Application Of siRNA Research Articles

The Role of CD40 in Allergic Rhinitis and Airway Remodelling.

Background Allergic rhinitis (AR) affects millions of people and is lack of effective treatment. CD40 is an important costimulatory molecule in immunity. However, few studies have focused on the role of CD40 in AR. Methods In this study, we built mouse model of chronic AR. The mice were divided into the AR, control, intravenous CD40 siRNA, and nasal CD40 siRNA groups (n = 6 each). We detected OVA-sIgE, IL-4, IL-5, IL-13, IL-10, IFN-γ, and TGF-β levels in serum and supernatant by ELISA, CD40+ splenic DCs, and Foxp3+ Tregs by flow cytometry and CD40 mRNA by RT2-PCR. We also used PAS and MT stains to assess tissue remodelling. Results (1) The OVA-sIgE, IL-4, IL-5, and IL-13 levels in the serum or supernatant of nasal septal membrane of AR mice were significantly higher than control. After treated with CD40 siRNA, those indicators were significantly decreased. The IFN-γ, IL-10, and TGF-β levels in AR mice were significantly lower than that in control and were increased by administration of CD40 siRNA. (2) AR mice had significantly fewer Foxp3+ Tregs in the spleen than control mice. After treated with CD40 siRNA, AR mice had significantly more Foxp3+ Tregs. (3) AR mice exhibited a significantly higher CD40 mRNA levels than control. Administration of CD40 siRNA significantly reduced the CD40 mRNA level. (4) The AR mice showed significantly greater collagen deposition than the control in MT staining. Applications of CD40 siRNA significantly reduced the collagen deposition in AR mice. Conclusion CD40 siRNA therapy shows promise for chronic AR as it significantly attenuated allergic symptoms and Th2-related inflammation and upregulated Foxp3+ Tregs. CD40 plays a role in tissue remodelling in AR, which can be inhibited by CD40 siRNA application.

Leukemia inhibitory factor regulates Schwann cell proliferation and migration and affects peripheral nerve regeneration

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that stimulates neuronal development and survival. Our previous study has demonstrated that LIF mRNA is dysregulated in the peripheral nerve segments after nerve injury. Here, we show that LIF protein is abundantly expressed in Schwann cells after rat sciatic nerve injury. Functionally, suppressed or elevated LIF increases or decreases the proliferation rate and migration ability of Schwann cells, respectively. Morphological observations demonstrate that in vivo application of siRNA against LIF after peripheral nerve injury promotes Schwann cell migration and proliferation, axon elongation, and myelin formation. Electrophysiological and behavior assessments disclose that knockdown of LIF benefits the function recovery of injured peripheral nerves. Differentially expressed LIF affects the metabolism of Schwann cells and negatively regulates ERFE (Erythroferrone). Collectively, our observations reveal the essential roles for LIF in regulating the proliferation and migration of Schwann cells and the regeneration of injured peripheral nerves, discover ERFE as a downstream effector of LIF, and extend our understanding of the molecular mechanisms underlying peripheral nerve regeneration.

Preparation Optimization of Bovine Serum Albumin Nanoparticles and Its Application for siRNA Delivery.

