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Choroidal neovascularization (CNV) is the key pathological change caused by irreversible blindness resulting from neovascular AMD (nAMD). However, the pathological mechanisms underlying CNV remain largely unknown. Here, we aimed to investigate the role of miR-146a-5p in CNV formation.At the cellular level, we overexpressed or downregulated miR-146a-5p in an umbilical vein endothelial cell line (EA.hy926) by transfecting cells with either a miR-146a-5p mimic or an inhibitor. CCK8, wound healing, and Matrigel assays were performed to examine the proliferation, migration, and tube formation of endothelial cells (EA.hy926). Target relationship between miR-146a-5p and OTUD7B was verified using a double luciferase reporter experiment. An experimental CNV model was established by treating fundi of male C57BL/6 J mice with 810 nm laser. Fundus fluorescein angiography (FFA) was performed to evaluate the leakage of CNV on day 7 after miR-146a-5p antagomir intravitreal injection. The CNV volume was measured using Choroidal Flatmounts in a confocal study. The expression levels of VEGF, ICAM1, and NF-κB (p50 and p65) were detected both in vitro and in vivo.The expression of miR-146a-5p was increased in LPS-stimulated endothelial cells and in experimental CNV RPE-choroidal complexes in mouse models. LPS-induced proliferation, migration, and tube formation were inhibited by the miR-146a-5p inhibitor. The miR-146a-5p antagomir attenuated CNV formation and fluorescent leakage in the vivo CNV model. In the LPS-stimulated endothelial cells and the CNV mouse model, the NF-κB signaling pathway was activated and the expression of VEGF and ICAM1 increased. Conversely, downregulation of miR-146a-5p inactivated the NF-κB signaling pathway and reduced the expression of VEGF and ICAM1.Our results indicated that downregulation of miR-146a-5p inhibited experimental CNV formation via inactivation of the NF-κB signaling pathway.

Nasopharyngeal carcinoma (NPC) progression is regulated by genetic, epigenetic, and epitranscript modulation. As one of the epitranscript modifications, the role of N6-Methyladenosine (m

Inflammation and tissue infiltration by various immune cells play a significant role in the pathogenesis of neurons suffering the central nervous systems diseases. Although brachial plexus root avulsion (BPRA) leads to dramatic motoneurons (MNs) death and permanent loss of function, however, the knowledge gap on cytokines and glial reaction in the spinal cord injury is still existing. The current study is sought to investigate the alteration of specific cytokine expression patterns of the BPRA injured spinal cord during an acute and subacute period. The cytokine assay, transmission electron microscopy, and histological staining were utilized to assess cytokine network alteration, ultrastructure morphology, and glial activation and MNs loss within two weeks post-injury on a mouse unilateral BPRA model. The BPRA injury caused a progressively spinal MNs loss, reduced the alpha-(α) MNs synaptic inputs, whereas enhanced glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule-1 (IBA-1), F4/80 expression in ipsilateral but not the contralateral spinal segments. Additionally, cytokine assays revealed BPRA significantly altered the level of CXCL1, ICAM1, IP10, MCP-5, MIP1-α, and CD93. Notably, the elevated MIP1-α was mainly expressed in the injured spinal MNs. While the re-distribution of CD93 expression, from the cytoplasm to the nucleus, occasionally occurred at neurons of the ipsilateral spinal segment after injury. Overall, these findings suggest that the inflammatory cytokines associated with glial cell activation might contribute to the pathophysiology of the MNs death caused by nerve roots injury.

