Charge Neutralization Research Articles

Coagulative removal of polystyrene microplastics from aqueous matrices using FeCl3-chitosan system: Experimental and artificial neural network modeling

Effluent from sewage treatment plants (STPs) is a significant source of microplastics (MPs) re-entry into the environment. Coagulation-flocculation-sedimentation (CFS) process as an initial tertiary treatment step requires investigation for coagulative MPs removal from secondary-treated sewage effluents. In this study, experiments were conducted on synthetic water containing 25 mg/L polystyrene (PS) MPs using varying dosages of FeCl3 (1–10 mg/L) and chitosan (0.25–9 mg/L) to assess the effect of process parameters, such as pH (4−8), stirring speed (0–200 rpm), and settling time (10–40 min). Results revealed that ∼89.3% and 21.4% of PS removal were achieved by FeCl3 and chitosan, respectively. Further, their combination resulted in a maximum of 99.8% removal at favorable conditions: FeCl3: 2 mg/L, chitosan: 7 mg/L, pH: 6.3, stirring speed: 100 rpm, and settling time: 30 min, with a statistically significant (p < 0.05) effect. Artificial neural network (ANN) validated the experimental results with RMSE = 1.0643 and R2 = 0.9997. Charge neutralization, confirmed by zeta potential, and adsorption, ascertained by field-emission scanning electron microscope (FESEM) and Fourier-transform infrared spectroscopy (FTIR), were primary mechanisms for efficient PS removal. For practical considerations, the application of the FeCl3-chitosan system on the effluents from moving bed biofilm reactor (MBBR) and sequencing batch reactor (SBR)-based STPs, spiked with PS microbeads, showed > 98% removal at favorable conditions.

Bistability between π-diradical open-shell and closed-shell states in indeno[1,2-a]fluorene.

Indenofluorenes are non-benzenoid conjugated hydrocarbons that have received great interest owing to their unusual electronic structure and potential applications in nonlinear optics and photovoltaics. Here we report the generation of unsubstituted indeno[1,2-a]fluorene on various surfaces by the cleavage of two C-H bonds in 7,12-dihydroindeno[1,2-a]fluorene through voltage pulses applied by the tip of a combined scanning tunnelling microscope and atomic force microscope. On bilayer NaCl on Au(111), indeno[1,2-a]fluorene is in the neutral charge state, but it exhibits charge bistability between neutral and anionic states on the lower-workfunction surfaces of bilayer NaCl on Ag(111) and Cu(111). In the neutral state, indeno[1,2-a]fluorene exhibits one of two ground states: an open-shell π-diradical state, predicted to be a triplet by density functional and multireference many-body perturbation theory calculations, or a closed-shell state with a para-quinodimethane moiety in the as-indacene core. We observe switching between open- and closed-shell states of a single molecule by changing its adsorption site on NaCl.

Rna Buffering Fluorogenic Probe for Nucleolar Morphology Stable Imaging And Nucleolar Stress-Generating Agents Screening.

In the realm of cell research, membraneless organelles have become a subject of increasing interest. However, their ever-changing and amorphous morphological characteristics have long presented a formidable challenge when it comes to studying their structure and function. In this paper, a fluorescent probe Nu-AN is reported, which exhibits the remarkable capability to selectively bind to and visualize the nucleolus morphology, the largest membraneless organelle within the nucleus. Nu-AN demonstrates a significant enhancement in fluorescence upon its selective binding to nucleolar RNA, due to the inhibited twisted intramolecular charge-transfer (TICT) and reduced hydrogen bonding with water. What sets Nu-AN apart is its neutral charge and weak interaction with nucleolus RNA, enabling it to label the nucleolus selectively and reversibly. This not only reduces interference but also permits the replacement of photobleached probes with fresh ones outside the nucleolus, thereby preserving imaging photostability. By closely monitoring morphology-specific changes in the nucleolus with this buffering fluorogenic probe, screenings for agents are conducted that induce nucleolar stress within living cells.

