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ARDS is an inflammatory diffused form of lung injury that leads to increased pulmonary vascular permeability, resulting in secretion of interstitial fluids in the alveolar space and their collapse, making oxygen exchange difficult or impossible [ 2 ]. Patients with ARDS tend to progress through three pathologic stages: the early exudative phase, the fibroproliferative phase, and the fibrotic phase.
The lipopolysaccharide LPS insult model stimulates the exudative stage, which includes a nonspecific reaction to lung injury, interstitial edema, accompanied by acute and chronic inflammation [ 2 , 6 , 7 ]. Inflammation involves the activation of numerous immune cell types including neutrophils and macrophages [ 1 , 8 , 9 ]. Moreover, in response to such stimuli, macrophages release reactive oxygen species ROS and produce nitric oxide NO , which regulate inflammatory cells and protect the organism from potential dangers [ 14 , 15 ].
NO levels were used as an additional parameter for macrophage activation in vitro. The detection of cytokines in bronchoalveolar lavage BAL fluid is well corelated with the severity of inflammation in disease models, as verified by histological analyses [ 16 ]. Glucocorticoids, such as methylprednisolone MPS are used widely for treating lung inflammation [  ,  ,  ]. Glucocorticoids diffuse through cell membranes leveraging amphipathic properties and cellular receptors.
In the cytoplasm glucocorticoids bind glucocorticoid receptors reducing proinflammatory cytokine synthesis and inhibiting fibroblast proliferation and collagen synthesis via non-genomic and genomic pathways [ 20 , 21 ]. In addition, steroids decrease NO synthase in macrophages, leading to decreased NO secretion [ 22 ]. However, systemic and prolonged administration of glucocorticoids is associated with severe side effects, such as interference with wound healing process, inhibition of effector cells in fighting active infections and other myopathic, cardiovascular and dermatologic side effects [ 23 , 24 ].
Additionally, a potential agent we tested for treating ARDS is N -acetyl cysteine NAC to treat mucus hypersecretion during inflammation that impairs blood-gas exchange. NAC is a mucolytic agent that reduces connecting disulfide bonds in mucus, thereby reducing mucus viscosity and accelerating mucus plugs removal [  ,  ,  ]. Drug concentrations at the target site following intravenous IV administration can be below the therapeutic window, therefore, to increase drug concentrations and bioavailability at the target site, decrease side effects, and prolonged the retention time of the drug at the target tissue we encapsulated drugs in nm liposomes [ 34 ].
Liposomal MPS uptake by macrophages and neutrophils can be mediated by adsorption, endocytosis, and phagocytosis [ 35 ], where drug release occurs intra- or extracellular. The dipalmitoyl-phosphatidylcholine DPPC was chosen as the main component. DPPC is a naturally occurring pulmonary surfactant, that reduces surface tension at the air-liquid interface of the alveolus, thus preventing alveolar collapse associated with inflammation [  ,  ,  ].
DPPC exerts its protective effect by integrating into plasma membrane of airway epithelium and immune cells affecting membrane integrity [ 38 ]. DPPC also acts as an immunomodulator by inhibiting cytokine release from innate immune cells in response to LPS [ 38 ]. Liposomes are already used in clinic for treating lung disorders and injuries [  ,  ,  ]. For example, an Amikacin liposome inhalation suspension ALIS is used for treating pulmonary infections [ 42 ].
The proposed liposomal formulation was tested for its ability to reduce lung inflammation. Liposome's size was designed to be approximal nm in order to improve penetration in inflamed tissues cite [ 43 ]. The study compared between endotracheal ET instillation to the lungs and intravenous IV administration, or both routes simultaneously.
Previously, Garbuzenko et al. Results showed that liposomes administered intratracheally remained in the lungs for at least 72 h post-administration with low levels in other organs. However, liposomes administered by IV accumulated in lungs for the first hour and declined thereafter [ 44 ]. Methylprednisolone sodium succinate MPS, solu-medrol from Pfizer was used.
