Inverse Vulcanization Polymers with Enhanced Thermal Properties via Divinylbenzene Homopolymerization-Assisted Cross-Linking.

High-refractive-index sulfur-rich polymers with significantly improved thermal properties are prepared using divinylbenzene (DVB) as a comonomer in a modified, low-temperature inverse vulcanization with elemental sulfur. Differential scanning calorimetry and Fourier transform infrared studies reveal that under the modified inverse vulcanization conditions, homopolymerized DVB segments form, leading to high glass-transition temperatures (Tg > 100 °C) and thermal stability previously unattainable from the inverse vulcanization of bifunctional olefin comonomers. On the basis of the modified procedures, a three-step molding process of the inverse vulcanization product of DVB, poly(S-r-DVB), involving (1) prepolymer formation, (2) hot-press compression molding of the soft prepolymer, and (3) thermal annealing of the molded product is demonstrated. The molded high-sulfur-content poly(S-r-DVB) exhibits a high refractive index (n > 1.85), along with high midwave infrared transmittance. Combined with a high Tg, these properties render poly(S-r-DVB) with properties highly desirable in applications involving infrared optics.

Reducible Polyethylenimine Nanoparticles for Efficient siRNA Delivery in Corneal Neovascularization Therapy.

The aim of this study is to establish the safe and effective ocular delivery system of therapeutic small interfering RNA (siRNA) in corneal neovascularization therapy. The major hurdle present in siRNA-based corneal neovascularization (CNV) therapy is severe cytotoxicity caused by repetitive drug treatment. A reducible branched polyethylenimine (rBPEI)-based nanoparticle (NP) system is utilized as a new siRNA carrier as a hope for CNV therapy. The thiolated BPEI is readily self-crosslinked in mild conditions to make high molecular weight rBPEI thus allowing the creation of stable siRNA/rBPEI nanoparticles (siRNA-rBPEI-NPs). In the therapeutic region, the rBPEI polymeric matrix is effectively degraded into nontoxic LMW BPEI inside the reductive cytosol causing the rapid release of the encapsulated siRNA into the cytosol to carry out its function. The fluorescent-labeled siRNA-rBPEI-NPs can release siRNA into the entire corneal region after subconjuctival injection into the eye of Sprague Dawley rats thus confirming the proof of concept of this system.

Therapeutic Effect of Akt1 siRNA Nanoparticle Eluting Coronary Stent on Suppression of Post-Angioplasty Restenosis.

For effective treatment of restenosis, therapeutic genes are delivered locally from a coated stent at the site of injury, leading to inhibition of smooth muscle proliferation and neo-intimal hyperplasia while promoting re-endothelialization. In a previous study, we delivered Akt1 siRNA nanoparticles (ASNs) from a hyaluronic acid (HA)-coated stent surface to specifically suppress the pro-proliferative Akt1 protein in smooth muscle cells (SMCs). In the present study, therapeutic efficacy was investigated in a rabbit restenosis model after percutaneous implantation of an ASN-immobilized stent in a rabbit iliac artery. Quantitative and qualitative analyses of in-stent restenosis were investigated in an in vivo animal model by micro-CT imaging and SEM observation, respectively. Proliferation status and neo-intima formation of the vascular tissues located near ASN-immobilized stents were analyzed by immunohistochemical staining using anti-Akt1 and anti-Ki67 antibodies and histological analyses, such as hematoxylin and eosin staining and Verhoeff's elastic stain. Re-endothelialization after implantation of an ASN-immobilized stent was also analyzed via immunohistochemistry using an anti-CD31 antibody. To elucidate the molecular mechanism related to reducing SMC proliferation and subsequent inhibition of in-stent restenosis in vivo, protein and mRNA expression of Akt1 and downstream signaling proteins were analyzed after isolating SMC-rich samples from the treated vasculature. The implanted Akt1 siRNA-eluting stent efficiently mitigated in-stent restenosis without any side effects and can be considered a successful substitute to current drug-eluting stents.

Discovery of novel peptides targeting pro-atherogenic endothelium in disturbed flow regions -Targeted siRNA delivery to pro-atherogenic endothelium in vivo.