BackgroundsiRNA brings hope for cancer therapy. However, there are many obstacles for application of siRNA in clinical. Because of the excellent biocompatibility, non-toxicity and non-immunogenicity of bovine serum albumin (BSA), BSA-based nanoparticles have been widely designed as a drug carrier system.MethodsThe optimal formula for BSA NPs preparation was investigated by central composite design response surface methodology (CCD-RSM), BSA-based survivin-siRNA delivery system (BSA NPs/siRNA) was characterized by dynamic light scattering, atomic force microscope, transmission electron microscope and Bradford method. The in vitro anti-tumor effect and mechanism of BSA NPs were investigated by confocal microscopic imaging, MTT assay, RT-qPCR and ELISA analysis. Moreover, the anti-tumor effect, distribution and biosafety of BSA NPs were studied in vivo.ResultsThe optimal formula for BSA NPs was settled to be 20 mg/mL for BSA concentration, 9 for pH value, 136% for crosslinking degree and 1.6 mL/min for speed of ethanol addition. BSA NPs/siRNA could remain stable at 4°C for 4 weeks and protect siRNA from degradation by RNase A. Besides, BSA NPs/siRNA could maintain a sustained release of siRNA and promote the uptake of siRNA significantly. The survivin-mRNA level and the survivin-protein level were decreased by 55% ± 1.6% and 54% ± 1.6% separately. The in vivo tumor inhibition results suggested that the tumor inhibition rate of BSA NPs/siRNA-treated group was 54% ± 12% and was similar with that of DOX-treated group (57% ± 9.2%, P > 0.05). The biosafety results confirmed that BSA NPs/siRNA could not induce significant damages to the main organs and blood in vivo.ConclusionThese results demonstrated that CCD-RSM was an effective tool for preparation analysis, and the BSA NPs/siRNA was a promising system for siRNA-based gene therapy.

Estrogen and serotonin enhance stress-induced visceral hypersensitivity in female rats by up-regulating brain-derived neurotrophic factor in spinal cord.

We previously reported that female offspring of dams subjected to chronic prenatal stress (CPS) develop enhanced visceral hypersensitivity (VHS) following exposure to chronic stress in adult life that is mediated by up-regulation of spinal cord BDNF. The aims of this study were to examine the roles of estrogen receptor alpha (ERα) and an increase in spinal serotonin signaling in promoting this enhanced VHS in female rats and up-regulation of spinal cord BDNF transcription. Pregnant dams were exposed to chronic stress from E11 until delivery. At 8weeks, a chronic adult stress (CAS) protocol was applied for nine days. Ovariectomy before CAS or treatment with letrozole before and during CAS significantly prevented the development of enhanced VHS in female CPS+CAS rats. Intrathecal application of ERα siRNA significantly reduced VHS, decreased lumbar-sacral spinal cord expression of both ERα and BDNF, and reversed pro-transcriptional epigenetic modifications at BDNF promoter lX. Cerebrospinal fluid serotonin levels and 5HT3A receptor expression in the LS spinal cord were both significantly increased in female CPS+CAS rats. During CAS, intrathecal infusion of alosetron significantly decreased VHS, reduced BDNF and ERα expression in the LS spinal cord, and attenuated RNA pol II and ERα binding to the BNDF core promoter IX. Serotonin-mediated activation of 5HT3A receptors in the spinal cord drives the development of enhanced female-specific VHS in our two hit CPS+CAS through up-regulation of spinal cord ERα.

Tetrahedral Framework Nucleic Acids Loaded with Aptamer AS1411 for siRNA Delivery and Gene Silencing in Malignant Melanoma.

siRNA is found to effectively knock down the target gene in cells, which is considered a promising strategy for gene therapy. However, the application of siRNA is limited due to its low efficiency of the cellular uptake. Tetrahedral framework nucleic acids (tFNAs) are synthesized by four single-stranded DNAs and show multiple biological functions in recent studies, especially suitable for drug delivery. More than 60% of malignant melanomas are associated with Braf gene mutation, an attractive therapeutic target for RNA interference. In this study, we modified anti-Braf siRNA (siBraf) with tFNAs to downregulate the target gene. Meanwhile, we directly incorporated AS1411 (a DNA aptamer) to our nanostructure, which assists tFNAs to improve the cellular uptake efficacy of siBraf significantly. The results indicated that tFNAs-AS1411-siBraf exhibited more potent activity to cleave Braf mRNA than free siBraf. This study may provide a new idea for the combination therapy of siRNA and aptamers via DNA nanomaterials to achieve gene silencing.

