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ABSTRACT: We developed a new detection method for l-Dopa decarboxylase (DDC) activity using gold nanoparticles (AuNPs). When l-3,4-dihydroxyphenylalanine (l-Dopa) is decarboxylated to dopamine (DA) by DDC, DA induces the aggregation of AuNPs, and the color of the AuNPs changes from red to blue.
The Analyst 04/2013; · 4.23 Impact Factor
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ABSTRACT: The cardiomyocyte apoptosis plays a critical role in the development of myocardial injury after ischemia and reperfusion. Thus, alteration of the major apoptosis-regulatory factors during myocardial ischemia-reperfusion is expected to have favorable cardioprotective effects. Herein, we report ischemic-reperfused myocardial infarction (MI) repair with siRNA against Src homology region 2 domain-containing tyrosine phosphatase-1 (SHP-1), which is known as a key factor involved in regulating the progress of apoptosis in many cell types. A low molecular weight polyethyleneimine modified with deoxycholic acid (PEI1.8-DA)-based delivery strategy was suggested for the cardiac application of SHP-1 siRNA to overcome the poor gene delivery efficiency to myocardium due to the highly charged structures of the compact cardiac muscles. The PEI1.8-DA conjugates formed stable nanocomplexes with SHP-1 siRNA via electrostatic and hydrophobic interactions. The PEI1.8-DA/SHP-1 siRNA polyplexes effectively silenced SHP-1 gene expression in cardiomyocytes, leading to a significant inhibition of cardiomyocyte apoptosis under hypoxia. In comparison to conventional gene carriers, relatively large amounts of siRNA molecules remained after treatment with the PEI1.8-DA/SHP-1 siRNA polyplexes. Cardiac administration of the PEI1.8-DA/SHP-1 siRNA polyplexes resulted in substantial improvement in SHP-1 gene silencing, which can be explained by the enhancement of cardiac delivery efficiency of the PEI1.8-DA conjugates. In addition, in vivo treatment with the PEI1.8-DA/SHP-1 siRNA polyplexes induced a highly significant reduction in myocardial apoptosis and infarct size in rat MI models. These results demonstrate that the PEI1.8-DA/SHP-1 siRNA polyplex formulation is a useful system for efficient gene delivery into the compact myocardium that provides a fundamental advantage in treating ischemic-reperfused MI.
Journal of Controlled Release 03/2013; · 5.73 Impact Factor
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ABSTRACT: Indocyanine green (ICG) encapsulated hyaluronic acid (HA) nanogels were first studied for highly selective detection of specific cancers and lymph nodes via hyaluronidase sensitive switch-on of near infrared fluorescence as a long-lasting and stimuli-responsive imaging probe.
Chemical Communications 06/2012; 48(69):8628-30. · 6.17 Impact Factor
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Macromolecular Research 05/2012; · 1.15 Impact Factor
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ABSTRACT: 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).
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.
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.
The Cr PEI-DA/siRNA polyplex formulation may be a promising siRNA delivery system for the treatment of incurable genetic disorders.
Pharmaceutical Research 04/2012; 29(8):2213-24. · 4.09 Impact Factor
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ABSTRACT: Because of RNA's ability to encode structure and functional information, researchers have fabricated diverse geometric structures from this polymer at the micro- and nanoscale. With their tunable structures, rigidity, and biocompatibility, novel two-dimensional and three-dimensional RNA structures can serve as a fundamental platform for biomedical applications, including engineered tissues, biosensors, and drug delivery vehicles. The discovery of the potential of small-interfering RNA (siRNA) has underscored the applications of RNA-based micro- and nanostructures in medicine. Small-interfering RNA (siRNA), synthetic double-stranded RNA consisting of approximately 21 base pairs, suppresses problematic target genes in a sequence-specific manner via inherent RNA interference (RNAi) processing. As a result, siRNA offers a potential strategy for treatment of many human diseases. However, due to inefficient delivery to cells and off-target effects, the clinical application of therapeutic siRNA has been very challenging. To address these issues, researchers have studied a variety of nanocarrier systems for siRNA delivery. In this Account, we describe several strategies for efficient siRNA delivery and selective gene silencing. We took advantage of facile chemical conjugation and complementary hybridization to design novel siRNA-based micro- and nanostructures. Using chemical crosslinkers and hydrophobic/hydrophilic polymers at the end of siRNA, we produced various RNA-based structures, including siRNA block copolymers, micelles, linear siRNA homopolymers, and microhydrogels. Because of their increased charge density and flexibility compared with conventional siRNA, these micro- and nanostructures can form polyelectrolyte complexes with poorly charged and biocompatible cationic carriers that are both more condensed and more homogenous than the complexes formed in other carrier systems. In addition, the fabricated siRNA-based structures are linked by cleavable disulfide bonds for facile generation of original siRNA in the cytosol and for target-specific gene silencing. These newly developed siRNA-based structures greatly enhance intracellular uptake and gene silencing both in vitro and in vivo, making them promising biomaterials for siRNA therapeutics.