Atherosclerosis occurs preferentially in arterial regions exposed to disturbed blood flow. Targeting these pro-atherogenic regions is a potential anti-atherogenic therapeutic approach, but it has been extremely challenging. Here, using in vivo phage display approach and the partial carotid ligation model of flow-induced atherosclerosis in mouse, we identified novel peptides that specifically bind to endothelial cells (ECs) exposed to disturbed flow condition in pro-atherogenic regions. Two peptides, CLIRRTSIC and CPRRSHPIC, selectively bound to arterial ECs exposed to disturbed flow not only in the partially ligated carotids but also in the lesser curvature and branching point of the aortic arch in mice as well as human pulmonary artery branches. Peptides were conjugated to branched polyethylenimine-polyethylene glycol polymer to generate polyplexes carrying siRNA targeting intercellular adhesion molecule-1 (siICAM-1). In mouse model, CLIRRTSIC polyplexes carrying si-ICAM-1 specifically bound to endothelium in disturbed flow regions, reducing endothelial ICAM-1 expression. Mass spectrometry analysis revealed that non-muscle myosin heavy chain II A (NMHC IIA) is a protein targeted by CLIRRTSIC peptide. Further studies showed that shear stress regulates NMHC IIA expression and localization in ECs. The CLIRRTSIC is a novel peptide that could be used for targeted delivery of therapeutics such as siRNAs to pro-atherogenic endothelium.

Novel Fabrication of MicroRNA Nanoparticle-Coated Coronary Stent for Prevention of Post-Angioplasty Restenosis.

BACKGROUND AND OBJECTIVES: MicroRNA 145 is known to be responsible for cellular proliferation, and its enhanced expression reportedly inhibits the retardation of vascular smooth muscle cell growth specifically. In this study, we developed a microRNA 145 nanoparticle immobilized, hyaluronic acid (HA)-coated stent. MATERIALS AND METHODS: For the gene therapy, we used disulfide cross-linked low molecular polyethylenimine as the carrier. The microRNA 145 was labeled with YOYO-1 and the fluorescent microscopy images were obtained. The release of microRNA 145 from the stent was measured with an ultra violet spectrophotometer. The downstream targeting of the c-Myc protein and green fluorescent protein was determined by Western blotting. Finally, we deployed microRNA 145/ssPEI nanoparticles immobilized on HA-coated stents in the balloon-injured external iliac artery in a rabbit restenosis model. RESULTS: Cellular viability of the nanoparticle-immobilized surface tested using A10 vascular smooth muscle cells showed that MSN exhibited negligible cytotoxicity. In addition, microRNA 145 and downstream signaling proteins were identified by western blots with smooth muscle cell (SMC) lysates from the transfected A10 cell, as the molecular mechanism for decreased SMC proliferation that results in the inhibition of in-stent restenosis. MicroRNA 145 released from the stent suppressed the growth of the smooth muscle at the peri-stent implantation area, resulting in the prevention of restenosis at the post-implantation. We investigated the qualitative analyses of in-stent restenosis in the rabbit model using micro-computed tomography imaging and histological staining. CONCLUSION: MicroRNA 145-eluting stent mitigated in-stent restenosis efficiently with no side effects and can be considered a successful substitute to the current drug-eluting stent.

Polymeric biomaterials for the delivery of platinum-based anticancer drugs.

Since cisplatin, cis-diamminedichloroplatinum(ii), received FDA approval for use in cancer treatment in 1978, platinum-based drugs have been one of the most widely used drugs for the treatment of tumors in testicles, ovaries, head and neck. However, there are concerns associated with the use of platinum-based anticancer drugs, owing to severe side effects and drug resistance. In order to overcome these limitations, various drug-delivery systems have been developed based on diverse organic and inorganic materials. In particular, the versatility of polymeric materials facilitates the tuning of drug-delivery systems to meet their primary goals. This review focuses on the progress made over the last five years in the application of polymeric nanoparticles for the delivery of platinum-based anticancer drugs. The present article not only describes the fundamental principles underlying the implementation of polymeric nanomaterials in platinum-based drug delivery, but also summarizes concepts and strategies employed in the development of drug-delivery systems.

Enhanced tumor-targeted gene delivery by bioreducible polyethylenimine tethering EGFR divalent ligands.

This work demonstrates successful delivery of a gene to EGFR-overexpressed cancer cells by using a rationally designed branched GE11 peptide as a targeting ligand. In addition, we exploited the effect of the divalent structure of the branched GE11 peptide on the gene delivery and tumor targeting efficiency, compared to the monovalent GE11 peptide. The GE11 or branched GE11-tethered polymers were successfully synthesized. They are composed of a targeting peptide, disulfide crosslinked low molecular weight polyethylenimine and polyethylene glycol. Here, we evaluated the physicochemical properties, cytotoxicity and in vitro transfection efficiency and in vivo biodistribution of the GE11 and branched GE11 tethered polyplexes. Our results demonstrated that GE11 and bGE11-tethered gene delivery carriers showed efficient gene condensing ability, an enhanced transfection efficiency and targeting ability with low cytotoxicity. Interestingly, the branched GE11-tethered polymer showed the greater targeting ability to EGFR-overexpressed cancer cells in vivo than the GE11-tethered polymer. Therefore, this branched structure of targeting ligand has the potential for providing a novel strategy to design an efficient targeted delivery system.