Analysis of the 3.2–3.3 μm Interstellar Absorption Feature on Three Milky Way Sightlines

We report new analyses of spectra of the 3.2–3.3 μm absorption feature observed in the diffuse interstellar medium toward three Milky Way sources: 2MASS J17470898 − 2829561 (2M1747) and the Quintuplet Cluster, both located in the Galactic center, and Cygnus OB2-12. The 3.2–3.3 μm interval coincides with the CH-stretching region for compact polycyclic aromatic hydrocarbons (PAHs). We focus on the 2M1747 spectrum. Its published optical depth spectrum contains residual telluric transmission features, which arise from the 0.06 difference in mean airmasses between the observations of the source and its telluric standard star. We corrected the published spectrum by adding the airmass residual optical depth spectrum. The corrected spectrum is well fit by a superposition of four Gaussians. The absorption spectra of the other two sources were also fit by four Gaussians, with similar central wavelengths, widths, and relative peak opacities. We associate the three longer wavelength Gaussians covering the 3.23–3.31 μm interval with compact PAHs in positive, neutral, and negative charge states. We identify the shortest-wavelength Gaussian, near 3.21 μm, with irregularly shaped PAHs. Constraints imposed by spectral smoothness on the corrected 2M1747 spectrum, augmented by a PAH cluster formation model for post-asymptotic giant branch stars, suggests that >99% of the PAHs in the diffuse interstellar medium reside in small clusters. This study supports the PAH hypothesis, and it suggests that a family of primarily compact PAHs with a C66H20 (circumvalene) parent is consistent with the observed mid-infrared and ultraviolet interstellar absorption spectrum.

Trivelpiece–Gould modes and low-frequency electron–ion instability of non-neutral plasma

The frequency spectra of the Trivelpiece–Gould modes of a waveguide partially filled with non-neutral plasma are determined numerically by solving the dispersion equation. The modes having azimuthal number $m = 1$ are considered. The results are presented for the entire acceptable range of electron densities, magnetic field strengths, for different values of the charge neutralization coefficient. The Cherenkov resonance condition of an ion with a diocotron mode having a finite value of the longitudinal wave vector was studied. The characteristics of resonant low-frequency electron–ion instability caused by relative azimuth motion of electrons and ions in crossed fields and by the anisotropy of the distribution function of ions are discussed. Ions are created by ionization of residual gas in the plasma volume. Due to the anisotropy, instability occurs not only in the vicinity of the resonance, but also outside it. For typical values of plasma parameters in experiments, estimations of the frequency growth rate are given. A conclusion is drawn that this instability can be the cause of the low-frequency oscillations observed in linear devices with non-neutral plasma produced in an electron beam channel.

The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Gene Expression in a Cell-Free Transcription/Translation System.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a protein that plays a key role in the metabolism of low-density lipoprotein (LDL) cholesterol. The gain-of-function mutations of the PCSK9 gene lead to a reduced number of surface LDL receptors by binding to them, eventually leading to endosomal degradation. This, in turn, is the culprit of hypercholesterolemia, resulting in accelerated atherogenesis. The modern treatment for hypercholesterolemia encompasses the use of biological drugs against PCSK9, like monoclonal antibodies and gene expression modulators such as inclisiran-a short, interfering RNA (siRNA). Peptide nucleic acid (PNA) is a synthetic analog of nucleic acid that possesses a synthetic peptide skeleton instead of a phosphate-sugar one. This different structure determines the unique properties of PNA (e.g., neutral charge, enzymatic resistance, and an enormously high affinity with complementary DNA and RNA). Therefore, it might be possible to use PNA against PCSK9 in the treatment of hypercholesterolemia. We sought to explore the impact of three selected PNA oligomers on PCSK9 gene expression. Using a cell-free transcription/translation system, we showed that one of the tested PNA strands was able to reduce the PCSK9 gene expression down to 74%, 64%, and 68%, as measured by RT-real-time PCR, Western blot, and HPLC, respectively. This preliminary study shows the high applicability of a cell-free enzymatic environment as an efficient tool in the initial evaluation of biologically active PNA molecules in the field of hypercholesterolemia research. This cell-free approach allows for the omission of the hurdles associated with transmembrane PNA transportation at the early stage of PNA selection.

Organic Nucleation: Water Rearrangement Reveals the Pathway of Ibuprofen.

