Manufacturing considerations for the development of lipid nanoparticles using microfluidics

In the recent of years, the use of lipid nanoparticles (LNPs) for RNA delivery has gained considerable attention, with a large number in the clinical pipeline as vaccine candidates or to treat a wide range of diseases. Microfluidics offers considerable advantages for their manufacture due to its scalability, reproducibility and fast preparation. Thus, in this study, we have evaluated operating and formulation parameters to be considered when developing LNPs. Among them, the flow rate ratio (FRR) and the total flow rate (TFR) have been shown to significantly influence the physicochemical characteristics of the produced particles. In particular, increasing the TFR or increasing the FRR decreased the particle size. The amino lipid choice (cationic—DOTAP and DDAB; ionisable—MC3), buffer choice (citrate buffer pH 6 or TRIS pH 7.4) and type of nucleic acid payload (PolyA, ssDNA or mRNA) have also been shown to have an impact on the characteristics of these LNPs. LNPs were shown to have a high (>90%) loading in all cases and were below 100 nm with a low polydispersity index (≤0.25). The results within this paper could be used as a guide for the development and scalable manufacture of LNP systems using microfluidics

Cryoprotection of Platelets by Grafted Polymers

Unlike red blood cells (RBC) which are stored at 4°C, platelets are stored at 22–24°C (room temperature) due to biophysical and biochemical changes induced by cold temperatures aggregately known as the ‘cold storage lesion’ (CSL). However, 22°C storage greatly increases the risk of microbial growth, thus limiting the safe storage of platelets to only 5–7 days (versus 42 days for RBC). Consequent to the short shelf life of platelets, blood services face chronic shortages of these life-saving cells. To overcome both the risk of microbial contamination and the constrained supplies of platelets, renewed research into attenuating the CSL and/or determining where cold stored platelets are clinically suitable are ongoing. In this chapter, we show that the covalent grafting of methoxypolyethylene glycol (mPEG), a biocompatible polymer, to the membrane of platelets attenuates the CSL. Moreover, the grafted mPEG serves as a potent cryoprotectant allowing platelets to be stored at 4°C, or frozen at −20°C, while retaining normal platelet counts and biologic function. The successful development of platelet PEGylation may provide a means by which the cold storage of platelets can be achieved with a minimal loss of platelet quality while improving both platelet microbial safety and inventory

Vertical profiles of light absorption and scattering associated with black carbon particle fractions in the springtime Arctic above 79◦ N

Despite the potential importance of black carbon (BC) for radiative forcing of the Arctic atmosphere, ver- tically resolved measurements of the particle light scatter- ing coefficient (σsp ) and light absorption coefficient (σap ) in the springtime Arctic atmosphere are infrequent, espe- cially measurements at latitudes at or above 80◦ N. Here, re- lationships among vertically distributed aerosol optical prop- erties (σap, σsp and single scattering albedo or SSA), par- ticle microphysics and particle chemistry are examined for a region of the Canadian archipelago between 79.9 and 83.4◦ N from near the surface to 500 hPa. Airborne data collected during April 2015 are combined with ground- based observations from the observatory at Alert, Nunavut and simulations from the Goddard Earth Observing Sys- tem (GEOS) model, GEOS-Chem, coupled with the TwO- Moment Aerosol Sectional (TOMAS) model (collectively GEOS-Chem–TOMAS; Kodros et al., 2018) to further our knowledge of the effects of BC on light absorption in the Arctic troposphere. The results are constrained for σsp less than 15 Mm−1, which represent 98 % of the observed σsp, be- cause the single scattering albedo (SSA) has a tendency to be lower at lower σsp, resulting in a larger relative contribution to Arctic warming. At 18.4 m2 g−1, the average BC mass ab- sorption coefficient (MAC) from the combined airborne and Alert observations is substantially higher than the two aver- aged modelled MAC values (13.6 and 9.1 m2 g−1) for two different internal mixing assumptions, the latter of which is based on previous observations. The higher observed MAC value may be explained by an underestimation of BC, the presence of small amounts of dust and/or possible differences in BC microphysics and morphologies between the obser- vations and model. In comparing the observations and simulations, we present σap and SSA, as measured, and σap/2 and the corresponding SSA to encompass the lower modelled MAC that is more consistent with accepted MAC values. Me- dian values of the measured σap, rBC and the organic com- ponent of particles all increase by a factor of 1.8 ± 0.1, going from near-surface to 750 hPa, and values higher than the sur- face persist to 600 hPa. Modelled BC, organics and σap agree with the near-surface measurements but do not reproduce the higher values observed between 900 and 600 hPa. The dif- ferences between modelled and observed optical properties follow the same trend as the differences between the mod- elled and observed concentrations of the carbonaceous com- ponents (black and organic). Model-observation discrepan- cies may be mostly due to the modelled ejection of biomass burning particles only into the boundary layer at the sources. For the assumption of the observed MAC value, the SSA range between 0.88 and 0.94, which is significantly lower than other recent estimates for the Arctic, in part reflecting the constraint of σsp < 15 Mm−1. The large uncertainties in measuring optical properties and BC, and the large differ- ences between measured and modelled values here and in the literature, argue for improved measurements of BC and light absorption by BC and more vertical profiles of aerosol chemistry, microphysics and other optical properties in the Arctic

