Leveraging Machine Learning-Guided Molecular Simulations Coupled with Experimental Data to Decipher Membrane Binding Mechanisms of Aminosterols.

Understanding the molecular mechanisms of the interactions between specific compounds and cellular membranes is essential for numerous biotechnological applications, including targeted drug delivery, elucidation of the drug mechanism of action, pathogen identification, and novel antibiotic development. However, estimation of the free energy landscape associated with solute binding to realistic biological systems is still a challenging task. In this work, we leverage the Time-lagged Independent Component Analysis (TICA) in combination with neural networks (NN) through the Deep-TICA approach for determining the free energy associated with the membrane insertion processes of two natural aminosterol compounds, trodusquemine (TRO), and squalamine (SQ). These compounds are particularly noteworthy because they interact with the outer layer of neuron membranes, protecting them from the toxic action of misfolded proteins involved in neurodegenerative disorders, in both their monomeric and oligomeric forms. We demonstrate how this strategy could be used to generate an effective collective variable for describing solute absorption in the membrane and for estimating free energy landscape of translocation via on-the-fly probability enhanced sampling (OPES) method. In this context, the computational protocol allowed an exhaustive characterization of the aminosterol entry pathway into a neuron-like lipid bilayer. Furthermore, it provided accurate prediction of membrane binding affinities, in close agreement with the experimental binding data obtained by using fluorescently labeled aminosterols and large unilamellar vesicles (LUVs). The findings contribute significantly to our understanding of aminosterol entry pathways and aminosterol-lipid membrane interactions. Finally, the computational methods deployed in this study further demonstrate considerable potential for investigating membrane binding processes.

Clickable polyethyleneimine incorporated into triblock copolymeric micelles as an efficient platform in the delivery of siRNA to NSCLC cells.

The efficacy of transfection vectors to cross the endosomal membrane into the cytosol is a central aspect in the development of nucleic acid-based therapeutics. The challenge remains the same: Delivery, Delivery, Delivery. Despite a rational and appropriate construct of triblock polymeric micelles, which could serve as an ideal platform for the co-delivery of siRNAs and hydrophobic anticancer drugs, we show here its inability to properly convey oligonucleotides to their final destination. In order to overcome biological barriers, a linear PEI comprising two orthogonal groups was synthesized, holding an appropriate balance between safety and efficacy. Micellar carriers were then formulated with this polymer to enhance endosomal siRNA release. This chemical technology also addresses the two major challenges to consider when developing novel micellar products for siRNA delivery, namely cytotoxicity of polycations and endosomal escape. Herein, we demonstrate successful release of siRNA using a polymer tailoring strategy combined with a relevant in vitro approach, considering STAT3 as a promising target in the treatment of non-small cell lung cancer (NSCLC).

Influence of dexamethasone on the interaction between glucocorticoid receptor and SOX9: A molecular dynamics study.

The glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating the transcription of specific genes. GR transcriptional activity is modulated by a series of ligands and coenzymes, where a ligand can act as an agonist or antagonist. GR agonists, such as the glucocorticoids dexamethasone (DEX) and prednisolone, are widely prescribed to patients with inflammatory and autoimmune diseases. DEX is also used to induce osteogenic differentiation in vitro. Recently, it has been highlighted that DEX induces changes in the osteogenic differentiation of human mesenchymal stromal cells by downregulating the transcription factor SRY-box transcription factor 9 (SOX9) and upregulating the peroxisome proliferator-activated receptor γ (PPARG). SOX9 is fundamental in the control of chondrogenesis, but also in osteogenesis by acting as a dominant-negative of RUNX2. Many processes remain to be clarified during cell fate determination, such as the interplay between the key transcription factors. The main objective pursued by this work is to shed light on the interaction between GR and SOX9 in the presence and absence of DEX at an atomic level of resolution using molecular dynamics simulations. The outcome of this research could help the understanding of possible molecular interactions between GR and SOX9 and their role in the determination of cell fate. The results highlight the key residues at the interface between GR and SOX9 involved in the complexation process and shed light on the mechanism through which DEX modulates GR-SOX9 binding and exerts its biological activity.

Reorganization of the outer layer of a model of the plasma membrane induced by a neuroprotective aminosterol.

