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The gravity dependence of pharmacodynamics: the integration of lidocaine into membranes in microgravity

The gravity dependence of pharmacodynamics: the integration of lidocaine into membranes in... www.nature.com/npjmgrav ARTICLE OPEN The gravity dependence of pharmacodynamics: the integration of lidocaine into membranes in microgravity 1 2 Florian P. M. Kohn and Jens Hauslage To realize long-term manned space missions, e.g. to Mars, some important questions about pharmacology under conditions of different gravity will have to be answered to ensure safe usage of pharmaceuticals. Experiments on the International Space Station showed that the pharmacokinetics of drugs are changed in microgravity. On Earth, it is well known that the incorporation of substances into cellular membranes depends on membrane fluidity, therefore the finding that membrane fluidity is gravity dependent possibly has effects on pharmacodynamics of hydrophobic and amphiphilic substances in microgravity. To validate a possible effect of gravity on pharmacodynamics, experiments have been carried out to investigate the incorporation of lidocaine into plain lipid membranes under microgravity conditions. In microgravity, the induced increase in membrane fluidity associated with lidocaine incorporation is smaller compared to 1g controls. This experiment concerning the gravity dependence of pharmacodynamics in real microgravity clearly shows that the incorporation of amphipathic drugs into membranes is changed in microgravity. This might have significant impact on the pharmacology of drugs during long-term space missions and has to be investigated in more detail to be able to assess possible risks. npj Microgravity (2019) 5:5 ; https://doi.org/10.1038/s41526-019-0064-5 INTRODUCTION is surface pressure dependent. Furthermore, it was demon- strated that ligand–receptor interactions are membrane fluidity Long-lasting manned space missions beyond Earth's orbit, e.g., to dependent, using the nicotinic acetylcholine receptor as a model Mars are already planned by space agencies and private system. corporations. Then humans will have to live in closed environ- From experiments in clinorotation and in real microgravity ments under space conditions including microgravity for long 13,14 and hypergravity it is known that membrane fluidity itself is periods without immediate access to medical or pharmacological affected by gravity. In microgravity membrane fluidity is increased; support from Earth. To minimize the risk for astronauts, the effects in hypergravity it is decreased. of relevant drugs under space conditions will have to be Following that the incorporation of hydrophobic (and other) investigated in detail before such manned mission will start. To substances and ligand–receptor interactions depends on mem- assess whether the action of pharmaceuticals is different in brane fluidity, which in turn is gravity dependent, it can be weightlessness compared to Earth, pharmacokinetics (PKs) and concluded that a major part of pharmacological relevant pharmacodynamics (PDs) have to be investigated in more detail to 2,3 substances might have changed PDs in microgravity compared establish a “space pharmacology”. According to National to 1g conditions. Aeronautics and Space Administration, there is evidence for A major class of drugs in the above stated field of space inadequate treatment of astronauts during and after a mission. pharmacology are anesthetics, which partially act via membrane Limited data are available for PKs in real weightlessness, incorporation. They are of special interest as a change in their PD showing that, e.g., the peak concentration of acetaminophen in properties could have severe consequences for astronauts. the saliva is significantly smaller, and the time to reach it is 4,5 To assess whether PDs of hydrophobic anesthetics is indeed significantly longer compared to Earth. Unfortunately, no data affected by gravity, we have used lidocaine as a model substance, exist for PDs in real weightlessness yet. Up to now, only ground- as it is well known that it incorporates into the membrane, based experiments (“simulated microgravity”), e.g., from bed rest increasing membrane fluidity. The lidocaine-induced fluidization studies have been performed in 1g. Lidocaine is a local anesthetic of membranes was measured by fluorescence polarization (FP) in and antiarrhythmic drug included in the International Space 1g laboratory conditions and in the microgravity phase during a Station (ISS) pharmacy, with potential variability in its PKs due to sounding rocket flight. metabolism by polymorphic enzymes. For this drug, PK changes have been detected in subjects exposed to 4-day head down tilt tests. RESULTS On Earth, it is well known that the incorporation of hydrophobic, amphiphilic, and other similar drugs into mem- During the microgravity phase of a rocket flight, the incorporation branes depends on membrane fluidity, as well as it has been of 16 mM lidocaine into plain lipid vesicle membranes was shown that the incorporation of Alamethicin into lipid monolayers measured by FP as described in the Methods section. 1 2 Department of Membrane Physiology (230b), Institute of Physiology, University of Hohenheim, Stuttgart, Germany and German Aerospace Center (DLR), Institute of Aerospace Medicine, Gravitational Biology, Linder Hoehe Cologne, Germany Correspondence: Florian P. M. Kohn (florian.p.m.kohn@uni-hohenheim.de) Received: 18 September 2018 Accepted: 5 February 2019 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA F.P.M. Kohn and J. Hauslage The data from the microgravity experiment was compared with membrane fluidity was significantly decreased compared to the 1g ground reference experiment. In both experiments, 16 mM microgravity conditions. lidocaine decreased FP, which means that the fluidity of the To assess whether lidocaine incorporated into the lipid bilayer, membrane is increased (Fig. 1a). Compared to the 1g reference, an additional 1g experiment was executed after the rocket flight. the lidocaine-induced increase in membrane fluidity was smaller The vesicle size of the untreated vesicles (not flown but same in microgravity. vesicle preparation) and of the returned flight sample was After the deployment of the parachute (big spike in Fig. 1a), the measured by dynamic light scattering (DLS) in the laboratory. FP slightly increased, which means that membrane fluidity was The vesicle size of the returned flight sample (treated with 16 mM decreased. This was due to the increased gravity conditions lidocaine during the flight; no additional treatment on Earth) caused by the reentry into the atmosphere and the deployment of