Purpose: To investigate whether triptolide-nanoliposome-APRPG (TP-nanolip-APRPG), a novel sustained-release nano-drug delivery system that targets vascular endothelial cells, could enhance the inhibition of triptolide (TP) on laser-induced choroidal neovascularization (CNV). Methods: TP was encapsulated with or without APRPG (Ala-Pro-Arg-Pro-Gly) peptide-modified nanoliposomes. CNV was induced by laser photocoagulation in C57BL/6J mice. One microliter of 10 μg free TP monomer, TP-nanolip containing 10 μg TP, TP-nanolip-APRPG containing 10 μg TP, or an identical volume of PBS was intravitreally injected in mice immediately after laser photocoagulation. Seven days after laser photocoagulation, CNV volumes were calculated in each group. Infiltration of M2 macrophages as well as protein levels of vascular endothelial growth factor (VEGF) and inflammatory factors including ICAM-1 and MCP-1 in the RPE-choroid complex were determined. In vitro assays for cell proliferation, migration, and tube formation were also performed. Results: TP-nanolip-APRPG was successfully synthesized and exhibited good TP delivery and enhanced the cellular uptake of TP in vitro. In vitro studies showed that TP-nanolip-APRPG was a better inhibitor of cell proliferation (31.34 ± 3.89 % vs 41.25 ± 4.67 % vs 53.55 ± 5.76 %), migration (62.60 ± 8.88 vs 104.60 ± 13.32 vs 147.00 ± 13.15), and tube formation (681.26 ± 108.15 vs 926.75 ± 54.01 vs 1189.84 ± 157.14) than TP-nanolip or free TP (all P 0.05, n=6). Conclusions: TP-nanolip-APRPG, a novel sustained-release drug delivery system targeting endothelial cells of CNV lesions, could enhance TP inhibition of the development of CNV without toxicity in the retina, suggesting therapeutic potential for CNV-related diseases in future clinical practice.

Background Choroidal neovascularization (CNV) is a common cause of irreversible blindness in elderly patients in developed countries, and subretinal fibrosis is an advanced stage of CNV. Currently, there is no effective clinical treatment for subretinal fibrosis. Purpose To investigate whether intravitreal injection of triptolide (TP) could attenuate subretinal fibrosis and determine its underlying mechanisms. Methods CNV was induced by laser photocoagulation in C57BL/6J mice. Immediately after laser photocoagulation, 1 μl of free TP (10 μg), TP-nanolip-PEG (TP-loaded PEGylated nanoliposomes containing 10 μg TP), or the same volume of phosphate-buffered saline (PBS) was intravitreally administered to each respective group. Areas and ratios of subretinal fibrosis were calculated seven days after laser injury. Additionally, expression levels of M2 macrophage-related markers, molecules of the transforming growth factor (TGF)-β1/Smad signaling pathway, and markers for epithelial-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndoMT) were detected both in vitro and in vivo. Results The areas of subretinal fibrosis were significantly reduced in both the free TP (10993.87 ± 2416.90 μm2) and TP-nanolip-PEG (7695.32 ± 2121.91 μm2) groups when compared with the PBS group (15971.97 ± 3203.10 μm2) (p Conclusions TP could attenuate subretinal fibrosis by suppressing the polarization of M2 macrophages and TGF-β1 induced EMT/EndoMT. TP-nanolip-PEG enhanced the inhibitory effects of TP on subretinal fibrosis, suggesting its therapeutic potential for CNV-related subretinal fibrosis.

Stimuli-responsive gene delivery vectors based on physiologically triggered structure changing have been recently recognized as a new therapeutic agent for their excellent performance in vivo. Herein, we present an intelligent gene delivery system based on the octa-arginine peptides (R8)-conjugated polyamino acid derivatives noted as PPCRC (PVIm-(PAsp-Cystamine-R8)-Cholesteryl), which processed pH responsive, surface charge-switching, intracellular redox-responsive and enhanced nucleus import of gene together. Due to the imidazole group in the PPCRC backbone, the DNA/PPCRC polyplexes not only exhibited the enhanced buffering capacity in the endosome after endocytosis, but also displayed the reversible surface charge from negative to positive with decreasing the pH value form pH 7.4 to pH 6.5–6.8, which would promote the cell membrane binding and cellular uptake. The disulfide bond for R8 peptides conjugation in the polymer side chain could be rapidly cleaved under reductive conditions, facilitating DNA release in the cytoplasm. Subsequently, the DNA would be still associated with the R8 peptides, which would promote the intracellular nucleus import of DNA. The luciferase gene expression level of COS-7 cells transfected by DNA/PPCRC polyplexes was almost 2000 folds higher than cells transfected by DNA/PPCC polyplexes (without R8 peptides modification) in growth-arrested cell model. Nearly 10 folds enhanced gene transfection efficiency was found on human bone mesenchymal stem cells (hBMSCs) using the same strategy, which revealed that this intelligent vector can be also utilized in transfection of non-dividing cells. Intravenous injection of the DNA/PPCRC polyplexes also achieved the effective transfection in subcutaneous tumor model. Taken together, PPCRC vector has great potential for both dividing and non-dividing cells transfection and in vivo gene delivery application.