Trodusquemine is an amphipathic aminosterol that has recently shown therapeutic benefit in neurodegenerative diseases altering the binding of misfolded proteins to the cell membrane. To unravel the underlying mechanism, we studied the interactions between Trodusquemine (TRO) and lipid monolayers simulating the outer layer of the plasma membrane. We selected two different compositions of dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM), cholesterol (Chol) and monosialotetrahexosylganglioside (GM1) lipid mixture mimicking either a lipid-raft containing membrane (Ld+So phases) or a single-phase disordered membrane (Ld phase). Surface pressure-area isotherms and surface compressional modulus-area combined with Brewster Angle Microscopy (BAM) provided the thermodynamic and morphological information on the lipid monolayer in the presence of increasing amounts of TRO in the monolayer. Experiments revealed that TRO forms stable spreading monolayers at the buffer-air interface where it undergoes multiple reversible phase transitions to bi- and tri-layers at the interface. When TRO was spread at the interface with the lipid mixtures, we found that it distributes in the lipid monolayer for both the selected lipid compositions, but a maximum TRO uptake in the rafts-containing monolayer was observed for a Lipid/TRO molar ratio equal to 3:2. Statistical analysis of BAM images revealed that TRO induces a decrease in the size of the condensed domains, an increase in their number and in the thickness mismatch between the Ld and So phase. Experiments and MD simulations converge to indicate that TRO adsorbs preferentially at the border of the So domains. Removal of GM1 from the lipid Ld+So mixture resulted in an even greater TRO-mediated reduction of the size of the So domains suggesting that the presence of GM1 hinders the localization of TRO at the So domains boundaries. Taken together these observations suggest that Trodusquemine influences the organization of lipid rafts within the neuronal membrane in a dose-dependent manner whereas it evenly distributes in disordered expanded phases of the membrane model.

In silico investigation of cytochrome bc1 molecular inhibition mechanism against Trypanosoma cruzi.

Chagas' disease is a neglected tropical disease caused by the kinetoplastid protozoan Trypanosoma cruzi. The only therapies are the nitroheterocyclic chemicals nifurtimox and benznidazole that cause various adverse effects. The need to create safe and effective medications to improve medical care remains critical. The lack of verified T. cruzi therapeutic targets hinders medication research for Chagas' disease. In this respect, cytochrome bc1 has been identified as a promising therapeutic target candidate for antibacterial medicines of medical and agricultural interest. Cytochrome bc1 belongs to the mitochondrial electron transport chain and transfers electrons from ubiquinol to cytochrome c1 by the action of two catalytic sites named Qi and Qo. The two binding sites are highly selective, and specific inhibitors exist for each site. Recent studies identified the Qi site of the cytochrome bc1 as a promising drug target against T. cruzi. However, a lack of knowledge of the drug mechanism of action unfortunately hinders the development of new therapies. In this context, knowing the cause of binding site selectivity and the mechanism of action of inhibitors and substrates is crucial for drug discovery and optimization processes. In this paper, we provide a detailed computational investigation of the Qi site of T. cruzi cytochrome b to shed light on the molecular mechanism of action of known inhibitors and substrates. Our study emphasizes the action of inhibitors at the Qi site on a highly unstructured portion of cytochrome b that could be related to the biological function of the electron transport chain complex.

Characterization of the Interaction of Polymeric Micelles with siRNA: A Combined Experimental and Molecular Dynamics Study.

The simulation of large molecular systems remains a daunting challenge, which justifies the exploration of novel methodologies to keep computers as an ideal companion tool for everyday laboratory work. Whole micelles, bigger than 20 nm in size, formed by the self-assembly of hundreds of copolymers containing more than 50 repeating units, have until now rarely been simulated, due to a lack of computational power. Therefore, a flexible amphiphilic triblock copolymer (mPEG45-α-PLL10-PLA25) containing a total of 80 repeating units, has been emulated and synthesized to embody compactified nanoconstructs of over 900 assembled copolymers, sized between 80 and 100 nm, for siRNA complexing purposes. In this study, the tailored triblock copolymers containing a controlled number of amino groups, were used as a support model to address the binding behavior of STAT3-siRNA, in the formation of micelleplexes. Since increasingly complex drug delivery systems require an ever more optimized physicochemical characterization, a converging description has been implemented by a combination of experimentation and computational simulations. The computational data were advantageous in allowing for the assumption of an optimal N/P ratio favoring both conformational rigidifications of STAT3-siRNA with low competitive phenomena at the binding sites of the micellar carriers. These calculations were consistent with the experimental data showing that an N/P ratio of 1.5 resulted in a sufficient amount of complexed STAT3-siRNA with an electrical potential at the slipping plane of the nanopharmaceuticals, close to the charge neutralization.