The organic nucleation of the pharmaceutical ibuprofen is investigated, as triggered by the protonation of ibuprofen sodium salt at elevated pH. The growth and aggregation of nanoscale solution species by Analytical Ultracentrifugation and Molecular Dynamics (MD) simulations is tracked. Both approaches reveal solvated molecules, oligomers, and prenucleation clusters, their size as well as their hydration at different reaction stages. By combining surface-specific vibrational spectroscopy and MD simulations, water interacting with ibuprofen at the air-water interface during nucleation is probed. The results show the structure of water changes upon ibuprofen protonation in response to the charge neutralization. Remarkably, the water structure continues to evolve despite the saturation of protonated ibuprofen at the hydrophobic interface. This further water rearrangement is associated with the formation of larger aggregates of ibuprofen molecules at a late prenucleation stage. The nucleation of ibuprofen involves ibuprofen protonation and their hydrophobic assembly. The results highlight that these processes are accompanied by substantial water reorganization. The critical role of water is possibly relevant for organic nucleation in aqueous environments in general.

Preparation, characterization and application of a composite bioflocculant.

Composite flocculant PAFS-PDM was prepared from Polymeric aluminium ferric sulphate (PAFS) and Poly (diallyl dimethyl ammonium chloride) (PDM) in this study. A bacterium was selected from the soil near the shale gas exploitation platform as a bioflocculant-producing bacterium, and polysaccharide was extracted and combined with PAFS-PDM to obtain composite bioflocculant (CBF) to treat shale gas fracturing flowback fluid. The prepared CBF was characterized and the results showed that the prepared PAFS-PDM contained aluminium-iron hydroxyl polymer, which was a cationic flocculant. By measuring the turbidity removal rate and chemical oxygen demand (COD) removal efficiency, the function mechanism of CBF on the shale gas fracturing flowback fluid was discussed. The results showed that CBF had a stable treatment effect on fracturing flowback fluid when the pH value was about 7.0. With the increase of dosage, the coagulation efficiency increased first and then decreased. When the dosage of the CBF was 2500 mg·L-1, the treatment effect of shale gas fracturing flowback fluid was the best, and COD removal rate reached 89.43%. Through Zeta potential analysis, it was concluded that one of the coagulation mechanisms was electrical neutralization. According to the characterization results, it could be concluded that both adsorption bridging and charge neutralization mechanisms played important roles in the treatment of shale gas fracturing flowback fluids.

Nanoparticles targeting the intestinal Fc receptor enhance intestinal cellular trafficking of semaglutide

Semaglutide is the first oral glucagon-like peptide-1 (GLP-1) analog commercially available for the treatment of type 2 diabetes. In this work, semaglutide was incorporated into poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles (NPs) to improve its delivery across the intestinal barrier. The nanocarriers were surface-decorated with either a peptide or an affibody that target the human neonatal Fc receptor (hFcRn), located on the luminal cell surface of the enterocytes. Both ligands were successfully conjugated with the PLGA-PEG via maleimide-thiol chemistry and thereafter, the functionalized polymers were used to produce semaglutide-loaded NPs. Monodisperse NPs with an average size of 170 nm, neutral surface charge and 3% of semaglutide loading were obtained. Both FcRn-targeted NPs exhibited improved interaction and association with Caco-2 cells (cells that endogenously express the hFcRn), compared to non-targeted NPs. Additionally, the uptake of FcRn-targeted NPs was also observed to occur in human intestinal organoids (HIOs) expressing hFcRn through microinjection into the lumen of HIOs, resulting in potential increase of semaglutide permeability for both ligand-functionalized nanocarriers. Herein, our study demonstrates valuable data and insights that the FcRn-targeted NPs has the capacity to promote intestinal absorption of therapeutic peptides.

