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The clinical application of nanoparticles (NPs) to deliver RNA for therapy has progressed rapidly since the FDA approval of Onpattro® in 2018 for the treatment of polyneuropathy associated with hereditary transthyretin amyloidosis. The emergency use authorization or approval and widespread global use of two mRNA-NP based vaccines developed by Moderna Therapeutics Inc. and Pfizer-BioNTech in 2021 has highlighted the translatability of NP technology for RNA delivery. Furthermore, in clinical trials, a wide variety of NP formulations have been found to extend the half-life of RNA molecules such as microRNA, small interfering RNA, and messenger RNA, with limited safety issues. In this review, we discuss the NP formulations that are already used in the clinic to deliver therapeutic RNA and highlight examples of RNA-NPs which are currently under evaluation for human use. We also detail NP formulations that failed to progress through clinical trials, in hopes of guiding future successful translation of nanomedicine-based RNA therapeutics into the clinic.

The ability to cross the blood-brain barrier (BBB) is critical for targeted therapy of the central nerve system (CNS). Six peptide vectors were covalently attached to a 50 kDa poly(β-l-malic acid)-trileucine polymer forming P/LLL(40%)/vector conjugates. The vectors were Angiopep-2 (AP2), B6, Miniap-4 (M4), and d-configurated peptides D1, D3, and ACI-89, with specificity for transcytosis receptors low-density lipoprotein receptor-related protein-1 (LRP-1), transferrin receptor (TfR), bee venom-derived ion channel, and Aβ/LRP-1 related transcytosis complex, respectively. The BBB-permeation efficacies were substantially increased ("boosted") in vector conjugates of P/LLL(40%). We have found that the copolymer group binds at the endothelial membrane and, by an allosterically membrane rearrangement, exposes the sites for vector-receptor complex formation. The specificity of vectors is indicated by competition experiments with nonconjugated vectors. P/LLL(40%) does not function as an inhibitor, suggesting that the copolymer binding site is eliminated after binding of the vector-nanoconjugate. The two-step mechanism, binding to endothelial membrane and allosteric exposure of transcytosis receptors, is supposed to be an integral feature of nanoconjugate-transcytosis pathways.

The use of a variety of RNA molecules, including messenger RNA, small interfering RNA, and microRNA, has shown great potential for prevention and therapy of many pathologies. However, this therapeutic promise has historically been limited by short in vivo half-life, lack of targeted delivery, and safety issues. Nanoparticle (NP)-mediated delivery has been a successful platform to overcome these limitations, with multiple formulations already in clinical trials and approved by the FDA. Although there is a diversity of NPs in terms of material formulation, size, shape, and charge that have been proposed for biomedical applications, specific modifications are required to facilitate sufficient RNA delivery and adequate therapeutic effect. This includes optimization of (i) RNA incorporation into NPs, (ii) specific cell targeting, (iii) cellular uptake and (iv) endosomal escape ability. In this review, we summarize the methods by which NPs can be modified for RNA delivery to achieve optimal therapeutic effects.

Nanoparticles may provide a viable way for neuroprotective drugs to cross the blood-brain barrier (BBB), which limits the passage of most drugs from the peripheral circulation to the brain. Heterotelechelic polymer prodrugs comprising a neuroprotective model drug (adenosine) and a maleimide functionality were synthesized by the "drug-initiated" approach and subsequent nitroxide exchange reaction. Nanoparticles were obtained by nano-precipitation and exhibited high colloidal stability with diameters in the 162-185 nm range and narrow size distributions. Nanoparticles were then covalently surface-conjugated to different proteins (albumin, alpha 2-macroglobulin and fetuin A) to test their capability of enhancing BBB translocation. Their performances in terms of endothelial permeability and cellular uptake in an in vitro BBB model were compared to that of similar nano particles with surface-adsorbed proteins, functionalized or not with the drug. It was shown that bare NPs (i.e., NPs not surface-functionalized with proteins) without the drug exhibited significant permeability and cellular uptake, which were further enhanced by NP surface functionalization with alpha 2-macroglobulin. However, the presence of the drug at the polymer chain-end prevented efficient passage of all types of NPs through the BBB model, likely due to a decrease in the hydrophobicity of the nanoparticle surface and alteration of the protein binding/coupling, respectively. These results established a new and facile synthetic approach for the surface-functionalization of polymer nanoparticles for brain delivery purposes.