High-density EEG power topography and connectivity during confusional arousal.

Confusional arousal is the milder expression of a family of disorders known as Disorders of Arousal (DOA) from non-REM sleep. These disorders are characterized by recurrent abnormal behaviors that occur in a state of reduced awareness for the external environment. Despite frequent amnesia for the nocturnal events, when actively probed, patients are able to report vivid hallucinatory/dream-like mental imagery. Traditional (low-density) scalp and stereo-electroencephalographic (EEG) recordings previously showed a pathological admixture of slow oscillations typical of NREM sleep and wake-like fast-mixed frequencies during these phenomena. However, our knowledge about the specific neural EEG dynamics over the entire brain is limited. We collected 2 consecutive in-laboratory sleep recordings using high-density (hd)-EEG (256 vertex-referenced geodesic system) coupled with standard video-polysomnography (v-PSG) from a 12-year-old drug-naïve and otherwise healthy child with a long-lasting history of sleepwalking. Source power topography and functional connectivity were computed during 20 selected confusional arousal episodes (from -6 to +18 sec after motor onset), and during baseline slow wave sleep preceding each episode (from - 3 to -2 min before onset). We found a widespread increase in slow wave activity (SWA) theta, alpha, beta, gamma power, associated with a parallel decrease in the sigma range during behavioral episodes compared to baseline sleep. Bilateral Broadman area 7 and right Broadman areas 39 and 40 were relatively spared by the massive increase in SWA power. Functional SWA connectivity analysis revealed a drastic increase in the number and complexity of connections from baseline sleep to full-blown episodes, that mainly involved an increased out-flow from bilateral fronto-medial prefrontal cortex and left temporal lobe to other cortical regions. These effects could be appreciated in the 6 sec window preceding behavioral onset. Overall, our results support the idea that DOA are the expression of peculiar brain states, compatible with a partial re-emergence of consciousness.

A calibrated model with a single-generator simulating polysomnographically recorded periodic leg movements.

The aim of this study was to assess, with numerical simulations, if the complex mechanism of two (or more) interacting spinal/supraspinal structures generating periodic leg movements can be modelled with a single-generator approach. For this, we have developed the first phenomenological model to generate periodic leg movements in-silico. We defined the onset of a movement in one leg as the firing of a neuron integrating excitatory and inhibitory inputs from the central nervous system, while the duration of the movement was defined in accordance to statistical evidence. For this study, polysomnographic leg movement data from 32 subjects without periodic leg movements and 65 subjects with periodic leg movements were used. The proportion of single-leg and double-leg inputs, as well as their strength and frequency, were calibrated on the without periodic leg movements dataset. For periodic leg movements subjects, we added a periodic excitatory input common to both legs, and the distributions of the generator period and intensity were fitted to their dataset. Besides the many simplifying assumptions - the strongest being the stationarity of the generator processes during sleep - the model-simulated data did not differ significantly, to a large extent, from the real polysomnographic data. This represents convincing preliminary support for the validity of our single-generator model for periodic leg movements. Future model extensions will pursue the ambitious project of a supportive diagnostic and therapeutic tool, helping the specialist with realistic forecasting, and with cross-correlations and clustering with other patient meta-data.

Fragmented blind docking: a novel protein-ligand binding prediction protocol.

In the present paper we propose a novel blind docking protocol based on Autodock-Vina. The developed docking protocol can provide binding site identification and binding pose prediction at the same time, by a systematical exploration of the protein volume performed with several preliminary docking calculations. In our opinion, this protocol can be successfully applied during the first steps of the virtual screening pipeline, because it provides binding site identification and binding pose prediction at the same time without visual evaluation of the binding site. After the binding pose prediction, MM/GBSA re-scoring rescoring procedures has been applied to improve the accuracy of the protein-ligand bound state. The FRAD protocol has been tested on 116 protein-ligand complexes of the Heat Shock Protein 90 - alpha, on 176 of Human Immunodeficiency virus protease 1, and on more than 100 protein-ligand system taken from the PDBbind dataset. Overall, the FRAD approach combined to MM/GBSA re-scoring can be considered as a powerful tool to increase the accuracy and efficiency with respect to other standard docking approaches when the ligand-binding site is unknown.Communicated by Ramaswamy H. Sarma.

Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore.

Cell-penetrating peptides (CPPs) allow intracellular delivery of bioactive cargo molecules. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (-150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying dimensions in CPP co-entry experiments, the diameter of the water pores in living cells was estimated to be 2 (-5) nm, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPP cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membranes. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.