FERRIHYDRITE-CHITOSAN NANOCOMPOSITE AS A RECYCLABLE FLOCCULANT FOR PALM OIL MILL EFFLUENT

In the present study, ferrihydrite-chitosan nanocomposite (FCN) was successfully produced by co-precipitation method and used for the first time as a recyclable flocculant for pre-treatment of palm oil mill effluent (POME). The physicochemical properties of FCN were studied using Raman spectrometer, Scanning Electron Microscope (SEM) and Thermogravimetric Analyser (TGA). The feasibility of FCN to remove total suspended solids (TSS), turbidity, chemical oxygen demand (COD), and, oil and grease (O&amp;G) from POME was investigated using a jar test method. The optimum conditions for contaminant removal from POME were determined by varying the experimental parameters such as flocculant dosage, solution pH and settling time. The results obtained showed that FCN, at a dosage of 1.5 g/L, a contact time of 60 min and pH of 5.0 gave a highest reduction of turbidity, TSS, COD and O&amp;G levels by 72.38%, 77.32%, 71.60% and 53.40%, respectively. Besides that, FCN exhibited a better flocculation performance as compared to alum and chitosan. After three cycles of flocculation/deflocculation process, FCN retained satisfying flocculation efficiency and flocculants recovery in the range of 80-83% and 43.2-78.6%, respectively. Combination of charge neutralisation and polymer bridging was the main key mechanism of interaction between FCN and POME contaminants. The synergy effect between iron oxide/oxyhydroxide nanoparticle and chitosan has increased the physicochemical properties and flocculation performance of the FCN nanocomposite. Overall, FCN nanocomposite can be used an alternative flocculant for POME treatment.

Copolymers functionalized with quaternary ammonium compounds under template chain exhibit simultaneously efficient bactericidal and flocculation properties: Characterization, performance and mechanism

In this work, novel multifunctional cationic template copolymers with flocculation and sterilization capabilities were synthesized using a low-pressure ultraviolet (LP-UV) template polymerization method for the removal of kaolin and Escherichia coli (E. coli) from water. The influence of template agents on the structural performance of the copolymers was evaluated through characterization, which showed that template copolymer TPADM possesses a higher cationic charge density and a more complex rough surface, contributing to better flocculation performance than that of the non-template copolymer CPADM. Under optimal experimental conditions, TPADM-1 exhibited removal rates of 98.45% for kaolin and 99% for E. coli (OD600 =0.04), marginally outperforming the non-template copolymer. Simultaneously, TPADM-1 produced good adaptability to kaolin and E. coli wastewater in terms of wide pH, speculating that charge neutralization, adsorption bridging, patching, and sweeping simultaneously dominate the flocculation mechanism. Interestingly, SEM and 3D-EEM analysis confirm that the sterilization of E. coli occurs through two distinct functions: initially adsorption followed by subsequent cell membrane rupture and leakage of cellular contents, ultimately leading to cell death. This research further confirms the feasibility of the designed novel multifunctional copolymers for achieving simultaneous disinfection and turbidity removal, demonstrating practical applicability in real water treatment processes.

Enhanced removal of polyethylene microplastics from water through polymeric ferric sulfate with laminarin

Coagulation has been demonstrated as an effective method for eliminating microplastics from water. However, based on the existing literature, the use of a single coagulant has not yet yielded satisfactory results in microplastic removal. Hence, it is crucial to investigate the potential of enhanced coagulation techniques. In this study, laminarin (LA) was used as a novel coagulant aid to investigate its enhanced coagulation performance in the removal of polyethylene (PE) microplastics using polymeric ferric sulfate (PFS). The coagulation mechanism was investigated through zeta potential, Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). The results demonstrated that the coagulation efficiency of PFS on PE microplastics could be improved by adding an appropriate amount of LA. The removal rates of PE microplastics by single PFS and PFS-LA reached 48.5% and 93.8%, respectively, when the dosages of PFS and LA were 250 mg/L and 5 mg/L. The coagulation mechanisms of PFS and PFS-LA on PE microplastics were found to be the same, with the addition of LA significantly enhancing the effects of charge neutralization, adsorption bridging, and sweep flocculation of PFS. Various experiments conducted under different pH levels, microplastic particle sizes, coexisting ions, humic acid coexistence, and real water conditions demonstrated that the PFS-LA complex coagulation system exhibited better environmental adaptability.