Accounts of Chemical Research 03/2012; 45(7):1014-25. · 21.64 Impact Factor
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ABSTRACT: The first attempt to prepare biologically active siRNA-based microhydrogels is reported. The self-assembled microhydrogels were fabricated using sense/antisense complementary hybridization between single-stranded linear and Y-shaped trimeric siRNAs. The siRNA microhydrogels were condensed using a popular cationic polymer such as LPEI to form compact, stable siRNA/polymeric nanoparticles that exhibited superb cellular uptake efficiency and gene silencing activity.
Journal of the American Chemical Society 08/2011; 133(35):13914-7. · 9.91 Impact Factor
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ABSTRACT: Simultaneous silencing of multiple up-regulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. Herein we report that multimerized siRNA conjugate composed of two different siRNA sequences in the same backbone shows more efficient inhibition of the two corresponding target genes at one time than physically mixed multimerized siRNA conjugates. Two model siRNAs against VEGF and GFP gene were chemically crosslinked via cleavable and noncleavable linkages for the preparation of dual gene targeted multimeric siRNA conjugates (DGT multi-siRNA). Cleavable DGT multi-siRNA with reducible disulfide linkages exhibited significantly higher gene silencing efficiencies at mRNA and protein expression levels than noncleavable DGT multi-siRNA, the physical mixture of naked siRNA, and that of single gene targeted multimeric siRNA (SGT multi-siRNA) with eliciting negligible immune response. DGT multi-siRNAs against two therapeutic siRNAs, anti-survivin and anti-bcl-2 targeted siRNA, also showed greatly enhanced apoptotic effect. This approach for concurrent suppression of combinatorial therapeutic target genes using cleavable multimeric siRNA structure can be potentially used for improved therapeutic efficacy.
Biomaterials 03/2011; · 7.40 Impact Factor
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ABSTRACT: Biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) was conjugated to the 3' end of small interfering RNA (siRNA) via a disulfide bond to synthesize siRNA-PLGA hybrid conjugates. siRNA-PLGA conjugates were spontaneously self-assembled to form a spherical core/shell type micellar structure of ~20 nm in an aqueous environment, probably by hydrophobic interaction of PLGA blocks in the core surrounded by an siRNA shell layer. When linear polyethylenimine was added to the siRNA-PLGA micelles in aqueous solution, stable siRNA-PLGA/LPEI micelles with a size of ~30 nm were produced via ionic complexation between siRNA and LPEI in the outer shell. The cationic siRNA-PLGA/LPEI micelles showed superior intracellular uptake and enhanced gene silencing effect, compared to naked siRNA/LPEI complexes. The hybrid micelle structure based on siRNA and PLGA can be potentially used as an efficient siRNA delivery system for gene silencing.
Journal of Controlled Release 12/2010; 152(1):152-8. · 5.73 Impact Factor
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ABSTRACT: Di- and triblock siRNA/PEG copolymers were synthesized and complexed with cationic SLN for assessing their gene silencing efficiency as a function of PEG density. A sssiRNA and a sassiRNA were separately conjugated with PEG via a disulfide linkage. AB-type diblock and ABA-type triblock copolymers were successfully prepared by stoichiometric hybridization of sssiRNA-PEG conjugate with sassiRNA and sassiRNA-PEG conjugate, respectively. The resultant di- and triblock copolymers were characterized by means of GPC and gel electrophoresis. The serum stability of siRNA in the copolymers was enhanced as compared to that of naked siRNA. Using cationic SLN as a model carrier, the PEGylation density effect of the siRNA-PEG/SLN complexes on gene silencing and cellular uptake was analyzed.
Macromolecular Bioscience 11/2010; 11(3):410-8. · 3.89 Impact Factor
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ABSTRACT: Small interfering RNA (siRNA) was conjugated with poly(ethylene glycol) (PEG) at four different terminal ends (sense 3', sense 5', antisense 3', and antisense 5') via cleavable disulfide and noncleavable thioether linkages to evaluate their gene silencing efficiencies upon complexation with Lipofectamine2000. The PEGylation site at the four siRNA termini and PEG molecular weight were not critical factors to significantly affect gene silencing activities. Cleavable siRNA-PEG conjugates showed comparable gene silencing activities to naked siRNA, and exhibited sequence-specific degradation of a target mRNA. Interestingly, noncleavable siRNA-PEG conjugates were processed by Dicer, enabling to exert RNAi effect without showing a target sequence-specific manner. However, only cleavable siRNA-PEG conjugates significantly reduced the extent of INF-alpha release as compared to noncleavable siRNA-PEG conjugates, suggesting that they can be potentially used for therapeutic siRNA applications.