PLGA/PCADK composite microspheres containing hyaluronic acid–chitosan siRNA nanoparticles: A rational design for rheumatoid arthritis therapy

Myeloid cell leukemia-1 (Mcl-1), a member of the Bcl-2 anti-apoptotic family, is overexpressed in the synovial macrophages of patients with rheumatoid arthritis (RA). Small interfering RNA (siRNA) Mcl-1 can induce macrophage apoptosis in the joints and is a potential therapeutic target of RA. Nevertheless, the application of siRNA is limited owing to its instability and susceptibility to degradation in vivo. To address these shortcomings, we developed composite microspheres (MPs) loaded with hyaluronic acid (HA)–chitosan (CS) nanoparticles (NPs). First, we synthesized HA–CS/siRNA NPs (HCNPs) using ionotropic gelation process. Then, HCNPs, as an internal aqueous phase, were loaded into poly (D, L-lactide-co-glycolide) (PLGA) and poly (cyclohexane-1,4-diyl acetone dimethylene ketal) (PCADK) MPs using the double emulsion method. The NPs-in-MPs (NiMPs) composite system provided sustained release of NPs, protected siRNA against nuclease degradation in the serum, and could readily cross the cellular membrane. In addition, we evaluated the advantages of NiMPs in an adjuvant-induced arthritis rat model. Our experimental results demonstrate that NiMPs have greater pharmacodynamic effects than common MPs. Meanwhile, compared with HCNPs, NiMPs reduced the frequency of drug administration. Therefore, NiMPs are a promising and novel siRNA delivery vehicle for RA therapy.

Thymoquinone Prevents Dopaminergic Neurodegeneration by Attenuating Oxidative Stress Via the Nrf2/ARE Pathway

Studies have indicated that oxidative stress plays a crucial role in the development of Parkinson’s disease (PD) and other neurodegenerative conditions. Research has also revealed that nuclear factor erythroid 2-related factor 2 (Nrf2) triggers the expression of antioxidant genes via a series of antioxidant response elements (AREs), thus preventing oxidative stress. Thymoquinone (TQ) is the bioactive component of Nigella sativa, a medicinal plant that exhibits antioxidant and neuroprotective effects. In the present study we examined whether TQ alleviates in vivo and in vitro neurodegeneration induced by 1-methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by acting as an activator of the Nrf2/ARE cascade. We showed that TQ significantly reduced MPP+-mediated cell death and apoptosis. Moreover, TQ significantly elevated the nuclear translocation of Nrf2 and significantly increased the subsequent expression of antioxidative genes such as Heme oxygenase 1 (HO-1), quinone oxidoreductase (NQO1) and Glutathione-S-Transferase (GST). The application of siRNA to silence Nrf2 led to an abolishment in the protective effects of TQ. We also found that the intraperitoneal injection of TQ into a rodent model of PD ameliorated oxidative stress and effectively mitigated nigrostriatal dopaminergic degeneration by activating the Nrf2-ARE pathway. However, these effects were inhibited by the injection of a lentivirus wrapped Nrf2 siRNA (siNrf2). Collectively, these findings suggest that TQ alleviates progressive dopaminergic neuropathology by activating the Nrf2/ARE signaling cascade and by attenuating oxidative stress, thus demonstrating that TQ is a potential novel drug candidate for the treatment of PD.

CRGD peptide-conjugated polyethylenimine-based lipid nanoparticle for intracellular delivery of siRNA in hepatocarcinoma therapy

The effective delivery system plays an important role in the application of siRNA in the antitumor study. However, until now, researches on the delivery systems targeting hepatocarcinoma cells are still being explored. Here we designed and prepared a novel siRNA delivery system, cRGD-PSH-NP, which was based on a modified polyethyleneimine (PSH) and DSPE-PEG2000-cRGD. cRGD-PSH-NP loaded with survivin siRNA (cRGD-PSH-NP/S) was composed of egg phosphatidylcholine, cationic PSH, PEGylated lipids, survivin siRNA, and cRGD peptide as a targeting ligand. The formulations of cRGD-PSH-NP/S were optimized and characterized. In vitro investigations showed excellent gene silencing and antitumor activity compared with the unmodified nanoparticles in HepG2 cells. In vivo antitumor efficacy of cRGD-PSH-NP/S exhibited potent tumor inhibition (74.71%) in HepG2-bearing nude mice without inducing toxicity. These data suggested further research of cRGD-PSH-NP/S in hepatocarcinoma therapy.

