Role of Flow-Sensitive Endothelial Genes in Atherosclerosis and Antiatherogenic Therapeutics Development.

Atherosclerosis is a chronic inflammatory disease that is the underlying cause of cardiovascular disease which initiates from endothelial dysfunction from genetic and environmental risk factors, including biomechanical forces: blood flow. Endothelial cells (ECs) lining the inner arterial wall regions exposed to disturbed flow are prone to atherosclerosis development, whereas the straight regions exposed to stable flow are spared from the disease. These flow patterns induce genome- and epigenome-wide changes in gene expression in ECs. Through the sweeping changes in gene expression, disturbed flow reprograms ECs from athero-protected cell types under the stable flow condition to pro-atherogenic cell conditions. The pro-atherogenic changes induced by disturbed flow, in combination with additional risk factors such as hypercholesterolemia, lead to the progression of atherosclerosis. The flow-sensitive genes and proteins are critical in understanding the mechanisms and serve as novel targets for antiatherogenic therapeutics.

Current Nanomedicine for Targeted Vascular Disease Treatment: Trends and Perspectives.

Nanotechnology has been developed to deliver cargos effectively to the vascular system. Nanomedicine is a novel and effective approach for targeted vascular disease treatment including atherosclerosis, coronary artery disease, strokes, peripheral arterial disease, and cancer. It has been well known for some time that vascular disease patients have a higher cancer risk than the general population. During atherogenesis, the endothelial cells are activated to increase the expression of adhesion molecules such as Intercellular Adhesion Molecule 1 (ICAM-1), Vascular cell adhesion protein 1 (VCAM-1), E-selectin, and P-selectin. This biological activation of endothelial cells gives a targetability clue for nanoparticle strategies. Nanoparticle formation has a passive targeting pathway due to the increased adhesion molecule expression on the cell surface as well as increased cell activation. In addition, the VCAM-1-targeting peptide has been widely used to target the inflamed endothelial cells. Biomimetic nanoparticles using platelet and leukocyte membrane fragment strategies have been promising techniques for targeted vascular disease treatment. Cyclodextrin, a natural oligosaccharide with a hydrophobic cavity, increase the solubility of cholesterol crystals at the atherosclerotic plaque site and has been used to deliver the hydrophobic drug statin as a therapeutic in a targeted manner. In summary, nanoparticles decorated with various targeting molecules will be an effective and promising strategy for targeted vascular disease treatment.

Hypoxia inducible factor 1α inhibitor PX-478 reduces atherosclerosis in mice.

BACKGROUND AND AIMS: Hypoxia inducible factor 1α (HIF1α) plays a critical role in atherosclerosis as demonstrated in endothelial-targeted HIF1α -deficient mice. However, it has not been shown if specific pharmacological inhibitors of HIF1α can be used as potential drugs for atherosclerosis. PX-478 is a selective inhibitor of HIF1α, which was used to reduce cancer and obesity in animal models. Here, we tested whether PX-478 can be used to inhibit atherosclerosis. METHODS: We first tested PX-478 in human aortic endothelial cells (HAEC) and found that it significantly inhibited expression of HIF1α and its targets, including Collagen I. Next, two independent atherosclerosis models, C57BL/6 mice treated with AAV-PCSK9 and ApoE-/- mice, were used to test the efficacy of PX-478. Both mouse models were fed a Western diet for 3 months with bi-weekly treatment with PX-478 (40 mg/kg) or saline. RESULTS: PX-478 treatment reduced atherosclerotic plaque burden in the aortic trees in both mouse models, while plaque burden in the aortic sinus was reduced in the AAV-PCSK9 mouse model, but not in the ApoE-/- mice. Russell-Movat's Pentachrome and Picrosirius Red staining showed a significant reduction in extracellular matrix remodeling and collagen maturation, respectively, in the PX-478-treated mice. As expected, PX-478 treatment reduced diet-induced weight-gain and abdominal adipocyte hypertrophy. Interestingly, PX-478 reduced plasma LDL cholesterol by 69% and 30% in AAV-PCSK9 and ApoE-/- mice, respectively. To explore the cholesterol-lowering mechanisms, we carried out an RNA sequencing study using the liver tissues from the ApoE-/- mouse study. We found 450 genes upregulated and 381 genes downregulated by PX-478 treatment in the liver. Further, gene ontology analysis showed that PX-478 treatment upregulated fatty acid and lipid catabolic pathways, while downregulating lipid biosynthesis and plasma lipoprotein particle remodeling processes. Of interest, Cfd, Elovl3, and Insig2 were some of the most downregulated genes by PX-478, and have been implicated in fat storage, fatty acid elongation, and cholesterol metabolism. The downregulation of Cfd, Elovl3, and Insig2 was further validated by qPCR in the liver tissues of ApoE-/- mice treated with PX-478. CONCLUSIONS: These results suggest that PX-478 is a potential anti-atherogenic drug, which targets vascular endothelium and hepatic cholesterol pathways.