Journal of Controlled Release 03/2010; 144(3):306-13. · 5.73 Impact Factor
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ABSTRACT: Small interfering RNA (siRNA) with 19-21 base pairs has been recently recognized as a new therapeutic agent for effectively silencing a specific gene on a post-transcription level. For siRNA therapeutics, safe and efficient delivery issues are significant hurdles to clinical applications. Here we present a new class of biologically active siRNA structure based on chemically self-crosslinked and multimerized siRNA through cleavable disulphide linkages. The multimerized siRNA can produce more stable and compact polyelectrolyte complexes with less cytotoxic cationic carriers than naked siRNA because of substantially increased charge densities and the presence of flexible chemical linkers in the backbone. The cleavable and multimerized siRNA shows greatly enhanced gene-silencing efficiencies in vitro and in vivo through a target-messenger-RNA-specific RNA interference processing without significantly eliciting immune induction. This study demonstrates that the multimerized siRNA structure complexed with selected cationic condensing agents can serve as potential gene-silencing therapeutics for treating various diseases.
Nature Material 03/2010; 9(3):272-8. · 32.84 Impact Factor
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ABSTRACT: For therapeutic applications of small interfering RNA (siRNA), serum stability, enhanced cellular uptake, and facile endosome escape are key issues for designing carriers. In this study, green fluorescent protein (GFP) siRNA was conjugated to a six-arm polyethylene glycol (PEG) derivative via a reducible disulfide linkage (6PEG-siRNA). The 6PEG-siRNA conjugate was also functionalized with a cell penetrating peptide, Hph1 to enhance its cellular uptake property (6PEG-siRNA-Hph1). The 6PEG-siRNA-Hph1 conjugate was electrostatically complexed with cationic self-crosslinked fusogenic KALA peptide (cl-KALA) to form multifunctional polyelectrolyte complex micelles for gene silencing. The resultant siRNA complex formulation with multiple PEG chains showed superior physical stability and resistance to enzymatic degradation. The 6PEG-siRNA-Hph1/cl-KALA complexes exhibited enhanced GFP gene silencing efficiency for MDA-MB-435 cells in the serum containing condition. The current reducible and multifunctional polyelectrolyte complex micelles are expected to have high potential for efficient delivery of therapeutic siRNA.
Biotechnology Progress 11/2009; 26(1):57-63. · 2.34 Impact Factor
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ABSTRACT: Polyethylene glycol (PEG) has been chemically immobilized onto the surface of adenoviruses (ADVs) to reduce non-specific immune response and extend blood circulation time while maintaining the high transduction efficiency of a foreign gene into cells. In this study, ADVs encoding an exogenous green fluorescent protein (GFP) were physically coated with PEG grafted and blocked poly-L-lysine (PLL-g-PEG and PLL-b-PEG) copolymers via ionic interactions to comparatively evaluate their gene transduction efficiency. The surface immobilization of ADVs with the two types of PLL-PEG copolymers exhibited significantly increased GFP transduction activity, compared to that of naked ADVs. ADVs coated with PLL-b-PEG showed higher extent of GFP expression than those with PLL-g-PEG under serum conditions. For PLL-g-PEG copolymers, the substitution degree of PEG in the PLL backbone greatly influenced the gene expression level. Additionally, ADVs modified with PLL-b-PEG exhibited greater transduction efficiency for bone marrow derived human mesenchymal stem cells compared to naked or PLL coated ADVs in the serum condition. This study suggests that enhanced ADV gene transduction efficiency can be attained for various cells by simply coating PLL-b-PEG on the surface.
Journal of Controlled Release 11/2009; 142(2):238-44. · 5.73 Impact Factor
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ABSTRACT: Cationic polymers have been chemically modified with a variety of targeting molecules such as peptides, proteins, antibodies, sugars and vitamins for targeted delivery of nucleic acid drugs to specific cells. Stimuli-sensitive polymers exhibiting different size, charge and conformation in response to physiological signals from specific cells have also been utilized for targeted delivery. To achieve target-specific delivery of nucleic acids, conjugation chemistry is critical to produce stable nanosized polyplexes tethered with cell-recognizable ligands for facile cellular uptake via a receptor-mediated endocytic pathway. In this review, synthetic strategies of functional cationic polymers with various targeting ligands are presented.
