Phosphonium Polymethacrylates for Short Interfering RNA Delivery: Effect of Polymer and RNA Structural Parameters on Polyplex Assembly and Gene Knockdown.

Synthetic polymers containing quaternary phosphonium salts are an emerging class of materials for the delivery of oligo/polynucleotides. In this work, cationic phosphonium salt-containing polymethacrylates and their corresponding ammonium analogues were synthesized by reversible addition-fragmentation chain transfer polymerization. Both the nature of the charged heteroatom (N vs P) and the length of the spacer separating the cationic units along the polymer backbone (oxyethylene vs trioxyethylene) were systematically varied. Polymers efficiently bound short interfering RNA (siRNA) at N(+)/P(-) or P(+)/P(-) ratios of 2 and above. At a 20:1 ratio, small polyplexes (Rh: 4-15 nm) suitable for cellular uptake were formed that displayed low cytotoxicity. While siRNA polyplexes from both ammonium and phosphonium polymers were efficiently internalized by green fluorescent protein (GFP)-expressing 3T3 cells, no knockdown of GFP expression was observed. However, 65% Survivin gene knockdown was observed when siRNA was replaced with novel, multimerized long interfering RNA in HeLa cells, demonstrating the importance of RNA macromolecular architecture on RNA-mediated gene silencing.

Paclitaxel-eluting nanofiber-covered self-expanding nonvascular stent for palliative chemotherapy of gastrointestinal cancer and its related stenosis.

Self-expanding non-vascular metal stents (SEMS) is now a choice of treatment for tumor-induced obstructive symptoms of gastrointestinal tract. But in-growing tumor causes re-stenosis. Here, we studied a paclitaxel-eluting nanofiber-covered stent for palliative chemotherapy of gastrointestinal cancer and its related stenosis. In vivo and in vitro feasibility of nanofiber-covered nonvascular stent was evaluated in this study. Nanofiber-covered stent released paclitaxel (PTX) in controlled manner for 30 days. PTX-NFM significantly inhibited the growth of CT-26 colon cancer in comparison with PTX injection. PTX maintained higher tumor concentrations over 1.0 µg/ml for more than 14 days without systemic exposure. TUNEL and H&E staining proved locally concentrated PTX induced the higher apoptosis than PTX injection. In this way, PTX-eluting nanofiber-covered stent possibly inhibits in-growth of cancer and extends patency of stent. Clinical feasibility of PTX-eluting nanofiber nonvascular stent for cholangiocarcinoma and gastrointestinal cancers will be investigated in further studies.

Long dsRNA-mediated RNA interference and immunostimulation: a targeted delivery approach using polyethyleneimine based nano-carriers.

RNA oligonucleotides capable of inducing controlled immunostimulation combined with specific oncogene silencing via an RNA interference (RNAi) mechanism provide synergistic inhibition of cancer cell growth. With this concept, we previously designed a potent immunostimulatory long double stranded RNA, referred to as liRNA, capable of executing RNAi mediated specific target gene silencing. In this study, we developed a highly effective liRNA based targeted delivery system to apply in the treatment of glioblastoma multiforme. A stable nanocomplex was fabricated by complexing multimerized liRNA structures with cross-linked branched poly(ethylene imine) (bPEI) via electrostatic interactions. We show clear evidence that the cross-linked bPEI was quite effective in enhancing the cellular uptake of liRNA on U87MG cells. Moreover, the liRNA-PEI nanocomplex provided strong RNAi mediated target gene silencing compared to that of the conventional siRNA-PEI complex. Further, the bPEI modification strategy with specific ligand attachment assisted the uptake of the liRNA-PEI complex on the mouse brain endothelial cell line (b.End3). Such delivery systems combining the beneficial elements of targeted delivery, controlled immunostimulation, and RNAi mediated target silencing have immense potential in anticancer therapy.

Biocompatible Cu/NiMo Composite Electrocatalyst for Hydrogen Evolution Reaction in Microbial Electrosynthesis; Unveiling the Self-Detoxification Effect of Cu.

