Heparin-mimicking polymer-based hydrogel matrix regulates macrophage polarization by controlling cell adhesion.

The physicochemical properties of biomaterials influence cell adhesion, shape, and polarization of macrophages. In this study, we aimed to evaluate the polarization of macrophages in terms of the regulation of cell adhesion and how synthetic mimics for heparin and poly(sodium-4-styrenesulfonate) can regulate macrophage polarization by modulating cell shape, focal adhesion, cell traction force, and intracellular tension. Our initial findings showed that macrophages cultured with heparin-mimicking polymer-based hydrogel matrix showed reduced expression of cell adhesion markers such as integrins, vinculin, RhoA, and ROCK1/2 and reduced cell shape, elongation, cell-matrix traction force, and intracellular tension. Furthermore, we observed a significant decrease in cell adhesion in cells cultured on the hydrogel, resulting in the promotion of M1 polarization. These findings offer insights into the important roles of cell-matrix interactions in macrophage polarization and offer a platform for heparin-mimicking polymer-based hydrogel matrices to induce M1 polarization by inducing cell adhesion without classical activators.

Highly Prolonged Release of the Cancer Vaccine and Immunomodulator via a Two-Layer Biodegradable Microneedle for Prophylactic Treatment of Metastatic Cancer.

Simultaneous sustained release of cancer vaccines and immunomodulators may effectively trigger durable immune responses and avoid multiple administrations. Here, we established a biodegradable microneedle (bMN) based on a biodegradable copolymer matrix made of polyethylene glycol (PEG) and poly(sulfamethazine ester urethane) (PSMEU). This bMN was applied to the skin and slowly degraded in the epidermis/dermis layers. Then, the complexes composed of a positively charged polymer (DA3), cancer DNA vaccine (pOVA), and toll-like receptor 3 agonist poly(I/C) were synchronously released from the matrix in a pain-free manner. The whole microneedle patch was fabricated with two layers. The basal layer was formed using polyvinyl pyrrolidone/polyvinyl alcohol that could be rapidly dissolved upon applying the microneedle patch to the skin, whereas the microneedle layer was formed by complexes encapsulating biodegradable PEG-PSMEU, which was stuck at the injection site for sustained release of therapeutic agents. According to the results, 10 days is the time for the complexes to be completely released and express specific antigens in antigen-presenting cells in vitro and in vivo. It is noteworthy that this system could successfully elicit cancer-specific humoral immune responses and inhibit metastatic tumors in the lungs after a single shot of immunization.

Sustained Exosome-Guided Macrophage Polarization Using Hydrolytically Degradable PEG Hydrogels for Cutaneous Wound Healing: Identification of Key Proteins and MiRNAs, and Sustained Release Formulation.

Macrophages (Mφs) are characterized by remarkable plasticity, an essential component of chronic inflammation. Thus, an appropriate and timely transition from proinflammatory (M1) to anti-inflammatory (M2) Mφs during wound healing is vital to promoting resolution of acute inflammation and enhancing tissue repair. Herein, exosomes derived from M2-Mφs (M2-Exos), which contain putative key regulators driving Mφ polarization, are used as local microenvironmental cues to induce reprogramming of M1-Mφs toward M2-Mφs for effective wound management. As an injectable controlled release depot for exosomes, hydrolytically degradable poly(ethylene glycol) (PEG) hydrogels (Exogels) are designed and employed for encapsulating M2-Exos to maximize their therapeutic effects in cutaneous wound healing. The degradation time of the hydrogels is adjustable from 6 days or up to 27 days by controlling the crosslinking density and tightness. The localization of M2-Exos leads to a successful local transition from M1-Mφs to M2-Mφs within the lesion for more than 6 days, followed by enhanced therapeutic effects including rapid wound closure and increased healing quality in an animal model for cutaneous wound healing. Collectively, the hydrolytically degradable PEG hydrogel-based exosome delivery system may serve as a potential tool in regulating local polarization state of Mφs, which is crucial for tissue homeostasis and wound repair.