Removal of algae from ballast water by cationic flocculant-composite titanium xerogel coagulant: Differences in freshwater and seawater

The treatment of ballast water has been garnering increasing attention, with planktonic algae removal being a crucial part. Coagulation has been demonstrated as an effective method for algae removal, albeit few coagulants exhibit excellent coagulation performance in both freshwater and seawater. To address this issue, the cationic flocculant poly dimethyl diallyl ammonium chloride (PDADMAC) was composited with titanium xerogel coagulant (TXC), abbreviated as PT. Using the algae Cyclotella meneghiniana as the research object, the algal removal performance and mechanisms of PT in freshwater and seawater were investigated. Cell integrity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content of C. meneghiniana during floc storage were also evaluated. The PT composite coagulant exhibited excellent coagulation performance in both freshwater and seawater. Its algal removal ability was superior to TXC and the commercial coagulants polyaluminum chloride (PAC) and polymerized ferrous sulfate (PFS), with removal rates for C. meneghiniana reaching 98.3 % in freshwater and 98.4 % in seawater at a 80 mg/L dosage. PT also exhibited excellent removal performance over wide dosage and pH ranges. PT acted via distinct coagulation mechanisms in freshwater and seawater, with charge neutralization and bridging played major roles in freshwater and sweeping being important in seawater. The floc storage experiment indicated that the coagulation process did not directly lead to algal cell fragmentation, but PT accelerated and exacerbated oxidative stress and oxidative damage to algal cells during floc storage. Furthermore, PT also showed excellent removal performance for the marine diatom Skeletonema costatum and the freshwater cyanobacteria Cylindrospermopsis raciborskii. This work not only provides a novel approach for algae removal in ballast water, but also enhances our understanding of the coagulation behavior of titanium-based coagulants in different water environments.

New Cap-Holed AlP, GaP, and InP Nanotubes.

The structural, vibrational, and electronic properties of new inorganic X-phosphide nanotubes (ch-XPNT), with X = Al, Ga, or In and chirality of (5,5), are investigated. These new NTs display cap-hole ends, with the cap-hole features induced by the nonpassivated ends. Studies are based on density functional theory (DFT) using the M06-2X, PBE, and B3LYP functionals together with the LanL2DZ basis set. All nanostructures have been relaxed by minimizing the total energy, assuming a nonmagnetic nature and a total neutral charge. Note that the cap-hole NTs are terminated by a 10-atom ring, which in turn favors the geometrical ordering and yields stable structures. The (5,5) ch-XPNT are highly electrophilic and nonpolar, in addition to having high solvation energy values. Let us remark that solvation energies are produced by the intermolecular forces that involve the induced dipoles. Structural and vibrational results show that the X-P bonds are single bonds. Finally, results suggest that the inorganic nanotubes are structurally stable with semiconductor features, which means that their functionalization may yield interesting future applications.

Poly(acrylamide-co-sodium acrylate) Used as a Flocculation Aid Associated with Aluminum Polychloride in the Treatment of Low Turbidity Surface Water by Charge Neutralization

Poly(acrylamide-co-sodium acrylate) Used as a Flocculation Aid Associated with Aluminum Polychloride in the Treatment of Low Turbidity Surface Water by Charge Neutralization

Influence of ion and hydration atmospheres on RNA structure and dynamics: insights from advanced theoretical and computational methods.

RNA, a highly charged biopolymer composed of negatively charged phosphate groups, defies electrostatic repulsion to adopt well-defined, compact structures. Hence, the presence of positively charged metal ions is crucial not only for RNA's charge neutralization, but they also coherently decorate the ion atmosphere of RNA to stabilize its compact fold. This feature article elucidates various modes of close RNA-ion interactions, with a special emphasis on Mg2+ as an outer-sphere and inner-sphere ion. Through examples, we highlight how inner-sphere chelated Mg2+ stabilizes RNA pseudoknots, while outer-sphere ions can also exert long-range electrostatic interactions, inducing groove narrowing, coaxial helical stacking, and RNA ring formation. In addition to investigating the RNA's ion environment, we note that the RNA's hydration environment is relatively underexplored. Our study delves into its profound interplay with the structural dynamics of RNA, employing state-of-the-art atomistic simulation techniques. Through examples, we illustrate how specific ions and water molecules are associated with RNA functions, leveraging atomistic simulations to identify preferential ion binding and hydration sites. However, understanding their impact(s) on the RNA structure remains challenging due to the involvement of large length and long time scales associated with RNA's dynamic nature. Nevertheless, our contributions and recent advances in coarse-grained simulation techniques offer insights into large-scale structural changes dynamically linked to the RNA ion atmosphere. In this connection, we also review how different cutting-edge computational simulation methods provide a microscopic lens into the influence of ions and hydration on RNA structure and dynamics, elucidating distinct ion atmospheric components and specific hydration layers and their individual and collective impacts.

