Insights into the Nature of Synergistic Effects in Proton-Conducting 4,4−1H,1H-Bitriazole-Poly(ethylene oxide) Composites

A nitrogen-containing heterocycle (NCH), 4,4-1H-1H-bi-1,2,3-triazole (bitriazole), capable of mimicking the hydrogen bonding of water in the solid state is synthesized and its ability to conduct protons in the presence of poly(ethylene oxides) under anhydrous conditions is investigated. Bitriazole is shown to have sufficient thermal and electrochemical stability for fuel cell applications. The composites formed between bitriazole and poly(ethylene oxides) give proton conductivities that can be described by the Vogel−Tamman−Fulcher (VTF) equation. These characteristics suggest coupling between polymer segmental motion and ion transport. The bitriazole N-H proton is shown to be the source of conductivity, and bitriazole and poly(ethylene oxides) function synergistically through specific intermolecular interactions and polymer-induced segmental motion to create a pathway for proton transport via structural diffusion

Proton-Conducting Solid Electrolyte via Ozonolysis of Cationic Ammonium Organoalkoxysilane Surfactant-Templated MCM-41

A functional polymerizable surfactant has been synthesized and used as a template to prepare MCM-41. Ozonolysis of the olefin functional group in the composite material generates a nonporous proton-conducting solid acid that has a conductivity of ca. 6 mS cm^(-1) at 95 °C and an activation energy of 14.2 kJ/mol

Development of siRNA-probes for studying intracellular trafficking of siRNA nanoparticles

One important barrier facing the delivery of short interfering RNAs (siRNAs) via synthetic nanoparticles is the rate of nanoparticle disassembly. However, our ability to optimize the release kinetics of siRNAs from nanoparticles for maximum efficacy is limited by the lack of methods to track their intracellular disassembly. Towards this end, we describe the design of two different siRNA-based fluorescent probes whose fluorescence emission changes in response to the assembly state of the nanoparticle. The first probe design involves a redox-sensitive fluorescence-quenched probe that fluoresces only when the nanoparticle is disassembled in a reductive environment. The second probe design is based on a FRET-labeled siRNA pair that fluoresces due to the proximity of the siRNA pair when the nanoparticle is intact. In both approaches, the delivery vehicle need not be labeled. The utility of these probes was investigated with a lipidoid nanoparticle (LNP) as proof-of-concept in both extracellular and intracellular environments. Fluorescence kinetic data from both probes were fit to a two-phase release and decay curve and subsequently quantified to give intracellular disassembly rate constants. Quantitative analysis revealed that the rate constant of siRNA release measured via the fluorescence-quenched probe was almost identical to the rate constant for nanoparticle disassembly measured via the FRET-labeled probes. Furthermore, these probes were utilized to determine subcellular localization of LNPs with the use of automated high-resolution microscopy as they undergo dissociation. Interestingly, this work shows that large amounts of siRNA remain inside vesicular compartments. Altogether, we have developed new siRNA probes that can be utilized with multiple nanocarriers for quantitative and qualitative analysis of nanoparticle dissociation that may serve as a design tool for future delivery systems.National Institutes of Health (U.S.) (Grant R37-EB000244)National Institutes of Health (U.S.) (Grant R01-CA132091)National Institutes of Health (U.S.) (Grant R01-CA132091)National Institutes of Health (U.S.) (Postdoctoral Fellowship

Proton-Conducting Solid Electrolyte via Ozonolysis of Cationic Ammonium Organoalkoxysilane Surfactant-Templated MCM-41

A functional polymerizable surfactant has been synthesized and used as a template to prepare MCM-41. Ozonolysis of the olefin functional group in the composite material generates a nonporous proton-conducting solid acid that has a conductivity of ca. 6 mS cm^(-1) at 95 °C and an activation energy of 14.2 kJ/mol

Degradable Terpolymers with Alkyl Side Chains Demonstrate Enhanced Gene Delivery Potency and Nanoparticle Stability