A Novel Strategy for Creating an Antibacterial Surface Using a Highly Efficient Electrospray-Based Method for Silica Deposition.

Adhesion of bacteria on biomedical implant surfaces is a prerequisite for biofilm formation, which may increase the chances of infection and chronic inflammation. In this study, we employed a novel electrospray-based technique to develop an antibacterial surface by efficiently depositing silica homogeneously onto polyethylene terephthalate (PET) film to achieve hydrophobic and anti-adhesive properties. We evaluated its potential application in inhibiting bacterial adhesion using both Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria. These silica-deposited PET surfaces could provide hydrophobic surfaces with a water contact angle greater than 120° as well as increased surface roughness (root mean square roughness value of 82.50 ± 16.22 nm and average roughness value of 65.15 ± 15.26 nm) that could significantly reduce bacterial adhesion by approximately 66.30% and 64.09% for E. coli and S. aureus, respectively, compared with those on plain PET surfaces. Furthermore, we observed that silica-deposited PET surfaces showed no detrimental effects on cell viability in human dermal fibroblasts, as confirmed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide and live/dead assays. Taken together, such approaches that are easy to synthesize, cost effective, and efficient, and could provide innovative strategies for preventing bacterial adhesion on biomedical implant surfaces in the clinical setting.

Heparin-Mimicking Polymer-Based In Vitro Platform Recapitulates In Vivo Muscle Atrophy Phenotypes.

The cell-cell/cell-matrix interactions between myoblasts and their extracellular microenvironment have been shown to play a crucial role in the regulation of in vitro myogenic differentiation and in vivo skeletal muscle regeneration. In this study, by harnessing the heparin-mimicking polymer, poly(sodium-4-styrenesulfonate) (PSS), which has a negatively charged surface, we engineered an in vitro cell culture platform for the purpose of recapitulating in vivo muscle atrophy-like phenotypes. Our initial findings showed that heparin-mimicking moieties inhibited the fusion of mononucleated myoblasts into multinucleated myotubes, as indicated by the decreased gene and protein expression levels of myogenic factors, myotube fusion-related markers, and focal adhesion kinase (FAK). We further elucidated the underlying molecular mechanism via transcriptome analyses, observing that the insulin/PI3K/mTOR and Wnt signaling pathways were significantly downregulated by heparin-mimicking moieties through the inhibition of FAK/Cav3. Taken together, the easy-to-adapt heparin-mimicking polymer-based in vitro cell culture platform could be an attractive platform for potential applications in drug screening, providing clear readouts of changes in insulin/PI3K/mTOR and Wnt signaling pathways.

Bioinspired pH- and Temperature-Responsive Injectable Adhesive Hydrogels with Polyplexes Promotes Skin Wound Healing.

Despite great potential, the delivery of genetic materials into cells or tissues of interest remains challenging owing to their susceptibility to nuclease degradation, lack of permeability to the cell membrane, and short in vivo half-life, which severely restrict their widespread use in therapeutics. To surmount these shortcomings, we developed a bioinspired in situ-forming pH- and temperature-sensitive injectable hydrogel depot that could control the delivery of DNA-bearing polyplexes for versatile biomedical applications. A series of multiblock copolymer, comprised of water-soluble poly(ethylene glycol) (PEG) and pH- and temperature-responsive poly(sulfamethazine ester urethane) (PSMEU), has been synthesized as in situ-forming injectable hydrogelators. The free-flowing PEG-PSMEU copolymer sols at high pH and room temperature (pH 8.5, 23 °C) were transformed to stable gel at the body condition (pH 7.4, 37 °C). Physical and mechanical properties of hydrogels, including their degradation rate and viscosity, are elegantly controlled by varying the composition of urethane ester units. Subcutaneous administration of free-flowing PEG-PSMEU copolymer sols to the dorsal region of Sprague-Dawley rats instantly formed hydrogel depot. The degradation of the hydrogel depot was slow at the beginning and found to be bioresorbable after two months. Cationic protein or DNA-bearing polyplex-loaded PEG-PSMEU copolymer sols formed stable gel and controlled its release over 10 days in vivo. Owing to the presence of urethane linkages, the PEG-PSMEU possesses excellent adhesion strength to wide range of surfaces including glass, plastic, and fresh organs. More importantly, the hydrogels effectively adhered on human skin and peeled easily without eliciting an inflammatory response. Subcutaneous implantation of PEG-PSMEU copolymer sols effectively sealed the ruptured skin, which accelerated the wound healing process as observed by the skin appendage morphogenesis. The bioinspired in situ-forming pH- and temperature-sensitive injectable adhesive hydrogel may provide a promising platform for myriad biomedical applications as controlled delivery vehicle, adhesive, and tissue regeneration.