Can pulsed electromagnetic fields trigger on-demand drug release from high-tm magnetoliposomes?

Recently, magnetic nanoparticles (MNPs) have been used to trigger drug release from magnetoliposomes through a magneto-nanomechanical approach, where the mechanical actuation of the MNPs is used to enhance the membrane permeability. This result can be effectively achieved with low intensity non-thermal alternating magnetic field (AMF), which, however, found rare clinic application. Therefore, a different modality of generating non-thermal magnetic fields has now been investigated. Specifically, the ability of the intermittent signals generated by non-thermal pulsed electromagnetic fields (PEMFS) were used to verify if, once applied to high-transition temperature magnetoliposomes (high-Tm MLs), they could be able to efficiently trigger the release of a hydrophilic model drug. To this end, hydrophilic MNPs were combined with hydrogenated soybean phosphatidylcholine and cholesterol to design high-Tm MLs. The release of a dye was evaluated under the effect of PEMFs for different times. The MNPs motions produced by PEMF could effectively increase the bilayer permeability, without affecting the liposomes integrity and resulted in nearly 20% of release after 3 h exposure. Therefore, the current contribution provides an exciting proof-of-concept for the ability of PEMFS to trigger drug release, considering that PEMFS find already application in therapy due to their anti-inflammatory effects

Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads

The success of Onpattro™ (patisiran) clearly demonstrates the utility of lipid nanoparticle (LNP) systems for enabling gene therapies. These systems are composed of ionizable cationic lipids, phospholipid, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced through rapid-mixing of an ethanolic-lipid solution with an acidic aqueous solution followed by dialysis into neutralizing buffer. A detailed understanding of the mechanism of LNP formation is crucial to improving LNP design. Here we use cryogenic transmission electron microscopy and fluorescence techniques to further demonstrate that LNP are formed through the fusion of precursor, pH-sensitive liposomes into large electron-dense core structures as the pH is neutralized. Next, we show that the fusion process is limited by the accumulation of PEG-lipid on the emerging particle. Finally, we show that the fusion-dependent mechanism of formation also applies to LNP containing macromolecular payloads including mRNA, DNA vectors, and gold nanoparticles

Technological challenges in the preclinical development of an HIV nanovaccine candidate

Despite a very active research in the field of nanomedicine, only a few nano-based drug delivery systems have reached the market. The “death valley” between research and commercialization has been partially attributed to the limited characterization and reproducibility of the nanoformulations. Our group has previously reported the potential of a peptide-based nanovaccine candidate for the prevention of SIV infection in macaques. This vaccine candidate is composed of chitosan/dextran sulfate nanoparticles containing twelve SIV peptide antigens. The aim of this work was to rigorously characterize one of these nanoformulations containing a specific peptide, following a quality-by-design approach. The evaluation of the different quality attributes was performed by several complementary techniques, such as dynamic light scattering, nanoparticle tracking analysis, and electron microscopy for particle size characterization. The inter-batch reproducibility was validated by three independent laboratories. Finally, the long-term stability and scalability of the manufacturing technique were assessed. Overall, these data, together with the in vivo efficacy results obtained in macaques, underline the promise this new vaccine holds with regard to its translation to clinical trialsThis work was supported by the European Union’s Horizon 2020 research program (NanoPilot project – grant agreement number 646142) and by Xunta de Galicia’s Grupos de referencia competitiva (grant number ED431C 2017/09). T.G. Dacoba acknowledges a predoctoral FPU grant from the Spanish Ministry of Education, Culture and Sports (grant number FPU14/05866)S