Calcium Acetate was purchased from Spectrum. The lipid solution was added to calcium acetate solution. After liposome were composed, liposomes were actively loaded with the drugs to compose the DPPC liposomal drugs Fig. After each encapsulation, dialysis was done to remove unencapsulated drugs.
A Liposomes loaded with two medicines methylprednisolone MPS , a steroid, and N -acetyl cysteine NAC , a mucus active agent were synthesized using a three-step formulation process. B To reach high loading levels of both drugs and a stable formulation process we followed the following optimization methodology. Optimization steps varied drug concentrations, temperature, drug loading time, intra- and extra-liposomal pH, and the Active Loading AL salt concentrations.
Selected conditions are labeled in yellow. Unfavorable outcomes are labeled in grey, and conditions leading to them in white. C Summary table of final DPPC liposomal drugs formation process conditions and drugs' encapsulation analysis. Particle concentration and size were measured using dynamic light scattering DLS. For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.
Samples were diluted in methanol. Buffers for MPS plan [ 45 ]: A Detection at wavelength nm. Buffers for NAC [ 46 ]: A: 0. Detection at wavelength and nm. Detection with ELSD. Specimens were transferred into a Gatan Specimens were examined in TEM nanoprobe mode using Volta phase plates for contrast enhancement. Imaging was performed at a low dose mode to minimize exposure of the imaged area to electrons.
Liposomes diluted x20 with saline. Since the system under study is aqueous, preparation was done in a temperature-controlled chamber with humidity at saturation, to prevent evaporation of volatiles [ 48 ]. A drop of the solution was placed on a carbon-coated perforated polymer film, supported on a nm mesh TEM grid, mounted on tweezers.
The drop was turned into a thin film preferably less than nm by blotting away excess solution with a metal strip covered with a filter paper. Drug concentrations were measured using HPLC. Raw Macrophages were cultured in well plates at a concentration of 0. To examine liposomes infiltration to the mucus, mucus and Cy5-labeled liposomes were imaged under confocal microscopy, with and without NAC treatment. Mucin from the porcine stomach was dissolved to a concentration of The mixture was let to achieve relaxation for 24 h.
A coverslip was placed on top with spacers that ensured that the coverslip was evenly spaced. PBS solution used as control. To examine in-vitro cells uptake efficiency under mucosal conditions, we developed an in-vitro mucus permeation assay using human colorectal adenocarcinoma Caco2 cell line. These cells are naturally covered with mucus layer in the intestine and therefore act as a good model for this purpose.
The cells were cultured separately in well plates for 5 days, with fresh medium replacement after 3 days, with RPMI medium. We also compared the uptake with 0. After 2 h, the mucus and medium were removed, and the cells were washed twice with PBS. Cells uptake was quantified by nuclei and liposomes fluorescence labling using Gen5 Image Prime software ver.
The syringe was then connected to the catheter, and if the tube was in the right place, the saline bubble moved at the rate of mice breathing. LPS from E. The experiments described here were approved by the ethics committee Ethical request No. IL and IL Square shows magnification of X exhibiting infiltration of lymphocytes into alveoli. The asterisk above bars connecting columns represents a significant difference between groups.
After different time points 1, 3, 6, 12, 24 and 48 h mice were euthanized by ketamine-xylazine overdose OD and organs were extracted and have been weighed. Gd concentration of each sample was measured using a standard calibration curve at the range of 0. Lung inflammation induced in mice. One hour after liposome administration, mice were euthanized by ketamine-xylazine overdose OD. Lungs were frozen using liquid nitrogen steam.
Leica DMI8 inverted fluorescent microscope and Confocal microscope were employed to examine liposome distribution in the lungs via two different administration pathways. The lower amount delivered ET was intended to prevent suffocation due to increased edema associated with lung inflammation treatments were DPPC liposomes, free drugs or DPPC liposomal drugs.