In Vitro and In Vivo Tumor Targeted Photothermal Cancer Therapy Using Functionalized Graphene Nanoparticles.

Despite the tremendous progress that photothermal therapy (PTT) has recently achieved, it still has a long way to go to gain the effective targeted photothermal ablation of tumor cells. Driven by this need, we describe a new class of targeted photothermal therapeutic agents for cancer cells with pH responsive bioimaging using near-infrared dye (NIR) IR825, conjugated poly(ethylene glycol)-g-poly(dimethylaminoethyl methacrylate) (PEG-g-PDMA, PgP), and hyaluronic acid (HA) anchored reduced graphene oxide (rGO) hybrid nanoparticles. The obtained rGO nanoparticles (PgP/HA-rGO) showed pH-dependent fluorescence emission and excellent near-infrared (NIR) irradiation of cancer cells targeted in vitro to provide cytotoxicity. Using intravenously administered PTT agents, the time-dependent in vivo tumor target accumulation was exactly defined, presenting eminent photothermal conversion at 4 and 8 h post-injection, which was demonstrated from the ex vivo biodistribution of tumors. These tumor environment responsive hybrid nanoparticles generated photothermal heat, which caused dominant suppression of tumor growth. The histopathological studies obtained by H&E staining demonstrated complete healing from malignant tumor. In an area of limited successes in cancer therapy, our translation will pave the road to design stimulus environment responsive targeted PTT agents for the safe eradication of devastating cancer.

Liposomal melatonin rescues methamphetamine-elicited mitochondrial burdens, pro-apoptosis, and dopaminergic degeneration through the inhibition PKCδ gene.

We have demonstrated that mitochondrial oxidative damage and PKCδ overexpression contribute to methamphetamine-induced dopaminergic degeneration. Although it is recognized that antioxidant melatonin is effective in preventing neurotoxicity induced by methamphetamine, its precise mechanism remains elusive. C57BL/6J wild-type mice exhibited a similar degree of dopaminergic deficit when methamphetamine was administered during light and dark phases. Furthermore, dopaminergic neuroprotection by genetic inhibition of PKCδ during the light phase was comparable to that during the dark phase. Thus, we have focused on the light phase to examine whether melatonin modulates PKCδ-mediated neurotoxic signaling after multiple high doses of methamphetamine. To enhance the bioavailability of melatonin, we applied liposomal melatonin. Treatment with methamphetamine resulted in hyperthermia, mitochondrial translocation of PKCδ, oxidative damage (mitochondria > cytosol), mitochondrial dysfunction, pro-apoptotic changes, ultrastructural mitochondrial degeneration, dopaminergic degeneration, and behavioral impairment in wild-type mice. Treatment with liposomal melatonin resulted in a dose-dependent attenuation against degenerative changes induced by methamphetamine in wild-type mice. Attenuation by liposomal melatonin might be comparable to that by genetic inhibition (using PKCδ((-/-)) mice or PKCδ antisense oligonucleotide). However, liposomal melatonin did not show any additional protective effects on the attenuation by genetic inhibition of PKCδ. Our results suggest that the circadian cycle cannot be a key factor in modulating methamphetamine toxicity under the current experimental condition and that PKCδ is one of the critical target genes for melatonin-mediated protective effects against mitochondrial burdens (dysfunction), oxidative stress, pro-apoptosis, and dopaminergic degeneration induced by methamphetamine.

