and biomarker discovery. ODNs, short sequences of nucleic acids, have been instrumental in unraveling complex biological mechanisms. They serve as probes for studying gene expression, protein interactions, and cellular pathways, providing invaluable insights into fundamental biological processes. By examining the synergistic interplay between nucleic acids as powerful biomaterials and ODNs as indispensable tools for biological studies and biomarkers, this review highlights the transformative impact of these molecules on biomedical research. Their versatile applications not only deepen our understanding of biological systems but also are the driving force for innovation in diagnostics and therapeutics, ultimately advancing the field of biomedicine.

RNA interference (RNAi) is a conserved molecular mechanism that plays a critical role in post-transcriptional gene silencing across diverse organisms. This review delves into the role of RNAi in plant functional genomics and its applications in crop improvement, highlighting its mechanistic insights and practical implications. The review begins with the foundational discovery of RNAi\'s mechanism, tracing its origins from petunias to its widespread presence in various organisms. Various classes of regulatory non-coding small RNAs, including siRNAs, miRNAs, and phasiRNAs, have been uncovered, expanding the scope of RNAi-mediated gene regulation beyond conventional understanding. These RNA classes participate in intricate post-transcriptional and epigenetic processes that influence gene expression. In the context of crop enhancement, RNAi has emerged as a powerful tool for understanding gene functions. It has proven effective in deciphering gene roles related to stress resistance, metabolic pathways, and more. Additionally, RNAi-based approaches hold promise for integrated pest management and sustainable agriculture, contributing to global efforts in food security. This review discusses RNAi\'s diverse applications, such as modifying plant architecture, extending shelf life, and enhancing nutritional content in crops. The challenges and future prospects of RNAi technology, including delivery methods and biosafety concerns, are also explored. The global landscape of RNAi research is highlighted, with significant contributions from regions such as China, Europe, and North America. In conclusion, RNAi remains a versatile and pivotal tool in modern plant research, offering novel avenues for understanding gene functions and improving crop traits. Its integration with other biotechnological approaches such as gene editing holds the potential to shape the future of agriculture and sustainable food production.

A number of functional nucleic acids, including plasmid DNA (pDNA) and small interfering RNA (siRNA), have been attracting increasing attention as new therapeutic modalities worldwide. Dry pDNA and siRNA powder formulations for inhalation are considered practical in clinical applications for respiratory diseases. However, physical stresses in the powder-forming process may destabilize nucleic acids, particularly when vectors with stabilizing effects are not used. We herein compare the stability of naked pDNA and siRNA through various physical treatments and two powder-forming processes. The structural and functional integrities of pDNA were markedly reduced via sonication, heating, and atomization, whereas those of siRNA were preserved throughout all of the physical treatments investigated. Spray-dried and spray-freeze-dried powders of siRNA maintained their structural and functional integrities, whereas those of pDNA did not. These results demonstrate that siRNA is more suitable for powder formation in the naked state than pDNA due to its higher stability under physical treatments. Furthermore, a spray-freeze-dried powder with a high content of naked siRNA (12% of the powder) was successfully produced that preserved its structural and functional integrities, achieving high aerosol performance with a fine particle fraction of approximately 40%.

Indirect response (IDR) and turnover with inactivation (TI) comprise two arrays of mechanism-based pharmacodynamic (PD) models widely used to describe delayed drug effects. IDR Model-IV (stimulation of response loss) and TI (irreversible loss) have been described with discerning \"signature\" profiles; classical IDR-IV response-time profiles display slow declines where peak response shifts later with increasing dose, whereas TI profiles feature steep response declines with earlier-shifting nadirs. Herein, we demonstrate mathematical convergence of IDR-IV and TI models upon implementation with identical linear versus nonlinear pharmacologic effect terms. Time of peak response in IDR-IV can in fact shift earlier or later depending on PK or PD parameters (e.g., kel, Smax) and effect type. A generalized dynamic model linking mRNA and protein turnover is proposed. Applicability of IDR-IV and TI, with either linear or nonlinear terms acting on degradation/catabolism/loss of response, is demonstrated through model-fitting PK-PD effects of three proteolysis-targeting chimeras (PROTACs) and two ligand-conjugated small interfering RNAs (siRNA). This work clarifies mathematical properties, convergence, and expected responses of IDR-IV and TI, demonstrates their applicability for targeted gene-silencing and protein-degrading agents, and illustrates how well-designed in vivo studies covering broad dose ranges with richly sampled time-points can influence PK-PD model structure and parameter resolution.