H2-driven microbial electrosynthesis (MES) is an emerging bioelectrochemical technology that enables the production of complex compounds from CO2. Although the performance of microbial fermentation in the MES system is closely related to the H2 production rate, high-performing metallic H2-evolving catalysts (HEC) generate cytotoxic H2O2 and metal cations from undesirable side reactions, severely damaging microorganisms. Herein, a novel design for self-detoxifying metallic HEC, resulting in biologically benign H2 production, is reported. Cu/NiMo composite HEC suppresses H2O2 evolution by altering the O2 reduction kinetics to a four-electron pathway and subsequently decomposes the inevitably generated H2O2 in sequential catalytic and electrochemical pathways. Furthermore, in situ generated Cu-rich layer at the surface prevents NiMo from corroding and releasing cytotoxic Ni cations. Consequently, the Cu/NiMo composite HEC in the MES system registers a 50% increase in the performance of lithoautotrophic bacterium Cupriavidus necator H16, for the conversion of CO2 to a biopolymer, poly(3-hydroxybutyrate). This work successfully demonstrates the concept of self-detoxification in designing biocompatible materials for bioelectrochemical applications as well as MES systems.

Enhanced intracellular delivery and multi-target gene silencing triggered by tripodal RNA structures.

BACKGROUND: The development of gene interfering RNA (iRNA) molecules such as small interfering RNAs (siRNAs) and antagomirs provides promising therapeutic modalities for targeting specific mRNAs and microRNAs (miRNAs) involved in disease mechanisms. Therapeutic iRNA strategy against cancer or hypermutable viruses prefers targeting multiple genes simultaneously to achieve synergistic inhibition and to prevent resistance. METHODS: In the present study, we report chemically synthesized, multi-target gene interfering RNA structures based upon branched, tripodal interfering RNAs (termed T-tiRNAs). RESULTS: The T-tiRNAs could simultaneously inhibit up to three different mRNAs or miRNAs by harboring three siRNA or antagomir units. Moreover, when complexed with cationic delivery vehicles, T-tiRNAs showed enhanced gene interfering activity over conventional siRNAs or antagomirs as a result of increased intracellular delivery. CONCLUSIONS: The data obtained in the present study provide an example of synthetic multi-functional RNA structures that enable multiple gene interference in mammalian cells, which could become powerful tools for an efficient combinatorial iRNA strategy.

Core-shell hybrid nanostructured delivery platforms for advanced RNAi therapeutics.

AIM: Study was aimed at combining the advantages of nonclassical RNAi-triggering oligonucleotides with nanoparticle-based advanced delivery platforms for developing efficient therapeutic systems. MATERIALS & METHODS: We utilized a core-shell hybrid nanostructured platform for effectively delivering nonclassical RNAi triggers, namely long double stranded interfering RNA and tripodal interfering RNA. Core-shell structure was prepared by stably anchoring thiol-modified cationic polymer on the surface of growing crystal gold (Au) seeds, and the resulting particles were further complexed with nonclassical RNAi candidates via electrostatic interactions. RESULTS: Our studies clearly demonstrated that the unique combination of nonclassical RNAi structures with an advanced core-shell hybrid nanostructured platform is an effective module for advanced RNAi-based therapeutic development.

Efficacy of a Multiplex Paclitaxel Emission Stent Using a Pluronic® Mixture Membrane versus a Covered Metal Stent in Malignant Biliary Obstruction: A Prospective Randomized Comparative Study.

BACKGROUND/AIMS: A drug-eluting stent for unresectable malignant biliary obstruction was developed to increase stent patency by preventing tumor ingrowth. The safety and efficacy of a new generation of metallic stents covered with a paclitaxel-incorporated membrane using a Pluronic® mixture (MSCPM-II) were compared prospectively with those of covered metal stents (CMSs) in patients with malignant biliary obstructions. METHODS: This study was initially designed as a prospective randomized trial but was closed early because of a high incidence of early occlusion. Therefore, the data were analyzed using the intent-to-treat method. A total of 72 patients with unresectable distal malignant biliary obstructions were prospectively enrolled. RESULTS: The two groups did not differ significantly in basic characteristics and mean follow-up period (MSCPM-II 194 days vs CMS 277 days, p=0.063). Stent occlusion occurred in 14 patients (35%) who received MSCPM-II and in seven patients (21.9%) who received CMSs. Stent patency and survival time did not significantly differ between the two groups (p=0.355 and p=0.570). The complications were mild and resolved by conservative management in both groups. CONCLUSIONS: There were no significant differences in stent patency or patient survival in MSCPM-II and CMS patients with malignant biliary obstructions.