Coal gasification crude slag based complex flocculants by two-step acid leaching process: synthesis, flocculation and mechanisms.

Coal gasification crude slag (CGCS) is the side-product of the coal gasification process, and its effective utilization has attracted great attention. A novel flocculant of poly-aluminum-ferric-acetate-chloride (PAFAC) was synthesized based on the recovery of CGCS by a two-step acid leaching process, namely HCl-acid leaching and HAc-acid leaching, which was optimized by an acid leaching liquor volume ratio of HCl to HAc of 3 : 2, polymerization pH of 3.5, and reaction temperature and time of 70 °C and 3.0 h, respectively. The performance of PAFAC was further evaluated by kaolin simulated wastewater, domestic sewage, river water, and aquaculture wastewater. The results revealed that PAFAC was feasible for the removal of turbidity, chemical oxygen demand (COD) and total phosphorus (TP). Moreover, PAFAC was characterized by X-ray Diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), X-ray fluorescence spectrometry (XRF) and scanning electron microscopy (SEM), which proved that PAFAC was a kind of amorphous polyionic composite. Additionally, the acid leaching kinetics and flocculation mechanisms were further investigated. It was found that the acid leaching process was followed by the unreacted shrinkage core model, and the flocculation process was dominated by charge neutralization, adsorption bridging and precipitation net trapping. The work is expected to develop a new method for the safe disposal of CGCS and provide a novel way for the preparation of Fe-Al composite flocculants, especially, offering a potential strategy for the promotion of the additional value of the coal chemical industry.

In silico and in vitro assessment of an optimized QbD-guided myoinositol and metformin-loaded mucus-penetrating particle-based gel for the amelioration of PCOS.

Polycystic ovarian syndrome (PCOS) is a multi-factorial endocrine disorder affecting women of reproductive age. However, its high prevalence and the unsuccessful translation of conventional modalities have made PCOS a pharmaco-therapeutic challenge. In the present study, we explored bi-formulations (comprising metformin-loaded mucus-penetrating nanoparticles, MTF-MPPs, and myoinositol-loaded mucus-penetrating particles, MI-MPPs) incorporated in a carbomer gel tailored for intravaginal administration. For the development and optimization of the MPPs-gel, a QbD (quality by design) approach was employed, including the initial and final risk assessment, central composite design of experts, and method validation. The optimized MTF-MPPs and MI-MPPs possessed an optimum nanometric particle size (195.0 nm and 178.8 nm, respectively) and a PDI of 0.150 and 0.123, respectively, together with a negligible negative zeta potential (-5.19 mV and -6.19 mV, respectively) through the vaginal mucus. It was observed that the MPPs are small and monodisperse with a neutral surface charge. It was observed that the MPPs-gel formulations released approximately 69.86 ± 4.65% of MTF and 67.14 ± 5.74% of MI within 120 h (5 days), which was observed to be sustained unlike MFT-MI-gel with approximately 94.89 ± 4.17% of MTF and 90.91 ± 15% of MI drugs released within 12 h. The confocal microscopy study of rhodamine-loaded MPPs indicated that they possessed a high fluorescence intensity at a depth of 15 μm, while as the penetration trajectory in the vaginal tissue increased to 35 μm, their intensity was reduced, appearing to be more prominent in the blood vessels. The analyzed data of MPPs-gel suggest that the optimized MPPs-gel formulation has potential to reach the targeted area via the uterovaginal mucosa, which has a wide network of blood vessels. Subsequently, in vivo studies were conducted and the results revealed that the proposed MPPs-gel formulation could regulate the estrous cycle of the reproductive system compared to the conventional formulation. Moreover, the formulation significantly reduced the weight of the ovaries compared to the control and conventional vaginal gel. Biochemical estimation showed improved insulin and sex hormone levels. Thus, the obtained data revealed that the deep penetration and deposition of MTF and MI on the targeted area through intravaginal delivery resulted in better therapeutic effects than the conventional vaginal gel. The obtained results confirmed the amelioration of PCOS upon treatment using the prepared MPPs-gel formulation. According to the relevant evaluation studies, it was concluded that MPPs-gel was retained in the vaginal cavity for systemic effects. Also, the sustained and non-irritating therapeutic effect meets the safety aspects. This work serves as a promising strategy for intravaginal drug delivery.