A size-shrinkable matrix metallopeptidase-2-sensitive delivery nanosystem improves the penetration of human programmed death-ligand 1 siRNA into lung-tumor spheroids

Given the maturation of small-interfering RNA (siRNA) techniques with nanotechnology, and because overexpression of human programmed death-ligand 1 (PD-L1) is crucial for T cell inactivation and immunosuppression of the tumor microenvironment, application of siRNA–PD-L1 has demonstrated positive progress in preclinical studies; however, the limited penetration of this compound into solid tumors remains a challenge. To decrease PD-L1 expression and increase the penetration efficacy of solid tumors, we synthesized a novel tumor-microenvironment-sensitive delivery polymer by conjugating hyaluronic acid (HA) to polyethyleneimine (PEI), with a matrix metalloproteinase-2 (MMP-2)-sensitive peptide acting as the linker (HA-P-PEI), for use in delivery of PD-L1–siRNA. Concurrent synthesis of a linker-less HA-PEI compound allowed confirmation that negatively charged siRNA can be complexed onto the positively charged HA-PEI and HA-P-PEI compounds to form nanoparticles with the same particle size and uniform distribution with serum stability. We found that the size of the HA-P-PEI/siRNA nanoparticles decreased to <10 nm upon addition of MMP-2, and that H1975 cells overexpressing CD44, PD-L1, and MMP-2 aided confirmation of the delivery efficacy of the HA-P-PEI/siRNA nanocomplexes. Additionally, the use of HA-P-PEI caused less cytotoxicity than PEI alone, demonstrating its high cellular uptake. Moreover, pretreatment with MMP-2 increased nanocomplex tumor permeability, and western blot showed that HA-P-PEI/PD-L1–siRNA efficiently downregulated the PD-L1 expression in H1975 cells. These results demonstrated a novel approach for siRNA delivery and tumor penetration for future clinical applications in cancer treatment.

Sixteen Different Types of Lipid-Conjugated siRNAs Containing Saturated and Unsaturated Fatty Acids and Exhibiting Enhanced RNAi Potency.

SiRNAs are strong gene-silencing agents that function in a target sequence-specific manner. Although siRNAs might one day be used in therapy for intractable diseases such as cancers, a number of problems with siRNAs must first be overcome. In this study, we developed 16 different types of lipid-conjugated siRNAs (lipid-siRNAs) that could effectively inhibit the expression of target genes. We determined the hybridization properties, cellular uptake efficacies, and RNAi potencies of the resulting lipid-siRNAs. The lipid-siRNAs exhibited a mild interaction with Lipofectamine RNAiMAX (LFRNAi) as a transfection reagent, and a high membrane permeability was observed in all lipid-siRNAs-LFRNAi complexes; the conjugate siRNAs composed of 16-18 carbon chains as fatty acids showed an especially good cellular uptake efficacy. The in vitro RNAi effect of lipid-siRNAs targeted to a β-catenin gene exhibited a strong RNAi potency compared with those of unmodified siRNAs. In particular, the conjugate siRNAs composed of 16-18 carbon chains as fatty acids showed excellent RNAi potencies with prolonged effectivities. Interestingly, the RNAi potencies of conjugate siRNAs containing 18 carbon chains with a trans-form (elaidic acid and trans-vaccenic acid) were inferior to those of the carbon chains with a cis-form (oleic acid and cis-vaccenic acid). These lipid-siRNAs can solve the many problems hindering the clinical application of siRNAs.