Alternative approaches to reduce type I collagen degradation

Orientadores: Fernanda Miori Pascon, Marcelo GianniniTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Os objetivos nesta tese foram: (1) investigar o efeito de FeCl3 na atividade da Catepsina K (CT-K) e capacidade de prevenir degradação do colágeno solúvel (CS) e insolúvel (CI); (2) comparar os efeitos do ácido fosfórico a 35% (AF) e soluções condicionantes contendo ácido cítrico (AC) e FeCl3 na resistência da união a dentina e degradação do colágeno. A tese foi apresentada em dois artigos: (1) para avaliar a atividade enzimática da CT-K e efeito de FeCl3 utilizou-se a espectrofluorimetria. Para avaliar a degradação do CS e CI (fibras de tendões de cauda de camundongo - FTCCs), os substratos foram incubados com CT-K e concentrações de 0,02% a 2% de FeCl3. O sobrenadante foi submetido à eletroforese de proteínas. Os experimentos foram feitos em triplicata. As FTCCs foram caracterizadas em microscopia eletrônica de varredura (MEV). A quantidade de Fe absorvida foi determinada pela espectroscopia de energia dispersiva de raios-X (EDX). Os dados foram submetidos à análise de variância (Brown-Forsythe), normalidade (Shapiro-Wilk ou Anderson-Darling), ANOVA um fator + Holm-Sidak ou Tukey (?=5%). (2) para avaliar a resistência da união a microtração (?TBS), 78 dentes foram distribuídos em 13 grupos de soluções contendo AC e/ou FeCl3 nas concentrações: 10%-1,8%, 10%-0,6%, 10% AC, 5%-1,8%, 5%-0,6%, 5% AC, 1%-1,8%, 1%-0,6%, 1% AC, 1,8% FeCl3, 0,6% FeCl3 (aplicadas por 15 segundos) e com AF (aplicado por 5 ou 15 segundos). Utilizou-se o sistema adesivo Adper Scotchbond Multi-Purpose, fotoativado por 20 segundos com o aparelho Bluephase G2 (1200Mw/cm2). A ?TBS foi avaliada em 24 horas e 9 meses de armazenamento. Para a degradação do colágeno dentinário, fatias de dentina (n=3) foram tratadas com e sem FeCl3 e incubadas com CT-K. O sobrenadante foi submetido à eletroforese de proteínas. A dentina foi caracterizada após a incubação utilizando MEV. Outras fatias foram submetidas às soluções condicionantes por 15 segundos e analisadas utilizando EDX. Os dados foram submetidos aos testes: normalidade, ANOVA dois fatores + Holm-Sidak, ANOVA um fator + Tukey ou Fisher (?=5%). A análise por espectrofluorimetria revelou que o FeCl3 inibiu a atividade de CT-K nas concentrações 0,005%-0,08%e suprimiu a atividade na concentração 0,08%. FeCl3 foi capaz de prevenir a degradação do CS e CI contra CT-K para as FTCCs e para a dentina desmineralizada. As micrografias revelaram desenovelamento das FTCCs na ausência de FeCl3 e integridade quando expostas a concentrações de 0,02%-0,8%de FeCl3. Observou-se interação Fe/colágeno. Os grupos 5-0,6 e 5-1,8 de AC/FeCl3 apresentaram maiores valores de ?TBS comparados ao AF, independentemente do tempo de armazenamento. Observou-se redução da ?TBS aos 9 meses para todos os grupos. A solução contendo 1,8% de FeCl3 preservou a estrutura da dentina. Observou-se pela análise de EDX que a imersão nas soluções condicionantes com FeCl3 por 15 segundos foi suficiente para que as fibrilas de colágeno adsorvessem Fe. Concluiu-se que o FeCl3 agiu como um indutor de reticulação do colágeno e como inibidor da atividade CT-K e estas duas habilidades atuaram em conjunto para a preservação do colágeno tipo I nas FTCCs e na dentina desmineralizadaAbstract: The aim of this thesis was (1) to investigate the effect of FeCl3 on Cathepsin K (CT-K) activity of and the ability to prevent degradation of soluble (SC) and insoluble collagen (IC), and (2) to compare the effects of phosphoric acid 35% and conditioning solutions containing citric acid (CA) and/or FeCl3 in dentine bond strength and collagen degradation. The thesis was presented in two articles: (1) spectrofluorimetry was used to evaluate the FeCl3 effect against CT-K enzymatic activity. To evaluate SC and IC (mouse tail tendon fibers-MTTF) degradation, the substrates were incubating with CT-K with different concentrations of FeCl3 (0.02% to 2%). The supernatant was submitted to protein electrophoresis. The experiments were done in triplicate. The fibers were characterized after incubation by scanning electron microscopy (SEM). The amount of Fe absorbed was determined by X-ray dispersive energy spectroscopy (EDX). Data were submitted to analysis of variance (Brown-Forsythe), normality test (Shapiro-Wilk or Anderson-Darling), and one-way ANOVA (Holm-Sidak or Tukey post-test) (?=5%). (2) for the microtensile bond strength (?TBS), 78 teeth were distributed into 13 groups of solutions containing citric acid (AC) and/or FeCl3 in different concentrations: 10%-1,8%, 10%-0,6%, 10% AC, 5%-1,8%, 5%-0,6%, 5% AC, 1%-1,8%, 1%-0,6%, 1% AC, 1,8% FeCl3, 0,6% FeCl3 (applied for 15 seconds) e com AF (applied for 5 and 15 seconds). The Adper Scotchbond Multi-Purpose adhesive system was used and light/cured for 20 seconds with Bluephase G2 (1200Mw/cm2). The ?TBS was evaluated at 24 hours and 9 months of storage. For the dentine collagen degradation, dentine slices (n=3) were treated with and without FeCl3 were incubated with CT-K. The supernatant was submitted to protein electrophoresis. Dentine was characterized after enzymatic treatment by SEM, and other slices were submitted to conditioning solutions and analyzed by EDX. The data were submitted to the tests: normality, two¿way ANOVA and post-test Holm-Sidak, one¿way ANOVA and Tukey or Fisher tests (?=5%). Spectrofluorimetry analysis revealed that FeCl3 inhibited CT-K activity at concentrations 0.005%-0.08% and suppressed activity when using 0.08%. FeCl3 was able to prevent the degradation of soluble collagen and collagen fibers against CT-K for the MTTF and the demineralized dentine. The micrographs revealed unraveling of collagen fibers in the absence of FeCl3 and showed fiber integrity exposed to 0.02%-2% FeCl3 concentrations. The Fe/collagen interaction was observed. AC/FeCl3 5-0.6 and 5-1.8 solutions had higher ?TBS values ??compared to the AF, regardless of storage time. A reduction of ?TBS was observed at 9 months for all groups. The treatment solution 1.8% FeCl3 preserved the dentine structure. The EDX analysis indicated that the 15 second immersion in the FeCl3 conditioning solutions was sufficient for the collagen fibrils to adsorb Fe. It was concluded that FeCl3 acted both as an inducer of collagen cross-linking and as inhibitor of CT-K activity, both abilities acted together for the preservation of collagen type I on the MTTF and on the demineralized dentineDoutoradoMateriais DentariosDoutora em Materiais Dentários99999.010711/2014-07, 1777-2014CAPE