Hyaluronic acid-siRNA conjugate/reducible polyethylenimine complexes for targeted siRNA delivery.

The clinical applications of therapeutic siRNA remain as a challenge due to the lack of efficient delivery system. In the present study, hyaluronic acid-siRNA conjugate (HA-SS-siRNA)/reducible polyethylenimine (BPEI1.2k-SS) complexes were developed to efficiently deliver the siRNA to HA receptor abundant region with the improved siRNA stability. HA and siRNA were conjugated with disulfide bonds, which are cleavable in cytoplasm. The synthesized HA-SS-siRNA was further complexed with BPEI1.2k-SS, resulting in the formation of spherical nanostructures with approximately 190 nm of size and neutral surface charge. HA-SS-siRNA/BPEI1.2k-SS complexes exhibited the improved stability against serum proteins or polyanions. These complexes were successfully translocated into intracellular region via HA receptor-mediated endocytosis, and silenced target gene expression.

The inhibitory effect of vitexin on the agonist-induced regulation of vascular contractility.

The present study was undertaken to investigate the influence of vitexin on vascular smooth muscle contractility and to determine the mechanism involved. Intact or denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Vitexin more significantly relaxed phorbol ester-induced vascular contraction than thromboxane A2 or fluoride-induced contraction suggesting as a possible anti-hypertensive on the agonist-induced vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, vitexin significantly inhibited phorbol ester-induced increases in pERK1/2 levels. On the other hand, it did not significantly inhibit thromboxane A2-induced increases in pMYPT1 levels suggesting the mechanism involving the primarily inhibition of MEK activity and the subsequent phosphorylation of ERK1/2. This study provides evidence regarding the mechanism underlying the relaxation effect of vitexin on agonist-induced vascular contraction regardless of endothelial function.

Inhibitory effect of doxycycline conjugated with deoxycholic acid and polyethylenimine conjugate on nasal fibroblast differentiation and extracellular production.

BACKGROUND: Chronic rhinosinusitis (CRS) is an inflammatory disease affecting the sinuses or nose. Persistent inflammatory responses can lead to tissue remodeling, which is a pathological characteristics of CRS. Activation of fibroblasts in the nasal mucosal stroma, differentiation and collagen deposition, and subepithelial fibrosis have been associated with CRS. OBJECTIVES: We aimed to assess the inhibitory effects of doxycycline and deoxycholic acid-polyethyleneimine conjugate (DA3-Doxy) on myofibroblast differentiation and extracellular matrix (ECM) production in nasal fibroblasts stimulated with TGF-ß1. METHODS: To enhance efficacy, we prepared DA3-Doxy using a conjugate of low-molecular-weight polyethyleneimine (PEI) (MW 1800) and deoxycholic acid (DA) and Doxy. The synthesis of the DA3-Doxy polymer was confirmed using nuclear magnetic resonance, and the critical micelle concentration required for cationic micelle formation through self-assembly was determined. Subsequently, the Doxy loading efficiency of DA3 was assessed. The cytotoxicity of Doxy, DA3, PEI, and DA-Doxy in nasal fibroblasts was evaluated using the WST-1 assay. The anti-tissue remodeling and anti-inflammatory effects of DA3-Doxy and DA3 were examined using real-time polymerase chain reaction (Real-time PCR), immunocytochemistry, western blot, and Sircol assay. RESULTS: Both DA3 and DA3-Doxy exhibited cytotoxicity at 10 µg/ml in nasal fibroblasts. Doxy partially inhibited α-smooth muscle actin, collagen types I and III, and fibronectin. However, DA3-Doxy significantly inhibited α-SMA, collagen types I and III, and fibronectin at 5 µg/ml. DA3-Doxy also modulated TGF-ß1-induced changes in the expression of MMP 1, 2, and 9. Nonetheless, TGF-ß1-induced expression of MMP3 was further increased by DA3-Doxy. The expression of TIMP 1 and 2 was partially reduced with 5 µg/ml DA3-Doxy. CONCLUSIONS: Although initially developed for the delivery of genetic materials or drugs, DA3 exhibits inhibitory effects on myofibroblast differentiation and ECM production. Therefore, it holds therapeutic potential for CRS, and a synergistic effect can be expected when loaded with CRS treatment drugs.