Advances in Targeting Cancer-Associated Genes by Designed siRNA in Prostate Cancer.

Simple SummaryDespite great advancements in early detection and therapeutic strategies, the 5-year survival rate for patients with metastatic prostate cancer remains low (i.e., ~30%). Targeting prostate cancer-associated genes has emerged as a promising treatment for this devastating disease. This review summarizes recent findings in silencing genes that are involved in prostate cancer pathogenesis. Moreover, novel nanotechnology-based platforms for effective delivery of therapeutic RNAs to prostate cancer cells have been discussed. Information provided in this review will benefit both researchers and clinicians to design and develop novel therapeutic approaches for patients suffering from prostate cancer.Short interfering RNAs (siRNAs) have provided novel insights into the field of cancer treatment in light of their ability to specifically target and silence cancer-associated genes. In recent years, numerous studies focus on determining genes that actively participate in tumor formation, invasion, and metastasis in order to establish new targets for cancer treatment. In spite of great advances in designing various siRNAs with diverse targets, efficient delivery of siRNAs to cancer cells is still the main challenge in siRNA-mediated cancer treatment. Recent advancements in the field of nanotechnology and nanomedicine hold great promise to meet this challenge. This review focuses on recent findings in cancer-associated genes and the application of siRNAs to successfully silence them in prostate cancer, as well as recent progress for effectual delivery of siRNAs to cancer cells.

Polyethylenimine-coated gold-magnetic nanoparticles for ADAM10 siRNA delivery in prostate cancer cells

For an effective medical application of therapeutic siRNA, a safe and an efficient delivery system are required. Herein, magnetic nanoparticles (MNPs) have been successfully used as siRNA delivery vehicles. Firstly, MNPs were coated with gold (Au) nanoparticles and then capped with PEI. To improve the biocompatibility of nanoparticles, hyaluronic acid (HA) was coated onto the surface of PEI-Au/Fe nanoparticles. The prepared HA-PEI-Au/Fe3O4 nanoparticles were characterized and found to be uniform and well segregated in TEM analysis. FTIR analysis confirmed that HA was successfully conjugated to PEI. The polymer content in these nanoparticles was relatively higher than PEG coated nanoparticles. Cell viability assay demonstrated that the nanoparticles were relatively biocompatible in nature. ADAM10 siRNA was loaded into the HA-PEI-Au/Fe3O4 nanoparticles and cytotoxicity to prostate cancer (PC3) cells was analyzed. The results indicate that ADAM10 siRNA loaded HA-PEI-Au/Fe3O4 suppress the PC3 cells growth in vitro. Clearly, it could be confirmed that HA-PEI coated Au/Fe3O4 nanoparticles with higher biocompatibility appear to be suitable for intracellular siRNA delivery.

IGF-1R stimulation alters microglial polarization via TLR4/NF-κB pathway after cerebral hemorrhage in mice

Intracerebral hemorrhage (ICH) is a common neurological disease, causing severe disability and even deaths. Stem cell transplantation has been increasingly used in stroke therapy, and neural stem cells (NSCs) are a good source for stem cell transplantation. However, the regulatory mechanism of NSCs remains unclear. In this study, we examined the impact of insulin-like growth factor-1 (IGF-1) secreted by NSCs on microglial polarization following ICH in adult C57BL/6 mice. Mouse models of ICH were established by collagenase injection. ICH mice received NSC transplantation 1 h after model establishment. Firstly, the changes of microglial polarization in cerebral tissues of ICH mice were detected by immunofluorescence and ELISA. Secondly, the molecular mechanism underlying the microglial polarization was evaluated repeatedly with the application of IGF-1R siRNA and IGF-1R-mediated inhibition. We assessed the brain water content and behavioral deficits of ICH mice 12, 24, 48, and 72 h after surgery. The survival of neurons in the brain was examined using Nissl staining and TUNEL staining at 72 h. The TLR4/NF-κB pathway implicated in ICH-induced inflammation was profiled by Western blot analysis and immunohistochemistry. Finally, the changes in microglia after ICH in mice were re-examined under different doses of rhIGF-1. In summary, NSC transplantation changed microglial polarization in ICH mice. IGF-1R inhibition and knockdown reversed the therapeutic effect of NSCs, and rhIGF-1 had an anti-inflammatory effect. The results of this study suggest that IGF-1R stimulation is a potential target for stem cell-based treatment of cerebral hemorrhage and may attenuate inflammatory response in the brain after bleeding.

Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair.

Homologous recombination (HR) is often used to achieve targeted gene integration because of its higher precision and operability compared with microhomology-mediated end-joining (MMEJ) or non-homologous end-joining (NHEJ). It appears to be inefficient for gene integration in animal cells and embryos due to occurring only during cell division. Here we developed genome-wide high-throughput screening and a subsequently paired crRNA library screening to search for genes suppressing homology-directed repair (HDR). We found that, in the reporter system, HDR cells with knockdown of SHROOM1 were enriched as much as 4.7-fold than those with control. Down regulating SHROOM1 significantly promoted gene integration in human and mouse cells after cleavage by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9), regardless of the donor types. The knock-in efficiency of mouse embryos could also be doubled by the application of SHROOM1 siRNA during micro-injection. The increased HDR efficiency of SHROOM1 deletion in HEK293T cells could be counteracted by YU238259, an HDR inhibitor, but not by an NHEJ inhibitor. These results indicated that SHROOM1 was an HDR-suppressed gene and that the SHROOM1 knockdown strategy may be useful for a variety of applications, including gene editing to generate cell lines and animal models for studying gene function and human diseases.

Suppression of Netrin-1 attenuates angiotension II-induced cardiac remodeling through the PKC/MAPK signaling pathway

BackgroundMyocardial remodeling caused by angiotensin II (Ang II) is essential for the pathological process of heart failure. Netrin-1, which is an axonal guidance cue, has been shown to be involved in the inflammatory response, tumorigenesis, and angiogenesis in non-neuronal tissues. However, the role of Netrin-1 in cardiac remodeling has not been fully elucidated. MethodsThe rat cardiomyocyte cell line H9c2 and primary neonatal rat cardiomyocytes were treated with Ang II. Cells were transfected with siRNA to silence Netrin-1 expression. Real-time polymerase chain reaction and Western blot analysis were used to detect the markers for fibrosis, apoptosis, and hypertrophy in cardiomyocytes. An Annexin V-EGFP/PI cell apoptosis detection kit was used to measure the level of apoptosis caused by angiotensin II. ResultsWe found that Netrin-1 expression was upregulated in the H9c2 cells and the neonatal rat cardiomyocytes stimulated by Ang II. The increased Netrin-1 expression was decreased by valsartan to block AT1R. Importantly, the application of Netrin-1 siRNA significantly alleviated the degrees of myocardial hypertrophy, fibrosis (reflected by Myhc, collagen I, and TGF-β) and apoptosis (reflected by the level of Caspase 3, Bax, and Bcl-2) induced by Ang II. In addition, the silencing of Netrin-1 substantially decreased the phosphorylation of PKCα, JNK, and P38. We treated H9c2 cells with LY317615, SP600125, and SB203580, inhibitors of PKCα, JNK, and P38, respectively, thereby resulting in a substantial decrease in hypertrophy, fibrosis, and apoptosis. ConclusionsAng II produces cardiac hypertrophy, fibrosis, and apoptosis through the upregulation of Netrin-1 and the activation of the AT1R/PKCα/MAPK (JNK, P38) pathway. Suppression of Netrin-1 can relieve Ang II-induced cardiac remodeling via inhibition of the PKCα/MAPK (JNK and P38) signaling pathway. Thus, Netrin-1 may be a novel therapeutic target for Ang II-mediated cardiac remodeling.