Nonvascular drug-eluting stent coated with sodium caprate-incorporated polyurethane for the efficient penetration of paclitaxel into tumor tissue.

To increase the therapeutic potency of nonvascular drug-eluting stents, sodium caprate was employed as a drug-penetration enhancer. A polytetrafluoroethylene-covered drug-eluting stent was coated with a mixture containing sodium caprate, paclitaxel, and polyurethane via the rolling coating technique. The coated stent has a smooth membrane surface with a 40-µm membrane thickness. Paclitaxel was released from the coated stent for two months. In the multilayered cell sheet model, sodium caprate in the polyurethane membrane (PUSC10) showed the possibility of enhancing the paclitaxel tissue penetration. The amount of penetrated paclitaxel for the sodium caprate-containing polyurethane membrane (PUSC10) was two times higher than that of sodium caprate-free polyurethane membrane. Additionally, the potential of sodium caprate was confirmed by a tumor-bearing small animal model. PUSC10 incorporated with Nile red (as a model fluorescence dye for visualization of drug penetration; PUSC10-Nile red) or PUSC10 incorporated with paclitaxel (PUSC10-paclitaxel) membrane was implanted at tumor sites in Balb/c mice. In the case of PUSC10-Nile red, the tissue penetration depth of Nile red was significantly increased from 30 µm (without sodium caprate) to 1060 µm (with sodium caprate). After seven days, an almost four times higher therapeutic area of PUSC10-paclitaxel was observed compared to that of polyurethane-paclitaxel (without sodium caprate) by a terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The results indicate that sodium caprate improves the penetration and therapeutic efficiencies of drugs in drug-eluting stents, and thus, it has potential for local stent therapy.

Efficient intracellular delivery and multiple-target gene silencing triggered by tripodal RNA based nanoparticles: a promising approach in liver-specific RNAi delivery.

RNA interference (RNAi) triggering oligonucleotides in unconventional structural format can offer advantages over conventional small interfering RNA (siRNA), enhanced cellular delivery and improved target gene silencing. With this concept, we present a well-defined tripodal-interfering RNA (tiRNA) structure that can induce simultaneous silencing of multiple target genes with improved potency. The tiRNA structure, formed by the complementary association of three single-stranded RNA units, was optimized for improved gene silencing efficacy. When combined with cationic polymers such as linear polyethyleneimine (PEI), tiRNA assembled to form a stable nano-structured complex through electrostatic interactions and induced stronger RNAi response over conventional siRNA-PEI complex. In combination with a liver-targeting delivery system, tripodal nucleic acid structure demonstrated enhanced fluorescent accumulation in mouse liver compared to standard duplex nucleic acid format. Tripodal RNA structure complexed with galactose-modified PEI could generate effective RNAi-mediated gene silencing effect on experimental mice models. Our studies demonstrate that optimized tiRNA structural format with appropriate polymeric carriers have immense potential to become an RNAi-based platform suitable for multi-target gene silencing.

Prospective evaluation of the partially covered nitinol "ComVi" stent for malignant non hilar biliary obstruction.

BACKGROUND: Biliary partially covered self-expandable metal stents (PC-SEMS) offer prolonged relief of symptoms of biliary obstruction but may induce complications including pancreatitis, cholecystitis and migration. AIMS: To assess efficacy and safety of the ComVi partially covered self-expandable metal stents as primary palliative treatment of distal malignant biliary obstruction. METHODS: Seventy patients (mean age 69.2 years) with distal malignant biliary strictures were prospectively included and underwent endoscopic retrograde cholangio-pancreatography and partially covered self-expandable metal stents placement. Follow-up was done for 12 months. self-expandable metal stents patency, survival and complication-rate after partially covered self-expandable metal stents placement were evaluated. RESULTS: Overall median survival time was 190 days (30-856). Forty-four patients (62.8%) died after median 175.5 days (30-614) without signs of stent dysfunction; 37 patients (52.8%) were alive after 6 months without signs of self-expandable metal stents occlusion. Survival rapidly dropped between 8 and 12 months after treatment. Survival was not influenced by sex (P = 0.1) or type of neoplasia (P = 0.178). Median survival was longer (254 days [44-836]) in patients who underwent chemotherapy (P < 0.0001). Partially covered self-expandable metal stents occlusion had 24 (35.7%) patients 154 days (35-485) after treatment. Median survival after re-treatment was 66 days (13-597). Cholecystitis occurred in one patient (1.7%). CONCLUSIONS: The ComVi partially covered self-expandable metal stents is effective for palliation of biliary obstruction secondary to distal malignant biliary strictures. Self-expandable metal stents patency during follow-up is satisfactory without significant complications.