Fabrication of stem cell heterospheroids with sustained-release chitosan and poly(lactic-co-glycolic acid) microspheres to guide cell fate toward chondrogenic differentiation.

Mesenchymal stem cell (MSC)-based therapies show great potential in treating various diseases. However, control of the fate of injected cells needs to be improved. In this work, we developed an efficient methodology for modulating chondrogenic differentiation of MSCs. We fabricated heterospheroids with two sustained-release depots, a quaternized chitosan microsphere (QCS-MP) and a poly (lactic-co-glycolic acid) microsphere (PLGA-MP). The results show that heterospheroids composed of 1 × 104 to 5 × 104 MSCs formed rapidly during incubation in methylcellulose medium and maintained high cell viability in long-term culture. The MPs were uniformly distributed in the heterospheroids, as shown by confocal laser scanning microscopy. Incorporation of transforming growth factor beta 3 into QCS-MPs and of dexamethasone into PLGA-MPs significantly promoted the expression of chondrogenic genes and high accumulation of glycosaminoglycan in heterospheroids. Changes in crucial metabolites in the dual drug depot-engineered heterospheroids were also evaluated using 1H NMR-based metabolomics analysis to verify their successful chondrogenic differentiation. Our heterospheroid fabrication platform could be used in tissue engineering to study the effects of various therapeutic agents on stem cell fate.

Target delivery and cell imaging using hyaluronic acid-functionalized graphene quantum dots.

This work demonstrates the way to achieve efficient and target specific delivery of a graphene quantum dot (GQD) using hyaluronic acid (HA) (GQD-HA) as a targeting agent. HA has been anchored to a GQD that accepts the fascinating adhesive properties of the catechol moiety, dopamine hydrochloride, conjugated to HA, which was confirmed by X-ray photoelectron spectroscopy. Transmission electron microscopy revealed a particle size of ∼20 nm, and the fluorescence spectra revealed significant fluorescence intensity even after the anchoring of HA. The prepared GQD-HA was applied to CD44 receptor overexpressed tumor-bearing balb/c female mice, and the in vivo biodistribution investigation demonstrated more bright fluorescence from the tumor tissue. In vitro cellular imaging, via a confocal laser scanning microscope, exhibited strong fluorescence from CD44 overexpressed A549 cells. Both in vivo and in vitro results showed the effectiveness of using HA as targeting molecule. The loading and release kinetics of the hydrophobic drug doxorubicin from a GQD under mildly acidic conditions showed that a GQD can be considered as a novel drug carrier, while the nontoxic behavior from the MTT assay strongly supports the identification of GQD-HA as a biocompatible material.

Enhanced transfection by antioxidative polymeric gene carrier that reduces polyplex-mediated cellular oxidative stress.

PURPOSE: To test the hypothesis in which polyplex-induced oxidative stress may affect overall transfection efficiency, an antioxidative transfection system minimizing cellular oxidative stress was designed for enhanced transfection. METHODS: An amphiphilic copolymer (PEI-PLGA) was synthesized and used as a micelle-type gene carrier containing hydrophobic antioxidant, α-tocopherol. Cellular oxidative stress and the change of mitochondrial membrane potential after transfection was measured by using a fluorescent probe (H2DCFDA) and lipophilic cationic probe (JC-1), respectively. Transfection efficiency was determined by measuring a reporter gene (luciferase) expression level. RESULTS: The initial transfection study with conventional PEI/plasmid DNA polyplex showed significant generation of reactive oxygen species (ROS). The PEI-PLGA copolymer successfully carried out the simultaneous delivery of α-tocopherol and plasmid DNA (PEI-PLGA/Toco/pDNA polyplex) into cells, resulting in a significant reduction in cellular ROS generation after transfection and helped to maintain the mitochondrial membrane potential (ΔΨ). In addition, the transfection efficiency was dramatically increased using the antioxidative transfection system. CONCLUSIONS: This work showed that oxidative stress would be one of the important factors that should be considered in designing non-viral gene carriers and suggested a possible way to reduce the carrier-mediated oxidative stress, which consequently leads to enhanced transfection.