Progress in the tumor environment (TME)-responsive nanocarriers for &amp;lt;italic&amp;gt;in vivo&amp;lt;/italic&amp;gt; siRNA delivery

Since the discovery of RNA interference (RNAi), RNAi technology has rapidly developed into an efficient tool for post-transcriptional gene silencing, which has been widely used for gene function analysis and clinical or preclinical treatment of various diseases (e.g., malignant tumor, Hepatitis B, obesity, andmultiple neuropathy). Small interfering RNA (siRNA) is the effector molecule of RNAi technology. As a biological macromolecule with negative charges, naked siRNA has a short half-life in vivo , and is easily attacked by nucleases and filtered by glomeruli. Therefore, the clinical application of naked siRNA is significantly limited. The key challenge for clinical translation of RNAi technology is to construct safe and efficient delivery carriers to improve the siRNA stability in vivo and gene silencing efficacy in target cells. Currently, numerous nanocarriers such as liposomes have been employed for in vivo siRNA delivery, which have achieved favorable therapeutic outcomes in clinical or preclinical disease treatment. It has been demonstrated that solid tumors show specific vascular structures and physiological microenvironment (e.g., weakly acidic environment, hypoxia, over-expression of specific chemicals and enzymes). Based on this fact, researches have recently paid great attention to designing and constructingtumor microenvironment (TME)-responsive nanocarriers to improve the accumulation of siRNA in tumor sites and enhance gene silencing in tumor cells. In this review, we summarize the design principles and functional characteristics of recently developed TME-responsive siRNA delivery nanocarriers, analyze their advantages and disadvantages, and explore the development trend of TME-responsive siRNA delivery nanocarriers in the future.

ROS-Activatable siRNA-Engineered Polyplex for NIR-Triggered Synergistic Cancer Treatment.

Small interfering RNA (siRNA) shows excellent pharmaceutical prospects in treating diverse life-threatening diseases. Photodynamic therapy (PDT) is a clinically employed noninvasive treatment method that can trigger selective damage toward targeted tissue and cells. However, insufficient delivery of siRNA and photosensitizer to cancer cells remarkably hindered the application of siRNA and PDT in the treatment of cancer. In this study, a unique reactive oxygen species (ROS)-activatable polyplex, which consists of the PEGylated cationic polymer, ROS-cleavable linker, photosensitizer Ce6, and RRM2-against siRNA, termed PPTC/siRNA, was engineered. Upon irradiation of near-infrared (NIR) light, the polyplex efficiently generated ROS, which triggered degradation of the ROS-sensitive linker, disassembling the complex, destabilization of the cell membrane, and significantly accelerated cellular entry and endosomal escape of siRNA. Besides achieving effective siRNA internalization and gene silence in cancer cells in vitro, PPTC/siRNA synergistically inhibited tumor growth in both cell line-derived xenograft and patient-derived xenograft hepatocellular carcinoma murine models by repressing the RRM2 expression (reducing cell proliferation) and triggering photodynamic killing (enhancing cell apoptosis). The proposed polyplex also showed ideal safety profiles both in cell line and in animal. It provides a novel strategy for NIR-triggered RNAi and PDT combinational cancer treatment.