Conditional Antisense Oligonucleotides Triggered by miRNA.

Antisense oligonucleotides (ASOs) are single-stranded short nucleic acids that silence the expression of target mRNAs and show increasing therapeutic potential. Since ASOs are internalized by many cell types, both normal and diseased cells, gene silencing in unwanted cells is a significant challenge for their therapeutic use. To address this challenge, we created conditional ASOs that become active only upon detecting transcripts unique to the target cell. As a proof-of-concept, we modified an HIF1α ASO (EZN2968) to generate miRNA-specific conditional ASOs, which can inhibit HIF1α in the presence of a hepatocyte-specific miRNA, miR-122, via a toehold exchange reaction. We characterized a library of nucleic acids, testing how the conformation, thermostability, and chemical composition of the conditional ASO impact the specificity and efficacy in response to miR-122 as a trigger signal. Optimally designed conditional ASOs demonstrated knockdown of HIF1α in cells transfected with exogenous miR-122 and in hepatocytes expressing endogenous miR-122. We confirmed that conditional ASO activity was mediated by toehold exchange between miR-122 and the conditional ASO duplex, and the magnitude of the knockdown depended on the toehold length and miR-122 levels. Using the same concept, we further generated another conditional ASO that can be triggered by miR-21. Our results suggest that conditional ASOs can be custom-designed with any miRNA to control ASO activation in targeted cells while reducing unwanted effects in nontargeted cells.

Effects of Decomplexation Rates on Ternary Gene Complex Transfection with α-Poly(l-Lysine) or ε-Poly(l-Lysine) as a Decomplexation Controller in An Easy-To-Transfect Cell or A Hard-To-Transfect Cell.

The tight binding of pDNA with a cationic polymer is the crucial requirement that prevents DNA degradation from undesired DNase attack to safely deliver the pDNA to its target site. However, cationic polymer-mediated strong gene holding limits pDNA dissociation from the gene complex, resulting in a reduction in transfection efficiency. In this study, to control the decomplexation rate of pDNA from the gene complex in a hard-to-transfect cell or an easy-to-transfect cell, either α-poly(l-lysine) (APL) or ε-poly(l-lysine) (EPL) was incorporated into branched polyethylenimine (bPEI)-based nanocomplexes (NCs). Compared to bPEI/pDNA NCs, the addition of APL or EPL formed smaller bPEI-APL/pDNA NCs with similar zeta potentials or larger bPEI-EPL/pDNA NCs with reduced zeta potentials, respectively, due to the different characteristics of the primary amines in the two poly(l-lysine)s (PLs). Interestingly, although both bPEI-APL/pDNA NCs and bPEI-EPL/pDNA NCs showed similar pDNA compactness to bPEI/pDNA NCs, the addition of APL or EPL resulted in slower or faster pDNA release, respectively, from the bPEI-PL/pDNA NCs than from the bPEI/pDNA NCs. bPEI-EPL/pDNA NCs with a decomplexation enhancer (i.e., EPL) improved the transfection efficiency (TE) in both a hard-to-transfect HepG2 cell and an easy-to-transfect HEK293 cell. However, although a decomplexation inhibitor (i.e., APL) reduced the TE of bPEI-APL/pDNA NCs in both cells, the degree of reduction in the TE could be compensated by PL-mediated enhanced nuclear delivery, particularly in HepG2 cells but not HEK293 cells, because both PLs facilitate nuclear localization of the gene complex per its cellular uptake. In conclusion, a decomplexation rate controller could be a potential factor to establish a high TE and design clinically available gene complex systems.

Effect of pH-Responsive Charge-Conversional Polymer Coating to Cationic Reduced Graphene Oxide Nanostructures for Tumor Microenvironment-Targeted Drug Delivery Systems.

