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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.

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.

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.

Ultrasound as an external stimulus for enhanced gene transfection represents a safe, noninvasive, cost-effective delivery strategy for gene therapy. Herein, we have developed an ultrasound-triggered phase-transition cationic nanodroplet based on a novel perfluorinated amphiphilic poly(amino acid), which could simultaneously load perfluoropentane (PFP) and nucleic acids. The heptadecafluoroundecylamine (C11F17-NH2) was chosen to initiate β-benzyl-L-aspartate N-carboxyanhydride (BLA-NCA) ring-opening polymerization to prepare C11F17-poly(β-benzyl-L-aspartate) (C11F17-PBLA). Subsequently, C11F17-poly{N-[N'-(2-aminoethyl)]aspartamide} [C11F17-PAsp(DET)] was synthesized by aminolysis reaction of C11F17-PBLA with diethylenetriamine (DET). PFP/pDNA-loaded nanodroplets PFP-TNDs [PFP/C11F17-PAsp(DET)/LucDNA/γ-PGA or poly(glutamic acid)-g-MeO-poly(ethylene glycol) (PGA-g-mPEG) ternary nanodroplets] were primarily formulated by an oil/water emulsification method, followed by surface modification with PGA-g-mPEG. The average diameter of PFP-TNDs ranged from 300 to 400 nm, and transmission electron microscopy images showed that the nanodroplets were nearly spherical in shape. The ζ potential of the nanodroplets dramatically decreased from +54.3 to +15.3 mV after modification with PGA-g-mPEG, resulting in a significant increase of the stability of the nanodroplets in the serum-containing condition. With ultrasound irradiation, the gene transfection efficiency was enhanced 14-fold on HepG2 cells, and ultrasound-triggered phase-transition cationic nanodroplets also displayed a good ultrasound contrast effect. These results suggest that the PFP/DNA-loaded phase-transition cationic nanodroplets can be utilized as efficient theranostic agents for targeting gene delivery.

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.

Ginkgo biloba, a traditional herbal remedy and a popular functional food is sold commercially in form of a standardized extract for its flavone glycosides content (24%) and terpene lactones (6%). The ginkgo terpene lactones are the unique components of ginkgo leaves unlike the flavone glycosides which are quite common in other botanical extracts. Therefore, flavone glycosides (and their respective aglycones) are the main targets of adulteration of ginkgo extracts to meet the marker levels in the commercially available raw materials in the market. We have investigated the typical quality of a wide variety of commercial ginkgo extracts as well as their potential adulterants. Based on our analytical results we have classified the commercially available extracts of G. biloba into three categories such as authentic, intermediate and adulterated/spiked. A combination of qualitative determination of the unhydrolyzed extracts by phytochemical fingerprinting as well as typical quantitative analysis for total flavone glycosides including Q/K/I (quercetin/kaempferol/isorhamnetin) peak area ratio on hydrolyzed samples is recommended to establish/track the authenticity of the available extracts for formulation purposes.

A method has been established and validated for identification and quantification of individual, as well as total, anthocyanins by HPLC and LC/ES-MS in botanical raw materials used in the herbal supplement industry. The anthocyanins were separated and identified on the basis of their respective M(+) (cation) using LC/ES-MS. Separated anthocyanins were individually calculated against one commercially available anthocyanin external standard (cyanidin-3-glucoside chloride) and expressed as its equivalents. Amounts of each anthocyanin calculated as external standard equivalent were then multiplied by a molecular-weight correction factor to afford their specific quantities. Experimental procedures and use of a molecular-weight correction factors are substantiated and validated using Balaton tart cherry and elderberry as templates. Cyanidin-3-glucoside chloride has been widely used in the botanical industry to calculate total anthocyanins. In our studies on tart cherry and elderberry, its use as external standard followed by use of molecular-weight correction factors should provide relatively accurate results for total anthocyanins, because of the presence of cyanidin as their major anthocyanidin backbone. The method proposed here is simple and has a direct sample preparation procedure without any solid-phase extraction. It enables selection and use of commercially available anthocyanins as external standards for quantification of specific anthocyanins in the sample matrix irrespective of their commercial availability as analytical standards. It can be used as a template and applied for similar quantification in several anthocyanin-containing raw materials for routine quality control procedures, thus providing consistency in analytical testing of botanical raw materials used for manufacturing efficacious and true-to-the-label nutritional supplements.