Intracellular trafficking kinetics of nucleic acid escape from lipid nanoparticles via fluorescence imaging.

Lipid nanoparticles (LNPs) are one of the most clinically advanced candidates for delivering nucleic acids to target cell populations, such as hepatocytes. Once LNPs are endocytosed, they must release their nucleic acid cargo into the cell cytoplasm. For delivering messenger RNA (mRNA), delivery into the cytosol is sufficient; however, for delivering DNA, there is an added diffusional barrier needed to facilitate nuclear uptake for transcription and therapeutic effect. Here, we use fluorescence microscopy to investigate the intracellular fate of different LNP formulations to determine the kinetics of localization to endosomes and lysosomes. LNPs used in the studies were prepared via self-assembly using a NanoAssemblr for microfluidic mixing. As the content of polyethylene glycol (PEG) within the LNP formulation influences cellular uptake by hepatocyte cells, the content and hydrocarbon chain length within the formulation were assessed for their impact on intracellular trafficking. Standard LNPs were then formed using three commercially available ionizable lipids, Dlin-MC3-DMA (MC3), Dlin-KC2-DMA (KC2), and SS-OP. Plasmid DNA (pDNA) and mRNA were used, more specifically with a mixture of Cyanine 3 (Cy3)-labeled and green fluorescence protein (GFP) producing plasmid DNA (pDNA) as well as Cy5-labeled GFP producing mRNA. After formulation, LNPs were characterized for the encapsulation efficiency of the nucleic acid, hydrodynamic diameter, polydispersity, and zeta potential. All standard LNPs were ~100 nm in diameter and had neutral surface charge. All LNPs resulted in encapsulation efficiency greater than 70%. Confocal fluorescence microscopy was used for the intracellular trafficking studies, where LNPs were incubated with HuH-7 hepatocyte cells at times ranging from 0-48 h. The cells were antibody-stained for subcellular components, including nuclei, endosomes, and lysosomes. Analysis was performed to quantify localization of pDNA to the endosomes and lysosomes. LNPs with 1.5 mol% PEG and a hydrocarbon chain C14 resulted in optimal endosomal escape and GFP production. Results from this study demonstrate that a higher percentage of C14 PEG leads to smaller LNPs with limited available phospholipid binding area for ApoE, resulting in decreased cellular uptake. We observed differences in the localization kinetics depending on the LNP formulation type for SS-OP, KC2, and MC3 ionizable lipids. The results also demonstrate the technique across different nucleic acid types, where mRNA resulted in more rapid and uniform GFP production compared to pDNA delivery. Here, we demonstrated the ability to track uptake and the sub-cellular fate of LNPs containing pDNA and mRNA, enabling improved screening prior to in vivo studies which would aid in formulation optimization.

Methodological and force field effects in the molecular dynamics-based prediction of binding free energies of host-guest systems.

As a contribution to the understanding and rationalization of methodological and modeling effects in recent host-guest SAMPL challenges, using an alchemical molecular dynamics technique we have examined the impact of force field parameterization and ionic strength in connection with guest charge neutralization on computed dissociation free energies in two typical SAMPL heavily charged macrocyclic hosts encapsulating small protonated amines with disparate binding affinities. We have shown that the methodological treatment for host neutralization, with explicit ions or with the background neutralizing plasma in the context of alchemical calculations under periodic boundary conditions, has a moderate effect on the calculated affinities. On the other hand, we have shown that seemingly small differences in the force field parameterization in highly symmetric hosts can produce systematic effects on the structural features that can have a significant impact on the predicted binding affinities.