Tumor tissue represents a slightly acidic pH condition compared to normal tissue due to the accumulation of lactic acids via anaerobic metabolism. In this work, pH-responsive charge-conversional polymer (poly(ethylene imine)-poly(l-lysine)-poly(l-glutamic acid), PKE polymer) was employed for endowing charge-conversional property and serum stability to poly(ethylene imine) conjugated reduced graphene oxide-based drug delivery system (PEI-rGO). Zeta-potential value of PEI-rGO coated with PK5E7 polymer (PK5E7(PEI-rGO)) was -10.9 mV at pH 7.4 and converted to 29.2 mV at pH 6.0, showing pH-responsive charge-conversional property. Sharp-edged plate morphology of PEI-rGO was transformed to spherical nanostructures with vague edges by PK5E7 coating. Size of PK5E7(PEI-rGO) was found to be smaller than that of PEI-rGO in the serum condition, showing its increased serum stability. Loaded doxorubicin (DOX) in PK5E7(PEI-rGO) could be released rapidly in lysosomal condition (pH 5.0, 5 mM glutathione). Furthermore, DOX-loaded PK5E7(PEI-rGO) showed enhanced anticancer activity in HeLa and A549 cells in the tumor microenvironment-mimicking condition (pH 6.0, serum), which would be mediated by non-specific cellular interaction with decorated serum proteins. These results indicate that the pH-responsive charge-conversional PKE polymer coating strategy of cationic rGO nanostructures possesses a potential for acidic tumor microenvironment-targeted drug delivery systems.

Effects of the Physicochemical, Colloidal, and Biological Characteristics of Different Polymer Structures between α-Poly(l-lysine) and ε-Poly(l-lysine) on Polymeric Gene Delivery.

Though α-poly(l-lysine) (APL) has been well-studied in gene delivery, ε-poly(l-lysine) (EPL) with same repeating unit of l-lysine but different structure has been rarely investigated. This study compared various effects of their different structures in gene delivery processes. EPL showed less cytotoxicity and more proton buffering capacity for endosomal release than APL. Also, EPL/pDNA polyplexes represented higher nucleus preference than APL/pDNA polyplexes. However, EPL had weaker affinities with pDNA than APL, leading to formation of larger EPL/pDNA complexes with less compactness and successively faster decomplexation. The resultant difference of their pDNA binding affinity caused lower cellular uptake and lower transfection efficiency of EPL/pDNA complexes than APL/pDNA complexes. Thus, this study confirmed that various effects of gene delivery processes are changed by chemical structure of polymeric gene carriers. Especially, despite the low transfection efficiency of EPL-based polyplexes, the study found potentials of EPL in cytocompatibility, endosomal release, and nuclear import.

pH-Responsive Charge-Conversional Poly(ethylene imine)-Poly(l-lysine)-Poly(l-glutamic acid) with Self-Assembly and Endosome Buffering Ability for Gene Delivery Systems.

Poly(ethylene imine)-poly(l-lysine)-poly(l-glutamic acid) (PKE) polymers with various glutamic acid portions were synthesized by ring opening polymerization of l-lysine N-carboxyanhydride (NCA) and l-glutamic acid NCA from poly(ethylene imine) 1.8 kDa (PEI1.8k) as a macroinitiator. It was found that their glutamic acid residues could buffer endosomal pH. PK5E9 polymer could form nanoparticles by self-assembly and nanosized polyplexes, possessing pH-responsive charge-conversion properties. PK5E9 or its polyplex nanoparticles showed polyhedral structures with bumpy surfaces. Its cytotoxicity was marginal at both pH 7.4 and 6.0, and its transfection efficiency was highly increased at pH 6.0. The improved transfection efficiency in acidic conditions was thought to be induced by elevated cellular uptake of the polyplexes via charge-conversion from negative to positive charges. Its transfection was also found to be mediated by endosomal escape through endosome buffering by bafilomycin A1-treated transfection. In conclusion, PK5E9 polymer with self-assembly and endosome buffering ability was found to possess potentials for efficient gene delivery systems in acidic conditions via charge conversion, which may be applied for tumor microenvironment-targeting.

Agmatine-Containing Bioreducible Polymer for Gene Delivery Systems and Its Dual Degradation Behavior.