Drug's concentrations were 4. Mice were euthanized. Slides were scanned using automatic slide scanner flash. Mice were euthanized by ketamine-xylazine OD and 22 g catheter was inserted into mouse's trachea. Cytokines were identified in BAL fluid supernatant or in macrophage medium to evaluate immune cell response.
DPPC and cholesterol were selected since they are a naturally occurring components of the pulmonary surfactant [ 52 ] Fig. For this, DPPC liposomes For each drug encapsulation process, different loading conditions, such as pH, drug concentration, temperature and time of incubation were examined, as detailed in Fig. Drug concentrations in the liposomes reached 4. Drug-to-lipid molar ratios are 7.
DPPC liposomal drugs particles concentration was 2. Comparing the free drug versus the liposomal treatment demonstrates benefit or equal therapeutic influence. Asterisk above bars connecting columns represents a significant difference between groups. C Confocal images of nm liposomes of NAC treated, or untreated, mucus's penetration. The ability of nanoparticles to cross the mucus layer and be taken up by the underlying cells was quantified over time using an automated microscope setup.
E Automated microscopy images of Caco-2 cellular uptake of Cy-7 fluorescently labeled nm liposomes. We examined the effect of NAC on liposomal mucus penetration. NAC treatments improved liposomes mucus penetration in comparison to untreated mucus Fig. The NAC, agent with thiol-amino structures which gives it the mucolytic feature, disrupts the disulfide bonds in the mucus [ 55 ]. To examine the effect of NAC treatment on liposomes cells uptake under mucosal conditions, we developed an in-vitro mucus permeation assay by spreading a mucus layer over Caco2 cells followed by automated imaging of the liposomal uptake by the cells undernieth the mucus [ 56 ] Fig.
The addition of NAC improved nanoparticle uptake by 1. The NAC dissolves the disulfide bonds of the mucus, has previously been used to remove the secreted mucus layer from cell cultures without cellular damagev [ 57 , 58 ]. Blinded pathologic assessment of mice lungs examined the inflammatory state of the lungs Fig. Compared to healthy lungs, LPS induced lungs had acute inflammatory infiltration with neutrophils filling the alveolar space.
Vascular congestion and extravasated RBCs were also seen. A LPS is administrated endotracheally to induce inflammation. Asterisk above bars connecting column represents significant difference between groups. We tested the liposomal biodistribution to different organs at different time points 1,3,6,12,24, and 48 h post IV-injection in healthy and acute LPS lung inflamed mice.
Whole-animal IVIS imaging demonstrates increased liposomal accumulation in the inflamed lungs compared to the healthy. This higher accumulation in inflamed tissues may occur due to increased vascular permeability at sites of inflammation as well [ 61 , 62 ] as increased mononuclear phagocytes system internalization of liposomes at inflammatory sites. This trend of higher accumulation of liposomes in LPS-induced mice was observed at all time points Fig.
C reaching up to 8-fold increase at 48 h Fig Sup. The enhanced accumulation of liposomes in inflamed lungs indicate a key potential of liposomes to target ARDS. A Ex-vivo representative images of healthy and inflamed lungs at varying time points after IV injection of rhodamine and Gd labeled liposomes taken by whole animal IVIS imaging system. B Biodistribution of rhodamine and Gd labeled liposomes at different time points from IV injection as measured using elemental analysis ICP-OES of the lungs, heart, liver, spleen, kidneys and blood.
E Confocal images of inflamed lung cryo-sections following Cy3-labeled liposomes administered via ET alongside Cy5-labeled liposomes administrated IV. Nuclei were stained using DAPI. Arrows exhibit high intensity of liposomes present. Arrowheads show liposomes in a blood vessel and arrows show liposomes in the airway. Gd content in lungs was normalized to the initial injected dose due to the apparent pulmonary edema observed in inflamed lungs which can alter weight measurements.