RPM peptide conjugated bioreducible polyethylenimine targeting invasive colon cancer.

CPIEDRPMC (RPM) peptide is a peptide that specifically targets invasive colorectal cancer, which is one of the leading causes of cancer-related deaths worldwide. In this study, we exploited RPM peptide as a targeting ligand to produce a novel and efficient gene delivery system that could potentially be used to treat invasive colon cancer. In order to achieve enhanced specificity to colon cancer cells, the RPM peptide was conjugated to a bioreducible gene carrier consisting of a reducible moiety of disulfide-crosslinked low molecular weight polyethylenimine, IR820 dye, and polyethylene glycol. Here, we examined the physiochemical properties, cytotoxicity, in vitro transfection efficiency, and in vivo biodistribution of the RPM-conjugated polyplex. Our results showed that the RPM-conjugated gene carrier formed a compact polyplex with pDNA that had low toxicity. Furthermore, the RPM-conjugated polymer not only had higher cellular uptake in invasive colon cancer than the non-targeted polymer, but also showed enhanced transfection efficiency in invasive colon cancer cells in vitro and in vivo.

Di-Sulfide Linked Polyethylenimine Coated Gold Nanoparticles as a Non-Viral Gene Delivery Agent in NIH-3T3 Mouse Embryonic Fibroblast.

Di-sulfide linked polyethylenimine coated gold nanoparticles (ssPEI-GNPs) of 20 nm size was prepared in order to deliver the genes to target site. DLS and TEM analysis demonstrated that the GNPs have average size of 13 nm in diameter. Upon coating the GNPs with ssPEI in the weight ratio of 1:3, the average hydrodynamic diameter of the ssPEI-GNPs was found to 19±1.14 nm and a zeta potential value 41±1.23 mV was observed. TEM analysis of ssPEI-GNPs demonstrated that the nanoparticles have spherical morphology. Thermogravemetric analysis of the prepared ssPEI-GNPs showed that the estimated composition of the ssPEI coated over the GNPs was approximately 5% (w/w). Gene expression capabilities of the nanoparticles were confirmed by fluorescent microscopy and luciferase assay, which demonstrated the transgene delivery capability of the ssPEI-GNPs. These results demonstrate that ssPEI-GNPs could be used as gene delivery agent.

Bioreducible guanidinylated polyethylenimine for efficient gene delivery.

Cationic polymers are known to afford efficient gene transfection. However, cytotoxicity remains a problem at the molecular weight for optimal DNA delivery. As such, optimized polymeric gene delivery systems are still a sought-after research goal. A guanidinylated bioreducible branched polyethylenimine (GBPEI-SS) was synthesized by using a disulfide bond to crosslink the guanidinylated BPEI (GBPEI). GBPEI-SS showed sufficient plasmid DNA (pDNA) condensation ability. The physicochemical properties of GBPEI-SS demonstrate that it has the appropriate size (~200 nm) and surface potential (~30 mV) at a nitrogen-to-phosphorus ratio of 10. No significant toxicity was observed, possibly due to bioreducibility and to the guanidine group delocalizing the positive charge of the primary amine in BPEI. Compared with the nonguanidinylated analogue, BPEI-SS, GBPEI-SS showed enhanced transfection efficiency owing to increased cellular uptake and efficient pDNA release by cleavage of disulfide bonds. This system is very efficient for delivering pDNA into cells, thereby achieving high transfection efficiency and low cytotoxicity.

Bioreducible polymer-delivered siRNA targeting MMP-9: suppression of granulation tissue formation after bare metallic stent placement in a rat urethral model.