Degradable, cationic poly(β-amino ester)s (PBAEs) with alkyl side chains are developed for non-viral gene delivery. Nanoparticles formed from these PBAE terpolymers exhibit significantly enhanced DNA transfection potency and resistance to aggregation. These hydrophobic PBAE terpolymers, but not PBAEs lacking alkyl side chains, support interaction with PEG-lipid conjugates, facilitating their functionalization with shielding and targeting moieties and accelerating the in vivo translation of these materials.National Heart, Lung, and Blood InstituteNational Institutes of Health (U.S.) (Program of Excellence in Nanotechnology (PEN) Award, Contract #HHSN268201000045C)National Institutes of Health (U.S.) (NIH Grant R01-EB000244-27)National Institutes of Health (U.S.) (NIH Grant 5-R01-CA132091-04)National Institutes of Health (U.S.) (NIH Grant R01-DE016516-03)National Science Foundation (U.S.) (Graduate Research Fellowship)Juvenile Diabetes Research Foundation International (Grant 17–2007-1063

Effect of molecular weight of amine end-modified poly(β-amino ester)s on gene delivery efficiency and toxicity

Amine end-modified poly(β-amino ester)s (PBAEs) have generated interest as efficient, biodegradable polymeric carriers for plasmid DNA (pDNA). For cationic, non-degradable polymers, such as polyethylenimine (PEI), the polymer molecular weight (MW) and molecular weight distribution (MWD) significantly affect transfection activity and cytotoxicity. The effect of MW on DNA transfection activity for PBAEs has been less well studied. We applied two strategies to obtain amine end-modified PBAEs varying in MW. In one approach, we synthesized four amine end-modified PBAEs with each at 15 different monomer molar ratios, and observed that polymers of intermediate length mediated optimal DNA transfection in HeLa cells. Biophysical characterization of these feed ratio variants suggested that optimal performance was related to higher DNA complexation efficiency and smaller nanoparticle size, but not to nanoparticle charge. In a second approach, we used preparative size exclusion chromatography (SEC) to obtain well-defined, monodisperse polymer fractions. We observed that the transfection activities of size-fractionated PBAEs generally increased with MW, a trend that was weakly associated with an increase in DNA binding efficiency. Furthermore, this approach allowed for the isolation of polymer fractions with greater transfection potency than the starting material. For researchers working with gene delivery polymers synthesized by step-growth polymerization, our data highlight the potentially broad utility of preparative SEC to isolate monodisperse polymers with improved properties. Overall, these results help to elucidate the influence of polymer MWD on nucleic acid delivery and provide insight toward the rational design of next-generation materials for gene therapy.Alnylam Pharmaceuticals (Firm)National Institutes of Health (U.S.) (Grant R01-EB000244-27)National Institutes of Health (U.S.) (Grant 5-R01-CA132091-04)National Science Foundation (U.S.). Graduate Research FellowshipNational Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (F32-EB011867

Associations between Cognition, Gender and Monocyte Activation among HIV Infected Individuals in Nigeria.

The potential role of gender in the occurrence of HIV-related neurocognitive impairment (NCI) and associations with markers of HIV-related immune activity has not been previously examined. In this study 149 antiretroviral-naïve seropositive subjects in Nigeria (SP, 92 women and 57 men) and 58 seronegative (SN, 38 women and 20 men) were administered neuropsychological testing that assessed 7 ability domains. From the neuropsychological test scores was calculated a global deficit score (GDS), a measure of overall NCI. Percentages of circulating monocytes and plasma HIV RNA, soluble CD163 and soluble CD14 levels were also assessed. HIV SP women were found to be younger, more educated and had higher CD4+ T cell counts and borderline higher viral load measures than SP men. On the neuropsychological testing, SP women were more impaired in speed of information processing and verbal fluency and had a higher mean GDS than SN women. Compared to SP men, SP women were also more impaired in speed of information processing and verbal fluency as well as on tests of learning and memory. Numbers of circulating monocytes and plasma sCD14 and sCD163 levels were significantly higher for all SP versus all SN individuals and were also higher for SP women and for SP men versus their SN counterparts. Among SP women, soluble CD14 levels were slightly higher than for SP men, and SP women had higher viral load measurements and were more likely to have detectable virus than SP men. Higher sCD14 levels among SP women correlated with more severe global impairment, and higher viral load measurements correlated with higher monocyte numbers and sCD14 and sCD14 levels, associations that were not observed for SP men. These studies suggest that the risk of developing NCI differ for HIV infected women and men in Nigeria and, for women, may be linked to effects from higher plasma levels of HIV driving activation of circulating monocytes

Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling

Despite substantial efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain largely unclear. Here we examined cellular uptake of siRNA delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy as well as defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR, and cathepsins. SiRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes/lysosomes and increased gene silencing of the target gene. Our data suggests that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways

Prevalence of surgically correctable conditions among children in a mixed urban-rural community in Nigeria using the SOSAS survey tool:Implications for paediatric surgical capacity-building

BackgroundIn many low- and middle-income countries, data on the prevalence of surgical diseases have been derived primarily from hospital-based studies, which may lead to an underestimation of disease burden within the community. Community-based prevalence studies may provide better estimates of surgical need to enable proper resource allocation and prioritization of needs. This study aims to assess the prevalence of common surgical conditions among children in a diverse rural and urban population in Nigeria.MethodsDescriptive cross-sectional, community-based study to determine the prevalence of congenital and acquired surgical conditions among children in a diverse rural-urban area of Nigeria was conducted. Households, defined as one or more persons 'who eat from the same pot' or slept under the same roof the night before the interview, were randomized for inclusion in the study. Data was collected using an adapted and modified version of the interviewer-administered questionnaire-Surgeons OverSeas Assessment of Surgical Need (SOSAS) survey tool and analysed using the REDCap web-based analytic application.Main resultsEight-hundred-and-fifty-six households were surveyed, comprising 1,883 children. Eighty-one conditions were identified, the most common being umbilical hernias (20), inguinal hernias (13), and wound injuries to the extremities (9). The prevalence per 10,000 children was 85 for umbilical hernias (95% CI: 47, 123), and 61 for inguinal hernias (95% CI: 34, 88). The prevalence of hydroceles and undescended testes was comparable at 22 and 26 per 10,000 children, respectively. Children with surgical conditions had similar sociodemographic characteristics to healthy children in the study population.ConclusionThe most common congenital surgical conditions in our setting were umbilical hernias, while injuries were the most common acquired conditions. From our study, it is estimated that there will be about 2.9 million children with surgically correctable conditions in the nation. This suggests an acute need for training more paediatric surgeons

Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles

Therapeutics that are designed to engage RNA interference (RNAi) pathways have the potential to provide new, major ways of imparting therapy to patients. Long, double-stranded RNAs were first shown to mediate RNAi in Caenorhabditis elegans, and the potential use of RNAi for human therapy has been demonstrated by the finding that small interfering RNAs (siRNAs; approximately 21-base-pair double-stranded RNA) can elicit RNAi in mammalian cells without producing an interferon response. We are at present conducting the first in-human phase I clinical trial involving the systemic administration of siRNA to patients with solid cancers using a targeted, nanoparticle delivery system. Here we provide evidence of inducing an RNAi mechanism of action in a human from the delivered siRNA. Tumour biopsies from melanoma patients obtained after treatment show the presence of intracellularly localized nanoparticles in amounts that correlate with dose levels of the nanoparticles administered (this is, to our knowledge, a first for systemically delivered nanoparticles of any kind). Furthermore, a reduction was found in both the specific messenger RNA (M2 subunit of ribonucleotide reductase (RRM2)) and the protein (RRM2) levels when compared to pre-dosing tissue. Most notably, we detect the presence of an mRNA fragment that demonstrates that siRNA-mediated mRNA cleavage occurs specifically at the site predicted for an RNAi mechanism from a patient who received the highest dose of the nanoparticles. Together, these data demonstrate that siRNA administered systemically to a human can produce a specific gene inhibition (reduction in mRNA and protein) by an RNAi mechanism of action