In lungs, a Gd concentration of 1. Moreover, a significant increase in Gd content in the healthy versus inflamed lungs was recorded 4 and 48 h after the administration 6. The Gd content in the heart, liver, spleen, kidneys, and blood was similar.
Both Rhodamine and Gd analysis exhibited a similar clearance profile Fig. Liposomes were present in inflamed lungs even 48 h post-injection, compared to 12 h in healthy lungs Fig. The biodistribution of liposomes administered by endotracheal intubation ET was assessed. IVIS images demonstrate the liposomes presence in the lungs at varying time points, in healthy and LPS-induced mice after endotracheal administration Fig.
One hour after administration, both Rhodamine and Gd analysis showed somewhat higher accumulation in healthy lungs compared to the inflamed lungs, however, thereafter the accumulation of the nanoparticles in the inflamed lung was greater Fig. We propose that mucus accumulation and breathing impairment among the LPS induced mice, led to reduced absorbance of the particles from the trachea. Six-hours post ET administration, healthy mice show a decrease in liposome accumulation compared to LPS-induced mice.
The fluorescent signal in inflamed lungs after 12 h is 4. To examine the liposomal accumulation in lungs, heart, liver, spleen, kidneys, and blood, quantitative Gd measurements were performed using ICP-OES Fig. Six, 12, 24 and 48 h after endotracheal administration, inflamed lungs showed higher Gd concentrations 1. In other organs, there was no significant difference in Gd concentration at all time points Fig.
In conclusion, administration of liposomes through ET shows higher retention rates in inflamed lungs compared to IV administration at all time points. Furthermore, the accumulation of liposomes is higher in inflamed versus healthy lungs. Lung inflammation is orchestrated by elevated cytokine levels in the blood secreted by multiple sites in the body. IV DPPC liposomal drugs have been shown to target multiple inflammatory sites simultaneously [ 43 ] for example the liver. Overall, the liposomal treatment decreased systemic cytokines levels in the lung and reduced the secretion of chemical mediators that induce inflammation.
We imaged the targeting of lung parenchyma from both the epithelium by ET administration , and endothelial side of the blood vessel cells via IV administration , 1 h after liposomes administration to inflamed lung Fig. Liposome administrated via IV were widely present throughout the lung, taking advantage of the spread capillary net in the lungs Cy5 liposomes, red.
In addition, high liposomal accumulation was observed in the endothelial blood vessels Fig. Liposomes administrated endotracheally, were widely present in lungs with higher intensity Cy3 liposomes, green compared to IV administered liposomes. However, via ET administration some distal regions of the lungs, did not have liposomal presence, possibly due to impaired ventilation to these sites in inflamed lung with spontaneous breathing.
High liposomal accumulation was observed in the epithelial airway Fig. The lower volume delivered ET was intended to prevent suffocation. The nanoparticle treatments reduced inflammation in the lungs, suggesting a positive effect for combined treatment Fig. Histologic sections were blindly analyzed demonstrating that the untreated group present a severe acute inflammatory reaction with neutrophils filling alveolar spaces.
Interestingly, intravenous administration of either free drugs or DPPC liposomal drugs resulted with milder inflammatory infiltrate compared to ET administration Fig. The percent of severe inflammation in the alveoli relative to the total lung were analyzed histopathologically to quantify the lungs injury, demonstrating that treatment with DPPC liposomal drugs was superior in reducing lung severity Sup. No significant decrease in cytokine levels was observed 26 h after free ET or combined ET plus IV administration of drugs treatment.
Following systemic IV administrations, liposome accumulation was more significant in inflamed lungs in comparison to healthy lungs, indicating that liposomes exhibit preferential targeting of the inflamed lung. Local administration of liposomes via the ET route leads to a greater amount and longer retention time of liposomes in inflamed lungs. An additional significant difference between IV and ET routes is that IV administered liposome distribution spanned across the whole lung, while ET liposomes were distributed mostly in lung parenchyma but were not found at partial distal areas of the lung.