Strategies and Approaches for siRNA Delivery

The present review discusses about RNA interference (RNAi) and its significance in gene therapy. The review mainly focuses on small interference RNA (siRNA) as a mediator of RNAi, its therapeutic benefits and various formulation strategies employed to overcome siRNA delivery hurdles. RNAi is a regulatory process which occurs endogenously within the cell wherein short double-stranded RNA (siRNA) effects sequence-specific posttranscriptional gene silencing. Even though siRNA assists researchers with its powerful therapeutic benefits, there are significant hurdles in developing efficient delivery systems for its systemic administration. These are extracellular and intracellular barriers for siRNA delivery. The present review addresses about pros and cons of gene therapy and superior advantages provided by siRNA over plasmid DNA in gene therapy. It also discloses about the discovery, mechanism of action, significance and applications of siRNA based gene therapies, challenges in its delivery and strategies for overcoming delivery hurdles. Furthermore, emphasis is provided on viral and non – viral vector based siRNA delivery and the significance of lipid based siRNA delivery, the lipoplexes over polymer based siRNA delivery - the polyplexes, followed by recent advances in siRNA based technologies directed against variety of diseases.&#x0D; Keywords: Endosomal escape, gene therapy, lipoplexes, polyplexes, siRNA, vectors.

Oxaliplatin-induced neuropathic pain involves HOXA6 via a TET1-dependent demethylation of the SOX10 promoter.

Chemotherapy-induced neuropathic pain is a common dose-limiting side effect of cancer treatment but the underlying mechanisms are largely unknown. Here, we used a whole-genome expression microarray and gene ontology analysis to identify the upregulation of a sequence-specific DNA-binding protein, HOXA6, in the spinal dorsal horn on Day 10 after injection of rats with oxaliplatin. Genetic disruption of HOXA6 with siRNAs alleviated mechanical allodynia after oxaliplatin administration. Reduced representation bisulfite sequencing assays indicated that oxaliplatin decreased the methylation levels of the SOX10 promoter but not of HOXA6. TET1 was also upregulated by oxaliplatin. Genetic disruption of TET1 with siRNA blocked the promoter demethylation of SOX10 and the upregulation of HOXA6 and SOX10. Importantly, inhibition of SOX10 by intrathecal application of SOX10 siRNA ameliorated the mechanical allodynia induced by oxaliplatin and downregulated the expression of HOXA6. Consistently, overexpression of SOX10 through intraspinal injection of AAV-SOX10-EGFP produced mechanical allodynia and upregulated the expression of spinal dorsal horn HOXA6. Moreover, chromatin immunoprecipitation assays demonstrated that oxaliplatin increased the binding of SOX10 to the promoter region of HOXA6. Taken together, our data suggest that HOXA6 upregulation through the TET1-mediated promoter demethylation of SOX10 may contribute to oxaliplatin-induced neuropathic pain.

Amlexanox exerts anti-inflammatory actions by targeting phosphodiesterase 4B in lipopolysaccharide-activated macrophages

Amlexanox, an anti-inflammatory agent, is widely used for treating aphthous ulcers. Recently, amlexanox has received considerable attention because of its efficacy in mitigating metabolic inflammation via directly suppressing IKKε/TBK1. However, because the knockdown of IKKε/TBK1 has no anti-inflammatory effect on lipopolysaccharide (LPS)-primed RAW264.7 cells, the mechanism of amlexanox against classical inflammation is independent of IKKε/TBK1. In this study, we aim to examine the effects of amlexanox on LPS-treated macrophages and in a mouse model of endotoxemia. We found that amlexanox significantly inhibited the production of pro-inflammatory mediators, both in vitro and in vivo, while increased interleukin-10 level in LPS-activated macrophages. Mechanistically, amlexanox down-regulated nuclear factor κB and extracellular signal-regulated kinase/activator protein-1 signaling by elevating intracellular 3′,5′-cyclic adenosine monophosphate (cAMP) level and subsequently activating protein kinase A. Molecular docking along with fluorescence polarization and enzyme inhibition assays revealed that amlexanox bound directly to phosphodiesterase (PDE) 4B to inhibit its activity. The anti-inflammatory effects of amlexanox could be abolished by the application of cAMP antagonist or PDE4B siRNA. In addition to PDE4B, the activities of PDE1C, 3A, and 3B were directly inhibited by amlexanox. Our results provide mechanistic insight into the clinical utility of amlexanox for the treatment of inflammatory disorders and might contribute to extending the clinical indications of amlexanox.