For successful gene therapy, it is imperative to accumulate therapeutic gene in tumor tissues followed by efficiently delivering gene into targeted cells. Ultrasound irradiation, as a noninvasive and cost-effective external stimulus, has been proved to be one of the most potential external-stimulating gene delivery strategies recently in further improving gene transfection. In this study, we developed tumor-targeting ultrasound-triggered phase-transition nanodroplets AHNP-PFP-TNDs comprising a perfluorinated poly(amino acid) C11F17–PAsp (DET) as a core for simultaneously loading perfluoropentane (PFP) and nucleic acids, and a polyanionic polymer PGA-g-PEG-AHNP as the shell for not only modifying the surface of nanodroplets but also introducing an anti-Her2/neu peptide (AHNP) aiming to targeted treatment of Her2-overexpressing breast cancer. The results showed the average diameter of AHNP-PFP-TNDs was below 400 nm, nearly spherical in shape. The modification of PGA-g-PEG-AHNP not only increased the serum s...

The neuronal mechanisms underlying brachial plexus roots avulsion-induced motoneuron death are unknown. Our previous studies showed that the avulsion induced obvious temporal and spatial expression of both degenerative and regenerative genes in the injured spinal cord tissue. Therefore, we hypothesized that lncRNAs (responsible for epigenetic molecular mechanisms) are altered (resulting in altered gene expression patterns) at days 3 and 14 after avulsion. In the present microarray study, 121 lncRNAs (83 up/38 down) and 844 mRNAs (726 up/118 down) were differentially expressed (ipsilateral vs contralateral) after avulsion. We further used qRT-PCR as a validation tool to confirm the expression patterns of 5 lncRNAs and 5 mRNAs randomly selected from our microarray analysis data. The gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to identify the critical biological processes and pathways. The noted downregulation of the AF128540 (which targets the nNOS gene) is consistent with the high expression of nNOS protein observed at day 14 post-avulsion. The downregulation of MRAK034299, whose target is the Adra1d gene, is consistent with the downregulation of Adra1d mRNA and protein at days 3 and 14 post avulsion. Immunofluorescence evaluation showed cytoplasmic translocation of ECEL1 after avulsion injury. Moreover, we also found that IL6 and Rac2 are the core genes at days 3 and 14 after unilateral brachial plexus roots avulsion, respectively. Overall, our present data suggest that the altered LncRNAs (avulsion-induced), via unknown epigenetic mechanisms, certainly contribute to the molecular mechanism underpinning motoneuron death or survival. Therefore, the avulsion-induced differentially expressed lncRNAs and mRNAs may offer potential diagnostic and therapeutic targets for BPRA.