Agmatine-containing bioreducible polymer, poly(cystaminebis(acrylamide)-agmatine) (poly(CBA-AG)) was synthesized for gene delivery systems. It could form 200-300 nm sized and positively charged polyplexes with pDNA, which could release pDNA in reducing the environment due to the internal disulfide bonds cleavage. Poly(CBA-AG) also showed a spontaneous degradation behavior in aqueous condition in contrast to the backbone polymer, poly(cystaminebis(acrylamide)-diaminobutane) (poly(CBA-DAB)) lacking guanidine moieties, probably due to the self-catalyzed hydrolysis of internal amide bonds by guanidine moieties. The cytotoxicity of poly(CBA-AG) was cell-dependent but minimal. Poly(CBA-AG) exhibited highly enhanced transfection efficiency in comparison with poly(CBA-DAB) and even higher transfection efficiency than PEI25k. However, cellular uptake efficiency of the polyplexes did not show positive correlation with the transfection efficiency. Confocal microscopy observation revealed that pDNA delivered by poly(CBA-AG) was strongly accumulated in cell nuclei. These results suggested that high transfection efficiency of poly(CBA-AG) may be derived from the efficient pDNA localization in cell nuclei by guanidine moieties and that the polyplexes dissociation via self-catalyzed hydrolysis as well as disulfide bonds cleavage in cytosol also may facilitate the transfection process. Finally, poly(CBA-AG)/pJDK-apoptin polyplex showed a high anticancer activity induced by apoptosis, demonstrating a potential of poly(CBA-AG) as a gene carrier for cancer gene therapy.

Crosslinked Polypropylenimine Dendrimers With Bioreducible Linkages for Gene Delivery Systems and Their Reductive Degradation Behaviors.

Crosslinked bioreducible polypropylenimine-cystaminebisacrylamides (PPI-CBAs) were synthesized for gene delivery systems. They formed nano-sized polyplexes with high stability even in reducing condition probably due to the re-crosslinking. PPI-CBAs displayed high transfection efficiency comparable to PEI25k in serum condition. However, cytotoxicity increase, glutathione (GSH) decrease, and reactive oxygen species (ROS) increase were observed in cells with the elevation of PPI-CBAs concentration and crosslinking degree. These results suggest that the cytotoxicity of bioreducible polymers may be closely related with their structures and that reduction of GSH by degradation of re-crosslinked bioreducible polymers and the following increase of ROS may induce cytotoxicity by oxidative stress.

Cationic methylcellulose derivative with serum-compatibility and endosome buffering ability for gene delivery systems.

In this work, methylcellulose was employed as a template polymer with graft of polyethylenimine 0.8 kDa (PEI0.8k) for gene delivery systems. Synthesized PEI-grafted oxidized methylcellulose (MC-PEI) could condense pDNA into positively charged and nano-sized particles, which could protect pDNA from serum nuclease. The cytotoxicity of MC-PEI was minimal in both serum-free and serum condition due to the biocompatibility of methylcellulose and low cytotoxicity of PEI0.8k. MC-PEI polyplex also showed low cytotoxicity in serum condition. In serum condition, MC-PEI showed less decreased transfection efficiency than PEI25k, meaning good serum-compatibility of MC-PEI. Bafilomycin A1-treated transfection results indicate that the transfection of MC-PEI is mediated via endosomal escape by endosome buffering ability. Flow cytometry results suggest that MC-PEI polyplex could be internalized into cells and efficiently deliver pDNA to cells due to its serum-compatibility. These results demonstrate that MC-PEI possesses a potential for efficient gene delivery systems.

Therapeutic gene delivery using bioreducible polymers.

Bioreducible polymers, which can be degraded in reducing environment due to the cleavage of internal disulfide bonds, have been developed for gene delivery systems. They show high stability in extracellular physiological condition and cytoplasm-specific release of genetic materials, as well as decreased cytotoxicity because cytoplasm is a reducing environment containing high level of reducing molecules such as glutathione. Based on these advantages, recently, many bioreducible polymers have been further investigated with therapeutic genes for the treatment of diseases and demonstrated promising results. This review will focus on the therapeutic gene delivery using bioreducible polymers and the evaluation of therapeutic efficacy for cancer, myocardial infarction, diabetes and miscellaneous diseases.

Anticancer activity of ferulic acid-inorganic nanohybrids synthesized via two different hybridization routes, reconstruction and exfoliation-reassembly.

We have successfully prepared nanohybrids of biofunctional ferulic acid and layered double hydroxide nanomaterials through reconstruction and exfoliation-reassembly routes. From X-ray diffraction and infrared spectroscopy, both nanohybrids were determined to incorporate ferulic acid molecules in anionic form. Microscopic results showed that the nanohybrids had average particle size of 150 nm with plate-like morphology. As the two nanohybridization routes involved crystal disorder and random stacking of layers, the nanohybrids showed slight alteration in z-axis crystallinity and particle size. The zeta potential values of pristine and nanohybrids in deionized water were determined to be positive, while those in cell culture media shifted to negative values. According to the in vitro anticancer activity test on human cervical cancer HeLa cells, it was revealed that nanohybrids showed twice anticancer activity compared with ferulic acid itself. Therefore we could conclude that the nanohybrids of ferulic acid and layered double hydroxide had cellular delivery property of intercalated molecules on cancer cell lines.