Macromolecular Bioscience 09/2009; 9(8):731-43. · 3.89 Impact Factor
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ABSTRACT: Polyethylene glycol (PEG)-immobilized quantum dot (QD) nanoparticles, which could be specifically dePEGylated in response to the presence of the matrix metalloprotease-2 (MMP-2) enzyme, were prepared. The degree of PEGylation (MW 3400) on the surface of 12 nm streptavidin-coated QDs was stoichiometrically controlled by varying the feed amount of a biotin-substrate-PEG conjugate, where the substrate contained an MMP-2 cleavable peptide sequence. A biotin-cell penetrating peptide (CPP) conjugate was also immobilized onto the surface of the PEGylated QD surface to enhance the cellular uptake after dePEGylation. It was found that more than nine PEG chains per single QD were required to effectively inhibit the cellular uptake of modified QD particles down to around 20%, as compared with that of QD without PEG chains. However, the treatment of MMP-2 enzyme in the medium resulted in a substantial enhancement in the extent of QD cellular uptake by dePEGylation with concomitant resurfacing of sterically hidden CPP moieties. This study analyzed the effects of surface PEGylation density and MMP-2 specific dePEGylation on the cellular uptake of CPP-QD nanoparticles in a quantitative manner.
Langmuir 01/2009; 25(3):1645-50. · 4.19 Impact Factor
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ABSTRACT: Small interfering RNA (siRNA) has been chemically conjugated to a variety of bioactive molecules, lipids, polymers, peptides, and inorganic nanostructured materials to enhance their pharmacokinetic behavior, cellular uptake, target specificity, and safety. To efficiently deliver siRNAs to the target cells and tissues, many different siRNA bioconjugates were synthesized and characterized, and their gene silencing efficiencies were tested in vitro and in vivo. In this review, recent developments of siRNA bioconjugates are summarized.
Bioconjugate Chemistry 12/2008; 20(1):5-14. · 4.93 Impact Factor
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ABSTRACT: A novel self-crosslinked and reducible peptide was synthesized for stable formation of nanoscale complexes with an siRNA-PEG conjugate to enhance transfection efficiency in serum containing condition without compromising cytotoxicity. A fusogenic peptide, KALA, with two cysteine residues at both terminal ends was crosslinked via disulfide linkages under mild DMSO oxidation condition. The reducible crosslinked KALA (cl-KALA) was used to form nano-complexes with green fluorescent protein (GFP) siRNA. Size and morphology of various polyelectrolyte complexes formulated with KALA and cl-KALA were comparatively analyzed. cl-KALA exhibited more reduced cell cytotoxicity and formed more stable and compact polyelectrolyte complexes with siRNA, compared with naked KALA and polyethylenimine (PEI), probably because of its increased charge density. The extent of gene silencing was quantitatively evaluated using MDA-MB-435 cells. cl-KALA/siRNA complexes showed comparable gene silencing efficiency with those of cytotoxic PEI. In a serum containing medium, cl-KALA/siRNA-PEG conjugate complexes exhibited superior gene inhibition because of the shielding effect of PEG on the surface. The formulation based on the self-crosslinked fusogenic peptide could be used as a biocompatible and efficient nonviral carrier for siRNA delivery.
Biopolymers 10/2008; 89(10):881-8. · 2.87 Impact Factor
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ABSTRACT: Reducible heparin nanogels cross-linked with disulfide linkages were developed for efficient cellular uptake of therapeutic heparin to induce apoptotic cell death. The heparin nanogels were synthesized by forming nanocomplexes between thiolated heparin and poly(ethylene glycol) in a selected organic solvent, and subsequently producing intermolecular disulfide bonds between thiolated heparin molecules by ultrasonication. The resultant heparin nanogels had a stable structure with an average diameter of 248.7+/-26.8nm in aqueous solution. However, they rapidly disintegrated and released free heparin molecules under reductive environments, such as intracellular cytosol, through the cleavage of disulfide cross-links within their network structure. Confocal laser scanning microscopy and flow cytometric analysis revealed that these heparin nanogels significantly inhibited proliferation of mouse melanoma cells by inducing caspase-mediated apoptotic cell death. The present study suggested that the reducible heparin nanogels exhibiting a remarkable apoptotic activity could be potentially applied for cancer cell targeted delivery when combined with various therapeutic and diagnostic agents.
Biomaterials 09/2008; 29(23):3376-83. · 7.40 Impact Factor
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ABSTRACT: Various biomacromolecules (BMs) such as proteins, DNA, and carbohydrates are extremely difficult to be dissolved in a single organic solvent phase for sustained release or targeted delivery formulation. In this study, three different BMs could be solubilized in selected organic solvents by forming poly(ethylene glycol) (PEG)-assisted nano-complexes while maintaining their structural integrity. Dynamic light scattering (DLS) and atomic force microscopy (AFM) analysis revealed that proteins, DNA, and carbohydrate polymers could be nano-complexed with PEG in various organic solvents. The diameter of nano-complexes decreased roughly from approximately 600 nm to approximately 100 nm with increasing weight ratio of PEG/BM. The present solubilization technique could be potentially applied for sustained release formulations of various therapeutic biological drugs.
International Journal of Pharmaceutics 06/2008; 356(1-2):306-13. · 3.35 Impact Factor