Thermodynamic calculations provide valuable insights into the reactions that drive the profound fluid transformations during serpentinization, where surface fluids are transformed into some of the ...

Klebsiella pneumoniae is among the leading bacteria that cause nosocomial infections. The capsule of this Gram-negative bacterium is a dominant virulence factor, with a prominent role in defense and biofilm formation. Bacteriophages, which are specific for one bacterial strain and its capsule type, can evoke the lysis of bacterial cells, aided by polysaccharide depolymerase enzymes. In this study, we isolated and characterized a bacteriophage against the nosocomial K. pneumoniae 52145 strain with K2 capsular serotype. The phage showed a narrow host range and stable lytic activity, even when exposed to different temperatures or detergents. Preventive effect of the phage in a nasal colonization model was investigated in vivo. Phlyogenetic analysis showed that the newly isolated Klebsiella phage B1 belongs to the Webervirus genus in Drexlerviridae family. We identified the location of the capsule depolymerase gene of the new phage, which was amplified, cloned, expressed, and purified. The efficacy of the recombinant B1dep depolymerase was tested by spotting on K. pneumoniae strains and it was confirmed that the extract lowers the thickness of the bacterium lawn as it degrades the protective capsule on bacterial cells. As K. pneumoniae strains possessing the K2 serotype have epidemiological importance, the B1 phage and its depolymerase are promising candidates for use as possible antimicrobial agents.

There are still major gaps in our understanding of the bacterial factors that influence the outcomes of human Campylobacter jejuni infection. The aim of this study was to compare the virulence-associated features of 192 human C. jejuni strains isolated from hospitalized patients with diarrhoea (150/192, 78.1%), bloody diarrhoea (23/192, 11.9%), gastroenteritis (3/192, 1.6%), ulcerative colitis (3/192, 1.5%), and stomach ache (2/192, 1.0%). Traits were analysed with genotypic and phenotypic methods, including PCR and extracellular matrix protein (ECMP) binding, adhesion, and invasion capacities. Results were studied alongside patient symptoms, but no distinct links with them could be determined. Since the capacity of C. jejuni to invade host epithelial cells is one of its most enigmatic attributes, a high throughput transcriptomic analysis was performed in the third hour of internalization with a C. jejuni strain originally isolated from bloody diarrhoea. Characteristic groups of genes were significantly upregulated, outlining a survival strategy of internalized C. jejuni comprising genes related (1) to oxidative stress, (2) to a protective sheath formed by the capsule, LOS, N-, and O- glycosylation systems, (3) to dynamic metabolic activity supported by different translocases and the membrane-integrated component of the flagellar apparatus, and (4) to hitherto unknown genes.

The deposition of amyloid-&beta, (A&beta, ) plaques in the brain is a significant pathological signature of Alzheimer&rsquo, s disease, correlating with synaptic dysfunction and neurodegeneration. Several compounds, peptides, or drugs have been designed to redirect or stop A&beta, aggregation. Among them, the trideca-peptide CWG-LRKLRKRLLR (mApoE), which is derived from the receptor binding sequence of apolipoprotein E, is effectively able to inhibit A&beta, aggregation and to promote fibril disaggregation. Taking advantage of Atomic Force Microscopy (AFM) imaging and fluorescence techniques, we investigate if the clustering of mApoE on gold nanoparticles (AuNP) surface may affect its performance in controlling A&beta, aggregation/disaggregation processes. The results showed that the ability of free mApoE to destroy preformed A&beta, fibrils or to hinder the A&beta, aggregation process is preserved after its clustering on AuNP. This allows the possibility to design multifunctional drug delivery systems with clustering of anti-amyloidogenic molecules on any NP surface without affecting their performance in controlling A&beta, aggregation processes.

Alzheimer's disease affects millions of people worldwide and this figure is continuously increasing. Currently, there is no resolutive cure for this disorder, but a valid contribution could be provided by nanomedicine, utilizing multi-functionalized nanodevices as drug vehicles with additional features of specific brain targeting. Nanomedicine may represent also a practicable strategy for the pharmaceutical industry that moved from small MW pharmaceuticals to larger biologicals, such as antibodies and nucleotides, as the next generation of drugs, leading to the challenge of effective drug delivery. This review provides a survey on the nano-based strategies for Alzheimer's disease diagnosis and treatment, aiming at enhancing the passage of candidate pharmaceuticals across the BBB, and at supporting the evaluation of new therapeutic agents targeting this disease.