Before a drug can have its desired effect, it must reach its target tissue or organ, and enter its cells. This is not easy because cells are surrounded by the plasma membrane, a fat-based barrier that separates the cell from its external environment. The plasma membrane contains proteins that act as channels, shuttling specific molecules in and out of the cell, and it also holds charge, with its inside surface being more negatively charged than its outside surface. Cell-penetrating peptides are short sequences of amino acids (the building blocks that form proteins) that carry positive charges. These positive charges allow them to cross the membrane easily, but it is not well understood how. To find out how cell-penetrating peptides cross the membrane, Trofimenko et al. attached them to dyes of different sizes. This revealed that the cell-penetrating peptides enter the cell through temporary holes called water pores, which measure about two nanometres across. The water pores form when the membrane becomes 'megapolarized', this is, when the difference in charge between the inside and the outside of the membrane becomes greater than normal. This can happen when the negative charge on the inside surface or the positive charge on the outer surface of the membrane increase. Megapolarization depends on potassium channels, which transport positive potassium ions outside the cell, making the outside of the membrane positive. When cell-penetrating peptides arrive at the outer surface of the cell near potassium channels, they make it even more positive. This increases the charge difference between the inside and the outside of the cell, allowing water pores to form. Once the peptides pass through the pores, the charge difference between the inside and the outside of the cell membrane dissipates, and the pores collapse. Drug developers are experimenting with attaching cell-penetrating peptides to drugs to help them get inside their target cells. Currently there are several experimental medications of this kind in clinical trials. Understanding how these peptides gain entry, and what size of molecule they could carry with them, provides solid ground for further drug development.

Extended diffusion theory: Recovering dynamics from biased/accelerated molecular simulations.

Dynamical properties are of great importance in determining the behavior of synthetic and natural molecules, but capturing them by computational methods is a nontrivial task. Very often the time scales of the relevant phenomena are far beyond the typical time windows accessible by classical Molecular Dynamics (MD) simulations, currently limited to the order of microseconds on standard laboratory workstations. On the other hand, biased and accelerated simulations allow for fast and thorough exploration of the molecular conformational space, but they lose the dynamic information. The problem of recovering dynamics from biased/accelerated simulations is a very active field of research, but no totally robust/reliable solutions have been given yet. In this paper it is shown how the Smoluchowski equation, in the framework of Diffusion Theory (DT), can be used to bridge this gap, and dynamical properties, in the form of time correlation functions (TCFs), can be extracted also from such kind of simulations. DT is first extended (EDT) to express the mobility tensors entering the Smoluchowski operator in terms of a recently introduced unified and regularized Rotne-Prager-Yamakawa approximation, [P. J. Zuk, E. Wajnryb, K. A. Mizerski, P. Szymczak, J. Fluid. Mech. 2014, 741, R5, 1-13] also involving mixed rotation-translation contributions, and rotation-rotation terms beside the classical translation-translation ones, so far used in DT. Then, the method is applied to recover the dynamics of a nontrivial example of a peptide in explicit water from the first 200 ns of a Replica Exchange Molecular Dynamics simulation, which is a popular computational method that destroys the long time dynamics. EDT dynamics were found to favorably compare against those coming from a standard MD simulation of the same system, requiring a time window of 30 µs to converge. This result shows that EDT is a tool of practical value to recover the long time dynamics of systems in diffusive regimes from biased/accelerated simulations, to be exploited in those cases when direct evaluation by standard MD is unfeasible.

TAT-RasGAP317-326 kills cells by targeting inner-leaflet-enriched phospholipids.

TAT-RasGAP317-326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP317-326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP2) and phosphatidylserine (PS). Decreasing the amounts of PIP2 in cells renders them more resistant to TAT-RasGAP317-326, while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP317-326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP2- and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP317-326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.

Making biological membrane resistant to the toxicity of misfolded protein oligomers: a lesson from trodusquemine.