Therefore, combining ET and IV administration modes stimulates lung accumulation in the lungs from both alveolar epithelial and endothelial blood vessel cells, allowing broad coverage of lungs. It can be concluded that DPPC liposomal drugs availability is significant since effect is seen. The DPPC liposomal system is a promising platform for treating lung diseases. Schroeder conceived the approach, supervised, and directed the research.
Arber Raviv and M. Alyan contributed equally to this work. Arber Raviv, M. Alyan, H. Korach-Rechtman, L. Koren, J. Shainsky, J. Shklover, designed the experiments. Alyan, E. Egorov, A. Zano, M. Yaskin Harush, A. Saadya, O. Doppelt Flikshtain, L. Mekies, and L. Koren performed the experiments. Korach-Rechtman, S. Farkash, O. Doppelt Flikshtain, and L. Koren analyzed the data. Kaneti, I. Nudelman, Y. Gal, E. Dor, and Y. Adir contributed to experimental design. Phone or email.
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This is the best most modern teams can afford anyway. Logging is an inevitable part of debugging. Well, at least in modern high-level programming languages and architectures. Sometimes we trace variables, but rarely. More often we just print them to console. Some logging may happen inside unit tests. Do we also leave them there or maybe not? Making constructors pre-process the arguments before encapsulating them seems to be bad practice. For this purpose I suggest using prestructors, which could be methods or stand-alone objects.
Read this and this , to get the full picture. However, until this day comes, here is some advice, to help you be a less annoying better head hunter. First, let me clarify what kind of conference we are talking about. There are two types: professional and academic.
The difference is huge. My understanding is that professional conferences are for practitioners , while academic ones are for researchers. ICCQ, which we organized this year, was an academic conference. Here is a more or less detailed description of the journey.
Feel free to learn from it and make a better conference yourself. We will try to make a better one next year, ICCQ The time of objectivity is fading out. Meritocracy is now a rude word. Metrics in management will soon be considered as harassment. Productivity is already a false objective. The era of imposters is coming! Here is a quick summary of key techniques to make you highly successful in any argument you may have in your flat democratic organizations of the future without any skills, knowledge, education, or real achievements.
Just pure love and emotions. There is a dataization function, which puts all three together in order to make an EO program alive. Here is how it works together with Java, for example. You have an objective, a budget, and a team. You are a manager and you want the project to be done. This article explains how CAPs work and summarizes the results of the experiment.
Here is a very simple management framework, which we have used in our teams for the last two years. This is the best most modern teams can afford anyway. Logging is an inevitable part of debugging. Well, at least in modern high-level programming languages and architectures. Sometimes we trace variables, but rarely. More often we just print them to console. Some logging may happen inside unit tests. Do we also leave them there or maybe not?
Making constructors pre-process the arguments before encapsulating them seems to be bad practice. For this purpose I suggest using prestructors, which could be methods or stand-alone objects. Read this and this , to get the full picture. However, until this day comes, here is some advice, to help you be a less annoying better head hunter.
First, let me clarify what kind of conference we are talking about. There are two types: professional and academic. The difference is huge. My understanding is that professional conferences are for practitioners , while academic ones are for researchers. ICCQ, which we organized this year, was an academic conference. Here is a more or less detailed description of the journey. Feel free to learn from it and make a better conference yourself. We will try to make a better one next year, ICCQ The time of objectivity is fading out.
Meritocracy is now a rude word. Metrics in management will soon be considered as harassment. Productivity is already a false objective. The era of imposters is coming! Here is a quick summary of key techniques to make you highly successful in any argument you may have in your flat democratic organizations of the future without any skills, knowledge, education, or real achievements. Just pure love and emotions. There is a dataization function, which puts all three together in order to make an EO program alive.
Here is how it works together with Java, for example.