Campylobacter jejuniis one of the most common food-borne bacteria that causes gastrointestinal symptoms. In the present study we have investigated the molecular basis of the anti-Campylobactereffect of peppermint essential oil (PEO), one of the oldest EO used to treat gastrointestinal diseases. Transcriptomic, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and proteomic, two-dimensional polyacryl amid gel electrophoresis (2D-PAGE) methods have revealed that, in the presence of a sublethal concentration of PEO, the expression of several virulence-associated genes was decreased (cheY0.84x;flhB0.79x;flgE0.205x;cadF0.08x;wlaB0.89x;porA0.25x;cbf24.3x) while impaired motility was revealed with a functional analysis. Scanning electron micrographs of the exposed cells showed that, unlike in the presence of other stresses, the originally curvedC. jejunicells straightened upon PEO exposure. Gaining insight into the molecular background of this stress response, we have revealed that in the presence of PEOC. jejunidominantly exerts a general stress response that elevates the expression of general stress genes likednaK,groEL,groES(10.41x, 3.63x, and 4.77x). The most important genesdps,sodB, andkatAinvolved in oxidative stress responses showed however moderate transcriptional elevations (1,58x, 1,55x, and 1,85x).

Thrombosis is central to the pathogenesis of many life-threatening diseases, including stroke and myocardial infarction. Current treatments for thrombosis, particularly anti-platelet agents that target GPIIb/IIIa, can cause serious bleeding complications. Unlike GPIIb/IIIa, GPIb initiates thrombus formation at the high shear stress that is prominent in atherosclerotic vessels. Therefore, this target could prevent pathological thrombosis while leaving normal haemostasis at low shear rates relatively unaffected. Anfibatide is a novel GPIb antagonist isolated from snake venom in China. Anfibatide will enter phase Ib-IIa trials in early 2015 and therefore, it is necessary to develop an assay to measure its efficacy in patients. We have developed a flow cytometry based receptor occupancy assay. In this assay, total GPIb receptor numbers are measured using an anti-GPIb monoclonal antibody, and the percentage of GPIb receptor occupancy by anfibatide is measured. Receptor occupancy by anfibatide correlated with dose-dependent inhibition of ristocetin-induced aggregation in whole blood by anfibatide. Functional studies were carried out using anfibatide. Anfibatide did not inhibit binding of two anti-GPIIb/IIIa antibodies. Anfibatide partially inhibited the anti-GPIb antibodies VM16d, and SZ 2, indicating that it binds to GPIbα between the thrombin-binding site and the sulfated tyrosine motif. Anfibatide did not cause platelet activation or loss of single platelets in vitro. Anfibatide had no effect on aggregation induced by low dose or high dose thrombin, ADP or arachidonic acid. Anfibatide inhibited Streptococcus sanguinis 133-79 induced aggregation by 50% in half of healthy donors, but had no effect on Staphylococcus aureus Newman aggregation. The receptor occupancy assay is optimised and available for use in Phase Ib-IIa clinical trials, and in future clinical use of anfibatide.

Escherichia (E.) coli K1 strains remain common causative agents of neonatal sepsis and meningitis. We have isolated a lytic bacteriophage (ΦIK1) against E. coli strain IHE3034 and tested its specificity in vitro, as well as distribution and protective efficacy in vivo. The phage was shown to be specific to the K1 capsular polysaccharide. In the lethal murine model, a high level of protection was afforded by the phage with strict kinetics. A single dose of 1 x 108 phage particles administered 10 and 60 minutes following the bacterial challenge elicited 100 % and 95 % survival, respectively. No mice could be rescued if phage administration occurred 3 hours postinfection. Tissue distribution surveys in the surviving mice revealed that the spleen was the primary organ in which accumulation of active ΦIK1 phages could be detected two weeks after phage administration. These results suggest that bacteriophages have potential as therapeutic agents in the control of systemic infections.