Human application of a metallic stent covered with a paclitaxel-incorporated membrane for malignant biliary obstruction: multicenter pilot study.

BACKGROUND: Paclitaxel, with its antitumor effect, may improve the function of metallic stents used for biliary drainage. However, clinical studies that use metallic stents covered with a paclitaxel-incorporated membrane (MSCPM) in the biliary tract of human beings have not been previously carried out. OBJECTIVE: To evaluate the safety and efficacy of an MSCPM for patients with malignant biliary obstruction. DESIGN AND SETTING: A case series that includes 4 endoscopy centers. PATIENTS: From July 2003 to August 2006, a total of 21 patients diagnosed with unresectable malignant biliary obstruction. INTERVENTION: Endoscopic placement of an MSCPM. MAIN OUTCOME MEASUREMENTS: Stent occlusion, complications, stent patency, patient survival, and the periodic mean concentration of paclitaxel in the blood. RESULTS: Occlusion of the MSCPM was observed in 9 patients and was caused by bile sludge or clog in 4, tumor overgrowth in 3, and tumor ingrowth in 2. Complications included obstructive jaundice in 6, cholangitis in 3, and 1 patient showed stent migration with cholecystitis. The mean patency of a MSCPM was 429 days (median 270 days, range 68-810 days) and cumulative patency rates at 3, 6, and 12 months were 100%, 71%, and 36%, respectively. The mean survival of patients was 350 days (median 281 days, range 68-811 days). The highest concentration of paclitaxel in the blood was found between 1 and 10 days after insertion. LIMITATIONS: Small number of patients and low rate of pathologic diagnosis. CONCLUSIONS: The endoscopic insertion of MSCPM is technically feasible, safe, and effective in patients with malignant biliary obstruction. In addition, MSCPM may exert local antitumor activity because of the steady release of paclitaxel.

The effect on porcine bile duct of a metallic stent covered with a paclitaxel-incorporated membrane.

BACKGROUND: Biliary metallic stents are covered with a membrane to prevent tumor ingrowth and to prolong patency. The only function of these stents is to promote biliary drainage; they have no antitumor effect. METHODS: A metallic stent was developed that is covered with a paclitaxel-incorporated membrane. The metallic stents were coated with one of 3 concentrations of paclitaxel (0, 10, and 20 % wt/v) and polyurethane. A stent with each concentration was surgically inserted in the bile duct of two pigs. Four weeks after insertion, the segment of bile duct containing the stent was examined histologically. To determine the efficacy of the drug release, stents were placed in phosphate buffered saline solution for 6 weeks, and the amount of paclitaxel released was measured by high-performance liquid chromatography. RESULTS: The histologic changes in the pig biliary epithelium were acceptable with respect to safety and included inflammatory cell infiltration and fibrous reactions. The changes corresponded to the amount of paclitaxel incorporated within the stent in contact with the bile duct. Epithelial denudation, mucin hypersecretion, and epithelial metaplasia were noted in the bile ducts that were in contact with stents containing 20 % wt/v paclitaxel. Transmural necrosis and perforation were not observed in any animal. In the in vitro experiment, the amounts of paclitaxel released over 1 week and over 6 weeks were similar, regardless of the concentration of paclitaxel incorporated in the stent. The stent with 10% (wt/v) paclitaxel in the covering membrane was found to be better than that with 20 % (wt/v) with respect to histologic changes and the effectiveness of drug release. CONCLUSIONS: A paclitaxel-incorporated metallic stent could serve as a basis for the development of a new and safe treatment modality for malignant biliary obstruction. Clinical trials of this stent with other adjuvant therapy are warranted.