The Mass-Concentration Relation and the Stellar-to-Halo Mass Ratio in the CFHT Stripe 82 Survey

We present a new measurement of the mass-concentration relation and the stellar-to-halo mass ratio over the halo mass range $5\times 10^{12}$ to $2\times 10^{14}M_{\odot}$. To achieve this, we use weak lensing measurements from the CFHT Stripe 82 Survey (CS82), combined with the central galaxies from the redMaPPer cluster catalogue and the LOWZ/CMASS galaxy sample of the Sloan Digital Sky Survey-III Baryon Oscillation Spectroscopic Survey Tenth Data Release. The stacked lensing signals around these samples are modelled as a sum of contributions from the central galaxy, its dark matter halo, and the neighboring halos, as well as a term for possible centering errors. We measure the mass-concentration relation: $c_{200c}(M)=A(\frac{M_{200c}}{M_0})^{B}$ with $A=5.24\pm1.24, B=-0.13\pm0.10$ for $0.2<z<0.4$ and $A=6.61\pm0.75, B=-0.15\pm0.05$ for $0.4<z<0.6$. These amplitudes and slopes are completely consistent with predictions from recent simulations. We also measure the stellar-to-halo mass ratio for our samples, and find results consistent with previous measurements from lensing and other techniques.Comment: 10 pages, 3 figures, 3 table