Gastric cancer (GC), particularly of the type with high expression of both human epidermal growth factor receptor 2 (Her2) and cluster determinant 44 (CD44), is one of the most malignant human tumors which causes a high mortality rate due to rapid tumor growth and metastasis. To develop effective therapeutic treatments, a dual-targeting hybrid nanoparticle (NP) system was designed and constructed to deliver the SN38 agent specifically to human solid gastric tumors bearing excessive Her2 and CD44. The hybrid NPs consist of a particle core made of the biodegradable polymer PLGA and a lipoid shell prepared by conjugating the AHNP peptides and n-hexadecylamine (HDA) to the carboxyl groups of hyaluronic acid (HA). Upon encapsulation of the SN38 agent in the NPs, the AHNP peptides and HA on the NP surface allow preferential delivery of the drug to gastric cancer cells (e.g., HGC27 cells) by targeting Her2 and CD44. Cellular uptake and in vivo biodistribution experiments verified the active targeting and prolonged in vivo circulation properties of the dual-targeting hybrid NPs, leading to enhanced accumulation of the drug in tumors. Furthermore, the anti-proliferation mechanism studies revealed that the inhibition of the growth and invasive activity of HGC27 cells was not only attributed to the enhanced cellular uptake of dual-targeting NPs, but also benefited from the suppression of CD44 and Her2 expression by HA and AHNP moieties. Finally, intravenous administration of the SN38-loaded dual-targeting hybrid NPs induced significant growth inhibition of HGC27 tumor xenografted in nude mice compared with a clinical antitumor agent, Irinotecan (CPT-11), and the other NP formulations. These results demonstrate that the designed dual-targeting hybrid NPs are promising for targeted anti-cancer drug delivery to treat human gastric tumors over-expressing Her2 and CD44.

We have developed new, efficient gene delivery systems based on PEGylated poly(lactone-co-β-amino ester) block copolymers that are biodegradable, stable and low in toxicity. The PEG-poly[PDL-co-3-(4-(methylene)piperidin-1-yl)propanoate] (PEG-PPM) diblock and PPM-PEG-PPM triblock copolymers with various compositions were synthesized in one step via lipase-catalyzed copolymerization of ω-pentadecalactone (PDL) and ethyl 3-(4-(hydroxymethyl)piperidin-1-yl)propanoate (EHMPP) with an appropriate PEG (MeO-PEG-OH or HO-PEG-OH). The amphiphilic block copolymers are capable of condensing DNA in aqueous medium via a self-assembly process to form polyplex micelle nanoparticles with desirable particle sizes (70-140 nm). These micelles possess low CMC values and are stable in the medium containing serum protein molecules (FBS). Among the PEG-PPM and PPM-PEG-PPM micelles, the PEG-PPM-15% PDL micelle particles exhibited high DNA-binding ability, the fastest cellular uptake rate and highest gene transfection efficacy. Flow cytometry analysis shows that LucDNA/PEG-PPM-15% PDL polyplex micelles can effectively escape from endosomal degradation after cellular uptake likely due to the presence of the tertiary amine groups in the copolymer chains that act as proton sponges. In vitro cytotoxicity and hemolysis assay experiments indicate that all copolymer samples are nonhemolytic and have minimal toxicity toward COS-7 cells within the polymer concentration range (≤200 μg/mL) used for the gene transfection. These results demonstrate that the PEGylated poly(lactone-co-β-amino ester) block copolymers are promising new vectors for gene delivery applications.

We have designed and constructed novel multifunctional nanoparticle drug-delivery systems that are stable under physiological conditions and responsive to tumor-relevant pH and intracellular reduction potential. The nanoparticles were fabricated from enzymatically synthesized poly(ethylene glycol) (PEG)-poly(ω-pentadecalactone-co-N-methyldiethyleneamine-co-3,3'-dithiodipropionate) (PEG-PPMD) and PEG-poly(ε-caprolactone-co-N-methyldiethyleneamine-co-3,3'-dithiodipropionate) (PEG-PCMD) block copolymers via self-assembly processes in aqueous solution. At acidic pH and in the presence of a reductant (e.g., d,l-dithiothreitol or glutathione), the nanosized micelle particles rapidly swell and disintegrate due to the protonation of amino groups and reductive cleavage of disulfide bonds in the micelle cores. Consistently, docetaxel (DTX)-loaded PEG-PPMD and PEG-PCMD micelles can be triggered synergistically by acidic endosomal pH and a high intracellular reduction potential to rapidly release the drug for efficient killing of cancer cells. The drug formulations based on PEG-PPMD and PEG-PCMD copolymers exhibited a substantially higher potency than free DTX in inhibiting tumor growth in mice, whereas their therapeutic effects on important organ tissues were minimal. These results demonstrate that PEG-PPMD and PEG-PCMD nanoparticles have a great potential to serve as site-specific, controlled drug-delivery vehicles for safe and efficient antitumor treatment.