Trodusquemine is an aminosterol known to prevent the binding of misfolded protein oligomers to cell membranes and to reduce their toxicity in a wide range of neurodegenerative diseases. Its precise mechanism of action, however, remains unclear. To investigate this mechanism, we performed confocal microscopy, fluorescence resonance energy transfer (FRET) and nuclear magnetic resonance (NMR) measurements, which revealed a strong binding of trodusquemine to large unilamellar vesicles (LUVs) and neuroblastoma cell membranes. Then, by combining quartz crystal microbalance (QCM), fluorescence quenching and anisotropy, and molecular dynamics (MD) simulations, we found that trodusquemine localises within, and penetrates, the polar region of lipid bilayer. This binding behaviour causes a decrease of the negative charge of the bilayer, as observed through ζ potential measurements, an increment in the mechanical resistance of the bilayer, as revealed by measurements of the breakthrough force applied with AFM and ζ potential measurements at high temperature, and a rearrangement of the spatial distances between ganglioside and cholesterol molecules in the LUVs, as determined by FRET measurements. These physicochemical changes are all known to impair the interaction of misfolded oligomers with cell membranes, protecting them from their toxicity. Taken together, our results illustrate how the incorporation in cell membranes of sterol molecules modified by the addition of polyamine tails leads to the modulation of physicochemical properties of the cell membranes themselves, making them more resistant to protein aggregates associated with neurodegeneration. More generally, they suggest that therapeutic strategies can be developed to reinforce cell membranes against protein misfolded assemblies.

The Impact of Natural Compounds on S-Shaped Aß42 Fibril: From Molecular Docking to Biophysical Characterization.

The pursuit for effective strategies inhibiting the amyloidogenic process in neurodegenerative disorders, such as Alzheimer's disease (AD), remains one of the main unsolved issues, and only a few drugs have demonstrated to delay the degeneration of the cognitive system. Moreover, most therapies induce severe side effects and are not effective at all stages of the illness. The need to find novel and reliable drugs appears therefore of primary importance. In this context, natural compounds have shown interesting beneficial effects on the onset and progression of neurodegenerative diseases, exhibiting a great inhibitory activity on the formation of amyloid aggregates and proving to be effective in many preclinical and clinical studies. However, their inhibitory mechanism is still unclear. In this work, ensemble docking and molecular dynamics simulations on S-shaped Aß42 fibrils have been carried out to evaluate the influence of several natural compounds on amyloid conformational behaviour. A deep understanding of the interaction mechanisms between natural compounds and Aß aggregates may play a key role to pave the way for design, discovery and optimization strategies toward an efficient destabilization of toxic amyloid assemblies.

Molecular and Coarse-Grained Modeling to Characterize and Optimize Dendrimer-Based Nanocarriers for Short Interfering RNA Delivery.

Dendrimer nanocarriers are unique hyper-branched polymers with biomolecule-like properties, representing a promising prospect as a nucleic acid delivery system. The design of effective dendrimer-based gene carriers requires considering several parameters, such as carrier morphology, size, molecular weight, surface chemistry, and flexibility/rigidity. In detail, the rational design of the dendrimer surface chemistry has been ascertained to play a crucial role on the efficiency of interaction with nucleic acids. Within this framework, advances in the field of organic chemistry have allowed us to design dendrimers with even small difference in the chemical structure of their surface terminals. In this study, we have selected two different cationic phosphorus dendrimers of generation 3 functionalized, respectively, with pyrrolidinium (DP) and morpholinium (DM) surface groups, which have demonstrated promising potential for short interfering RNA (siRNA) delivery. Despite DP and DM differing only for one atom in their chemical structure, in vitro and in vivo experiments have highlighted several differences between them in terms of siRNA complexation properties. In this context, we have employed coarse-grained molecular dynamics simulation techniques to shed light on the supramolecular characteristics of dendrimer-siRNA complexation, the so-called dendriplex formations. Our data provide important information on self-assembly dynamics driven by surface chemistry and competition mechanisms.

Aminoacid substitutions in the glycine zipper affect the conformational stability of amyloid beta fibrils.

The aggregation of amyloid-beta peptides is associated with the pathogenesis of Alzheimer's disease. The hydrophobic core of the amyloid beta sequence contains a GxxxG repeated motif, called glycine zipper, which involves crucial residues for assuring stability and promoting the process of fibril formation. Mutations in this motif lead to a completely different oligomerization pathway and rate of fibril formation. In this work, we have tested G33L and G37L residue substitutions by molecular dynamics simulations. We found that both protein mutations may lead to remarkable changes in the fibril conformational stability. Results suggest the disruption of the glycine zipper as a possible strategy to reduce the aggregation propensity of amyloid beta peptides. On the basis of our data, further investigations may consider this key region as a binding site to design/discover novel effective inhibitors.Communicated by Ramaswamy H. Sarma.

Steroid identification via deep learning retention time predictions and two-dimensional gas chromatography-high resolution mass spectrometry.