PURPOSE: To evaluate the effectiveness of small interfering RNA (siRNA) targeting matrix metalloproteinase 9 (MMP-9) in suppressing granulation tissue formation caused by bare metallic stent placement in a rat urethral model. MATERIALS AND METHODS: All experiments were approved by the committee of animal research. In 20 Sprague-Dawley male rats (weight range, 300-350 g), a self-expanding metallic bare stent was inserted in the urethra with fluoroscopic guidance. One group of 10 rats (group A) was treated with MMP-9 siRNA/bioreducible branched polyethylenimine-disulfide cross-linked-indocyanine green (bioreducible BPEI-SS-ICG), while the other group of 10 rats (group B) received control siRNA/bioreducible BPEI-SS-ICG treatment. All rats were sacrificed at 4 weeks. The therapeutic effectiveness of the MMP-9 siRNA/bioreducible BPEI-SS-ICG complex was assessed by comparing the two results of retrograde urethrography, histologic examination, and quantification of MMP-9 by using zymography and Western blot analysis between the two groups. The Mann-Whitney U test was used to evaluate differences. RESULTS: Stent placement was successful in all rats without a single case of migration at follow-up. Retrograde urethrography performed 4 weeks after stent placement demonstrated significantly larger luminal diameters of the urethra within the stents in group A compared with those in group B (P = .011). Histologic analysis revealed that the mean percentage of granulation tissue area (P < .001), mean number of epithelial layers (P < .001), and mean thickness of submucosal fibrosis (P < .001) were significantly decreased in group A compared with group B. Meanwhile, the mean density of inflammatory cell infiltration did not significantly differ between the two groups (P = .184). Quantitative analysis disclosed MMP-9 levels to be lower in group A relative to group B, indicating positive inhibition of MMP-9 by MMP-9 siRNA/bioreducible BPEI-SS-ICG. CONCLUSION: MMP-9 siRNA/bioreducible BPEI-SS-ICG is effective for inhibiting granulation tissue formation after bare metallic stent placement in a rat urethral model.

Photothermally triggered cytosolic drug delivery via endosome disruption using a functionalized reduced graphene oxide.

Graphene oxide has unique physiochemical properties, showing great potential in biomedical applications. In the present work, functionalized reduced graphene oxide (PEG-BPEI-rGO) has been developed as a nanotemplate for photothermally triggered cytosolic drug delivery by inducing endosomal disruption and subsequent drug release. PEG-BPEI-rGO has the ability to load a greater amount of doxorubicin (DOX) than unreduced PEG-BPEI-GO via π-π and hydrophobic interactions, showing high water stability. Loaded DOX could be efficiently released by glutathione (GSH) and the photothermal effect of irradiated near IR (NIR) in test tubes as well as in cells. Importantly, PEG-BPEI-rGO/DOX complex was found to escape from endosomes after cellular uptake by photothermally induced endosomal disruption and the proton sponge effect, followed by GSH-induced DOX release into the cytosol. Finally, it was concluded that a greater cancer cell death efficacy was observed in PEG-BPEI-rGO/DOX complex-treated cells with NIR irradiation than those with no irradiation. This study demonstrated the development of the potential of a PEG-BPEI-rGO nanocarrier by photothermally triggered cytosolic drug delivery via endosomal disruption.

Turn-on fluorescence detection of apoptotic cells using a zinc(II)-dipicolylamine-functionalized poly(diacetylene) liposome.

A liposome-based fluorescence sensing system for apoptotic cells has been developed from stimuli-responsive poly(diacetylene)-liposomes for the first time. The combination of the liposome components, a phosphatidylserine-binding Zn(II)-dipicolylamine component and an alcohol-terminated component in the ratio of 2:1, has led to an efficient detection system for apoptotic cells, as demonstrated by confocal fluorescence microscopy and FACS analysis. The liposome shows a color change from blue to reddish purple and emits fluorescence in the turn-on mode upon interaction with phosphatidylserine. The present system thus avoids the washing steps required for "always-on"-type sensing systems.

A brain-targeted rabies virus glycoprotein-disulfide linked PEI nanocarrier for delivery of neurogenic microRNA.

Recent advances in efficient microRNA (miRNA) delivery techniques using brain-targeted nanoparticles offer critical information for understanding the functional role of miRNAs in vivo, and for supporting targeted gene therapy in terms of treating miRNA-associated neurological diseases. Here, we report the rabies virus glycoprotein (RVG)-labeled non-toxic SSPEI nanomaterials capable of neuron-specific miR-124a delivery to neuron in vivo. The RVG-labeled BPEI-SS (RVG-SSPEI) nanocarrier showed less toxicity in acetylcholine receptor-positive Neuro2a cells, and electrostatic interaction of RVG-SSPEI with miR-124a exhibited optimal transfection efficacy. The RVG-SSPEI polymer specifically targeted Neuro2a using cy5.5-miR-124a mixed with RVG-SSPEI. The functional action of miR-124a oligomers released from polyplexes in the cytoplasmic region was evaluated by a reporter vector containing a miR-124a -binding sequence, and showed a significantly reduced reporter signal in a dose-dependent manner. Cy5.5-miR-124a/RVG-SSPEI- injected into mice via tail veins displayed the enhanced accumulation of miR-124a in the isolated brain. Hindrance of the efficient penetration of neuronal cells by size limitation of the miR-124a/RVG-SSPEI improved with the help of mannitol through blood-brain barrier disruption. These findings indicated that the RVG peptide combined with mannitol infusion using SSPEI polymer for neuron-specific targeting in vivo is sufficient to deliver neurogenic microRNA into the brain.