Escherichia (E.) coliK1 strains remain common causative agents of neonatal sepsis and meningitis. We have isolated a lytic bacteriophage (ΦIK1) againstE. colistrain IHE3034 and tested its specificityin vitro, as well as distribution and protective efficacyin vivo. The phage was shown to be specific to the K1 capsular polysaccharide. In the lethal murine model, a high level of protection was afforded by the phage with strict kinetics. A single dose of 1 x 108phage particles administered 10 and 60 minutes following the bacterial challenge elicited 100 % and 95 % survival, respectively. No mice could be rescued if phage administration occurred 3 hours postinfection. Tissue distribution surveys in the surviving mice revealed that the spleen was the primary organ in which accumulation of active ΦIK1 phages could be detected two weeks after phage administration. These results suggest that bacteriophages have potential as therapeutic agents in the control of systemic infections.

Aim: To investigate if and how the ability of liposomes, previously designed for Alzheimer's therapy, to reach the brain changes in aging/pathological conditions with respect to the healthy state. Methods: Biodistribution and pharmacokinetics of liposomes in young or aged healthy mice and in an Alzheimer's mouse model were measured by radiochemical techniques. The expression of brain receptors and structural proteins was evaluated by Western blot. Results: At equal blood levels, the amount and integrity of liposomes in the brain were dramatically lower in Alzheimer's or aged mice, with respect to young animals. These differences are likely attributable to molecular alterations in the brain vasculature. Conclusion: Brain alterations in pathology or aging should be considered in the design of drug delivery systems for brain targeting.

Purpose. The treatment of device-related infections is challenging and current anti-microbial compounds have poor anti-biofilm activity. We aimed to identify and characterize novel compounds effective in the eradication of Staphylococcus aureus biofilms. Methodology. Two novel compounds, MMV665953 {1-(3-chloro-4-fluorophenyl)-3-(3,4-dichlorophenyl)urea} and MMV665807{5-chloro-2-hydroxy-N-[3-(trifluoromethyl)phenyl]benzamide}, effective in killing S. aureus biofilms, were identified by screening of the open access 'malaria box' chemical library. The minimum bactericidal concentrations, half-maximal inhibition concentration (IC50) values and minimal biofilm killing concentrations effective in the killing of biofilm were determined against meticillin-resistant S. aureus and meticillin-sensitive S. aureus. Fibrin-embedded biofilms were grown under in vivo-relevant conditions, and viability was measured using a resazurin-conversion assay and confocal microscopy. The potential for the development of resistance and cytotoxicity was also assessed. Results. MMV665953 and MMV665807 were bactericidal against S. aureus isolates. The IC50 against S. aureus biofilms was at 0.15–0.58 mg l−1 after 24 h treatment, whereas the concentration required to eradicate all tested biofilms was 4 mg l−1, making the compounds more bactericidal than conventional antibiotics. The cytotoxicity against human keratinocytes and primary endothelial cells was determined as IC50 7.47 and 0.18 mg l−1 for MMV665953, and as 1.895 and 0.076 mg l−1 for MMV665807. Neither compound was haemolytic nor caused platelet activation. MMV665953 and MMV665807 derivatives with reduced cytotoxicity exhibited a concomitant loss in anti-staphylococcal activity. Conclusion. MMV665953 and MMV665807 are more bactericidal against S. aureus biofilms than currently used anti-staphylococcal antibiotics and represent a valuable structural basis for further investigation in the treatment of staphylococcal biofilm-related infections.

Hydrothermal fluids obtain organic compounds through diverse pathways. In submarine systems organic compounds are already dissolved in seawater that is heated and transformed into hydrothermal fluids through water-rock reactions. Microbes inhabiting hydrothermal systems produce metabolites that enter the fluids, and cells can be carried into the reaction zones by circulating fluids and pyrolyzed. Analogous sources of organic compounds can be anticipated in continental systems with the possible addition of novel plant- and soil-derived organic compounds from the surface. In addition, hydrothermal systems possess large potentials for abiotic organic synthesis that may add a novel suite of compounds. When sedimentary rocks are present, ancient biogenic organic matter can be mobilized or transformed by hot fluids. These transformations accompany the generation of petroleum, coal, and other fossil fuels, suggesting that expectations for hydrothermal transformations can be built on those that occur in sedimentary basins. Likewise, some types of ore deposition are accompanied by transformations of organic compounds, and metal-organic complexes may be involved in enhancing the transport of metals in ore-forming and other crustal fluids. With these thoughts in mind, this review starts with an inventory of the types of organic compounds found in hydrothermal systems and some ways that hydrothermal organic compounds are transformed. Methane can be generated biotically and abiotically from organic or inorganic reactants, and since it lacks a carbon-carbon bond, some researchers would not consider it to be an organic compound. Nevertheless, more data exist for methane in hydrothermal fluids than for any organic compound that fits the definition. Methane has been quantified in continental and submarine hydrothermal fluids, fumarolic gases associated with hydrothermal systems, oil-field brines, deep fluids in sedimentary basins and igneous basement rocks, fluids associated with active serpentinization, and fluid inclusions in minerals from ore deposits, sedimentary basins, and deep crustal settings (recent examples include: …