Bioreducible crosslinked polyelectrolyte complexes for MMP-2 siRNA delivery into human vascular smooth muscle cells.

PURPOSE: Bioreducible crosslinked polyplexes were prepared via disulfide bond formation after siRNA condensation with polyethylenimine-modified by deoxycholic acid (PEI-DA) to stabilize polyplex structure in an extracellular environment and to promote transfection efficiency in human smooth muscle cells (hSMCs). METHODS: The PEI-DA/siRNA polyplexes were further modified by crosslinking the primary amines of PEI with thiol-cleavable crosslinkers. The effect of disulfide crosslinked PEI-DA/siRNA (Cr PEI-DA/siRNA) polyplexes on target gene silencing was investigated by transfecting hSMCs with matrix metalloproteinase-2 (MMP-2) siRNA under serum conditions. The MMP-2 levels in the conditioned medium were examined using gelatin zymography. RESULTS: The Cr PEI-DA/siRNA polyplexes showed increased stability against heparin exchange reactions, while their disulfide linkages were successfully cleaved under reducing conditions. The polyplex crosslinking reaction led to a slight decrease in MMP-2 gene silencing activity in hSMCs due to the insufficient redox potential. However, the gene silencing efficiency of the Cr PEI-DA/siRNA polypexes was gradually improved in response to increasing intracellular reduction potential. The increased serum stability of the Cr PEI-DA/siRNA polyplexes resulted in significant enhancement of the intracellular delivery efficiency especially under serum conditions. CONCLUSION: The Cr PEI-DA/siRNA polyplex formulation may be a promising siRNA delivery system for the treatment of incurable genetic disorders.

Delivery of self-replicating messenger RNA into the brain for the treatment of ischemic stroke.

Ischemic stroke is caused by the occlusion of cerebral arteries. In the ischemic stroke, ischemia-reperfusion injury increases the damage in the brain after reperfusion. In the previous study, heme oxygenase-1 (HO1) mRNA was delivered into the ischemic brain, showing that HO1-mRNA had higher therapeutic effect and less side-effect than HO1-plasmid (pHO1). However, mRNA is degraded faster than plasmid DNA reducing the duration of gene expression. In this study, self-replicating mRNA (Rep-mRNA) was developed using a replicon system from Venezuelan Equine Encephalitis virus to compensate this disadvantage of mRNA delivery. Deoxycholic acid-conjugated polyethylenimine (DA-PEI) was used as a carrier of the mRNAs. The Rep-mRNA/DA-PEI complex had a size of around 90 nm and a zeta-potential of 33 mV. In the in vitro transfection assays, gene expression by the HO1-Rep-mRNA/DA-PEI complex persisted at least 14 days, while that by the HO1-mRNA/DA-PEI complex approached basal level at 3 days after transfection. Therapeutic effects of the HO1-Rep-mRNA/DA-PEI complexes were evaluated in the ischemic stroke animal model. The complexes were injected into the brain stereotaxically. HO1 expression by the HO1-Rep-mRNA/DA-PEI complex persisted at least 7 days after injection, but the pHO1/DA-PEI or HO1-mRNA/DA-PEI complex showed basal level of HO1-expression at 7 days after injection. Due to higher and longer expression of HO1, the apoptosis level and infarct size were decreased by the HO1-Rep-mRNA/DA-PEI complexes, compared with the pHO1/DA-PEI and HO1-mRNA/DA-PEI complex. These results suggest that HO1-Rep-mRNA/DA-PEI complex may have a potential as a long-lasting therapeutic system for the treatment of ischemic stroke.

Self-assembled and nanostructured siRNA delivery systems.