Untargeted steroid identification represents a great analytical challenge even when using sophisticated technology such as two-dimensional gas chromatography coupled to high resolution mass spectrometry (GC × GCHRMS) due to the chemical similarity of the analytes. Moreover, when analytical standards, mass spectral and retention index databases are not available, compound annotation is cumbersome. Hence, there is a need for the development of retention time prediction models in order to explore new annotation approaches. In this work, we evaluated the use of several in silico methods for retention time prediction in multidimensional gas chromatography. We use three classical machine learning (CML) algorithms (Partial Least Squares (PLS), Support Vector Regression (SVR) and Random Forest Regression (RFR)) and two deep learning approaches (dense neural network (DNN) and three-dimensional convolutional neural network (CNN)). Whereas molecular descriptors were utilized for the CLM and DNN algorithms, three-dimensional molecular representation based on the electrostatic potential (ESP) was studied as input data as is for the CNN. All the developed models showed similar performances with Q2 values over 0.9. However, among all CNN showed the best performance, resulting in average retention time prediction errors of 2% and 6% for the first and second separation dimension, respectively. Additionally, only the three-dimensional ESP representation coupled with CNN was able to extract the stereochemical information crucial for the separation of diastereomers. The combination of retention time prediction and high-resolution mass spectral data applied to clinical samples enabled the untargeted annotation of 12 steroid metabolites in the urine of new-borns.

Biofunctionalization of Silica Nanoparticles with Cell-Penetrating Peptides: Adsorption Mechanism and Binding Energy Estimation.

Nanoparticles represent one of the most promising materials as they have found application in bionanotechnology for enhanced imaging, diagnosis, and treatment of several diseases. Silica coating is widely used to improve colloidal stability and the binding affinity of nanoparticles for various organic molecules, such as cell-penetrating peptides (CPPs). The functionalization of the silica coating by CPPs is very promising, since it enhances the uptake of the nanoparticles due to the intrinsic ability of the CPPs to cross the cellular membrane. However, molecular level phenomena characterizing the CPPs interaction with silica-coated nanoparticles are not clarified yet. In this work, classical molecular dynamics has been used to shed light on the adsorption mechanism of several CPPs onto silica surfaces, in order to highlight the influence of the surface ionization's state on the adsorption mechanism. Our data highlight how the cationic peptides strongly interact with the anionic silica surfaces by H-bond formation between charged residues and the negatively charged siloxide groups, as well as ion pairing between the surfaces and the N-termini residues. Moreover, thanks to metadynamics simulations the CPP-silica binding affinity has been quantified. The free energy profile description allows providing insight into the relationship between ionization of silica nanoparticles and binding selectivity/specificity to CPP.

The synergistic effect of chlorotoxin-mApoE in boosting drug-loaded liposomes across the BBB.

We designed liposomes dually functionalized with ApoE-derived peptide (mApoE) and chlorotoxin (ClTx) to improve their blood-brain barrier (BBB) crossing. Our results demonstrated the synergistic activity of ClTx-mApoE in boosting doxorubicin-loaded liposomes across the BBB, keeping the anti-tumour activity of the drug loaded: mApoE acts promoting cellular uptake, while ClTx promotes exocytosis of liposomes.

Effect of Lactose Pseudopolymorphic Transition on the Aerosolization Performance of Drug/Carrier Mixtures.

Physico-chemical properties of lactose are key factors in adhesive mixtures used as dry powder inhaler (DPI). Despite the abundant literature on this topic, the effect of the polymorphism and pseudo-polymorphism of lactose has been seldom investigated and discussed although often lactose used in DPI is subjected to unit operations, which may alter its solid-state properties. Here, we studied the aerosolization performance of salbutamol sulphate (SS) or budesonide (BUD) formulations by investigating the effect of lactose pseudopolymorphism in ternary (coarse lactose/fine lactose/drug) and binary (coarse lactose/drug) mixtures. An improvement of the aerosolization performance of SS formulations with the increase of the amount of fine micronized lactose up to 30% (fine particle fraction (FPF) = 57%) was observed. Micronized lactose contained hygroscopic anhydrous α-lactose, which converted to α-lactose monohydrate at ambient conditions. This implied that the positive effect of fines on the aerosolization performance decreased and eventually disappeared with the formulation aging. Positive effect on SS deposition was observed also with binary mixtures with anhydrous lactose, whereas the opposite occurred with budesonide-containing formulations. The collected data demonstrated the crucial role of the carrier crystal form on the positive effect of fines on the deposition.