Even casual observations of continental hot springs reveal that photosynthesis has its limits. In an effort to explore the transition to photosynthesis, field measurements of temperature and pH were made at 996 hot spring locations at Yellowstone National Park ranging from 14° to 94 °C and pH from 0.8 to 9.7. In addition, sulfide measurements were made in 426 of these locations showing concentrations up to 8820 μg L − 1 total sulfide. These data indicate that the previously established upper temperature (73–75 °C) for the transition to photosynthesis is reached in many basic hot springs, but that the transition occurs at lower temperature with decreasing pH below ~ 6.5. As an example, no strong evidence for photosynthesis was found above 45 °C at pH ~ 2. In several locations, photosynthesis appears to be suppressed despite temperatures and pH values that permit photosynthesis elsewhere. Sulfide concentrations may be responsible for the suppression of photosynthesis at these sites. Total sulfide concentrations were observed to decrease downstream in hot spring outflow channels. Abiotic processes (degassing, oxidation, mineral precipitation, etc.) are too slow to account for these decreases, suggesting an explanation from microbial sulfide oxidation that is supported by field experiments. Microbial sulfide oxidation may determine the ultimate suitability of some hot springs for microbial photosynthesis.

Thesis (Ph. D.)--Joint Program in Chemical Oceanography (Massachusetts Institute of Technology, Dept. of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), 2011.

Engineered nanoparticles offer the chance to improve drug transport and delivery through biological barriers, exploiting the possibility to leave the blood circulation and traverse the endothelial vascular bed, blood-brain barrier (BBB) included, to reach their target. It is known that nanoparticles gather molecules on their surface upon contact with biological fluids, forming the "protein corona", which can affect their fate and therapeutic/diagnostic performance, yet no information on the corona's evolution across the barrier has been gathered so far. Using a cellular model of the BBB and gold nanoparticles, we show that the composition of the corona undergoes dramatic quantitative and qualitative molecular modifications during passage from the "blood" to the "brain" side, while it is stable once beyond the BBB. Thus, we demonstrate that the nanoparticle corona dynamically and drastically evolves upon crossing the BBB and that its initial composition is not predictive of nanoparticle fate and performance once beyond the barrier at the target organ.

Much evidence suggests a protective role of high-density lipoprotein (HDL) and its major apolipoprotein apoA-I, in Alzheimer's disease (AD). The biogenesis of nascent HDL derived from a first lipidation of apoA-I, which is synthesized by the liver and intestine but not in the brain, in a process mediated by ABCA1. The maturation of nascent HDL in mature spherical HDL is due to a subsequent lipidation step, LCAT-mediated cholesterol esterification, and the change of apoA-I conformation. Therefore, different subclasses of apoA-I-HDL simultaneously exist in the blood circulation. Here, we investigated if and how the lipidation state affects the ability of apoA-I-HDL to target and modulate the cerebral β-amyloid (Aβ) content from the periphery, that is thus far unclear. In particular, different subclasses of HDL, each with different apoA-I lipidation state, were purified from human plasma and their ability to cross the blood-brain barrier (BBB), to interact with Aβ aggregates, and to affect Aβ efflux across the BBB was assessed in vitro using a transwell system. The results showed that discoidal HDL displayed a superior capability to promote Aβ efflux in vitro (9 × 10-5 cm/min), when compared to apoA-I in other lipidation states. In particular, no effect on Aβ efflux was detected when apoA-I was in mature spherical HDL, suggesting that apoA-I conformation, and lipidation could play a role in Aβ clearance from the brain. Finally, when apoA-I folded its structure in discoidal HDL, rather than in spherical ones, it was able to cross the BBB in vitro and strongly destabilize the conformation of Aβ fibrils by decreasing the order of the fibril structure (-24%) and the β-sheet content (-14%). These data suggest that the extent of apoA-I lipidation, and consequently its conformation, may represent crucial features that could exert their protective role in AD pathogenesis.