A wide range of organic and inorganic materials have been used in the development of nano-scale self-assembling gene delivery systems to improve the therapeutic efficacy of nucleic acid drugs. Small interfering RNA (siRNA) has recently been recognized as a promising and potent nucleic acid medicine for the treatment of incurable genetic disorders including cancer; however, siRNA-based therapeutics suffer from the same delivery problems as conventional nucleic acid drugs such as plasmid DNA and antisense oligonucleotides. Many of the delivery strategies developed for nucleic acid drugs have been applied to siRNA therapeutics, but they have not produced satisfactory in vivo gene silencing efficiencies to warrant clinical trials. This review discusses recent progress in the development of self-assembled and nanostructured delivery systems for efficient siRNA-induced gene silencing and their potential application in clinical settings.

Reducible poly(amido ethylenimine)-based gene delivery system for improved nucleus trafficking of plasmid DNA.

In a nonviral gene delivery system, localization of a plasmid DNA in the nucleus is a prerequisite for expression of a desired therapeutic protein encoded in the plasmid DNA. In this study, a reducible polymer-based gene delivery system for improved intracellular trafficking and nuclear translocation of plasmid DNA is introduced. The system is consisted of two components, a plasmid DNA having repeated binding sequence for a karyophilic protein, NFkappaB, and a reducible polymer. A reducible poly(amido ethylenimine), poly(TETA-CBA), was synthesized by a Michael-type addition polymerization between cystamine bisacrylamide and triethyl tetramine. The polymer forming tight complexes with plasmid DNA could be degraded in the reductive cytosol to release the plasmid DNA. The triggered release mechanism in the cytosol could facilitate the interaction between cytosolic NFkappaB and the plasmid DNA having repeated NFkappaB biding motif. Upon activation of NFkappaB by interleukin-1beta (IL-1beta), most of the plasmid distributed in the cytoplasm was localized within the nucleus, resulting in significantly higher gene transfection efficiency than controls with nondegradable PEI. The current study suggests an alternative way of improving transfection efficiency by taking advantage of endogenous transport machinery for intracellular trafficking and nuclear translocation of a plasmid DNA.

Thermally triggered cellular uptake of quantum dots immobilized with poly(N-isopropylacrylamide) and cell penetrating peptide.

Thermally sensitive quantum dots (TSQDs) that exhibit an "on-demand" cellular uptake behavior via temperature-induced "shielding/deshielding" of cell penetrating peptides (CPP) on the surface were fabricated. Poly(N-isopropylacrylamide) (PNIPAAm) (M(w) = 11.5K) and CPP were biotinylated at their terminal ends and co-immobilized on to the surface of streptavidin-coated quantum dots (QDs-Strep) through biotin-streptavidin interaction. The cellular contact of CPP was sterically hindered due to hydrated PNIPAAm chains below the lower critical solution temperature (LCST). In contrast, above the LCST, grafted PNIPAAm chains were collapsed to make CPP moieties resurfaced, leading to increased cellular uptake of QDs. The temperature-controlled "shielding/deshielding" of CPP was further applied for a thermally triggered siRNA delivery system, where biotinylated siRNA was additionally conjugated to the surface of TSQDs. The level of gene silencing was significantly enhanced by increasing temperature above the LCST due to the surface exposure of CPP.

Protective effect of resveratrol on agonist-dependent regulation of vascular contractility via inhibition of rho-kinase activity.

The present study was undertaken to investigate the influence of resveratrol on vascular smooth muscle contractility and to determine the mechanism involved. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Resveratrol at a low concentration (0.03 mmol/l) relaxed directly and more markedly fluoride-induced vascular contraction than phorbol ester-induced contraction. Furthermore, resveratrol more markedly inhibited fluoride-induced increases in pMYPT1 levels than phorbol ester-induced increases. It also more markedly inhibited fluoride-induced increases in pMYPT1 levels than pERK1/2 levels, suggesting that the mechanism involved the inhibition of Rho-kinase activity and the subsequent phosphorylation of MYPT1. This study provides evidence regarding the mechanism underlying the relaxation effect of resveratrol on agonist-induced vascular contraction regardless of endothelial function.