increased by 10.6%. No time-dependent variation of vesicle size the parachute (Fig. 1b). was observed (Fig. 3). A statistical analysis of the flight and 1g data (Fig. 2) revealed that the increase in membrane fluidity of asolectin vesicles after DISCUSSION the addition of 16 mM lidocaine is significant, but more importantly, it showed that the increase was significantly less in There is experimental evidence that lidocaine affects neuronal microgravity compared to 1g. After the onset of gravity, sodium channels directly by binding to the D4-S6 region of the α- Fig. 1 a Normalized data of the changes in fluorescence polarization (FP) of the microgravity experiment and 1g ground reference. In both cases, upon addition of 16 mM lidocaine (start of mixing is indicated by arrow; mixing indicated by # ; artifacts caused by moving air bubbles are marked by # ), FP decreased. A reduced FP indicates a decrease in membrane viscosity or an increase in membrane fluidity. It is visible that, during microgravity, the increase membrane fluidity is less compared to 1g. The big spike was caused by the high g-load (24.2g) during the deployment of parachute. One datapoint (second 428) during the deployment was removed as an artifact due to the mechanical shock. b FP flight data compared to the acceleration profile (vector addition of all three axes x, y, and z). At the end of the flight, the average gravity increases again, the big spike indicates the opening of the parachute. With onset of gravity, FP slightly increased again. The dotted lines indicate 0g and 1g Fig. 2 Statistical analysis of the data from the flight and 1g experiment. In 1g and in microgravity, 16 mM lidocaine significantly reduced fluorescence polarization (FP), so membrane fluidity is increased. A comparison between the lidocaine-induced changes of FP reveals a significant smaller decrease in microgravity compared Fig. 3 The change in vesicle size after the addition of 16 mM to 1g. During the reentry phase, FP significantly increases again due lidocaine measured in 1g after the flight. The untreated vesicle to the general gravity dependence of membrane fluidity itself, but it preparation of the rocket mission (not flown) was compared to the does not recover to the values before the addition of lidocaine returned flight sample. The mean vesicle radius increased from as this decreases FP. n (from left to right) = 98, 287, 98, 287, 94. 72.51 nm before the addition to 80.18 nm afterwards. n = 20. Mean ± SD, Welch–analysis of variance and Games–Howell post hoc Boxplot: median, 25 and 75 percentile, whiskers min to max. test. *p < .0005 Unpaired t test, *p < .0001 npj Microgravity (2019) 5 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; F.P.M. Kohn and J. Hauslage subunit and indirectly by modification of membrane proper- (which are parts of PDs) or use hydrophobic substances have to be 19,20 ties. How and to what extent these direct and indirect effects conducted in real microgravity (and hypergravity). on ion channels are intertwined for the pharmacological effect is It is known that the properties of the nicotinic acetylcholine 15,17,21 still under discussion. receptor are affected by the biophysical properties of the The direct effect is preferred by many researchers, but it is well membrane. Following this, it can be postulated that also known that ion channel parameters, as the closed-state prob- receptor–ligand interactions and other signal cascades should be affected by gravity as most of the receptor proteins are ability of the nicotinic acetylcholine receptor, for example, clearly membrane proteins. From experiments during parabolic flight depend on membrane fluidity. Dissolved lidocaine exist in missions (generating real microgravity and hypergravity), electro- uncharged and positively charged form, the uncharged mole- physiological data are available, indicating that synaptic transmis- cules can diffuse through the membrane and interact with ion sion at the motor end plate is also affected. Of course, it has to channels directly, and the charged lidocaine accumulates in the be investigated in more detail to be able to make a definitive hydrophobic part of the lipid membrane. statement. By incorporation into the membrane, lidocaine increases 23,24 25 Coming back to the ascertained gravity dependence of membrane fluidity and decreases membrane thickness. This 23,26,27 lidocaine PDs, the question arises about the mechanism. One can be measured by optical methods as FP, for instance. plausible explanation is that the number of incorporated However, no absolute values of membrane viscosity can be molecules is reduced in microgravity due to the general gravity obtained with this technique. Nevertheless, it can be used for dependence of membrane fluidity. From Earth-bound experi- relative assessments, e.g., if the fluidity of a sample decreases or ments, we know that the increase in membrane fluidity depends increases or if ≥2 samples differ after treatment with different on lidocaine concentration. It is increased with rising lidocaine lidocaine concentrations. concentration. With these prerequisites, the presented experiment now clearly If the result of this experiment is interpreted to state that less shows two things. First, the incorporation of lidocaine into the lidocaine molecules are integrated into the membrane, this would membrane increases membrane fluidity (FP is decreased). The have a huge impact on space pharmacology. It would mean that increase in vesicle size after the addition of lidocaine (Fig. 3)is a in microgravity the dose of hydrophobic medication (as many strong support that lidocaine molecules really incorporate into the anesthetics are) must be increased. This could—in worst case— membrane. More molecules per membrane area could correlate negatively influence the therapeutic index. The same would be with an increase in vesicle size. true for many other drugs as outlined already shortly. Of course, There are also light scatter experiments indicating that the size 28 this has to be verified with additional experiments not only on the of spheroid cells is increased in microgravity. As a lidocaine- 25 pharmacokinetic but also on the pharmacodynamic level as well induced decrease in membrane thickness was found, an as with efficacy studies. interesting experiment would be to monitor membrane thickness Consequently, on long-lasting human space missions, this has in microgravity and hypergravity to evaluate whether this has an to be taken into account, and sufficient data have to be provided effect on the found general gravity dependence of membrane by setting up a research field of space pharmacology, which not fluidity. only focuses on PKs, where first results already have been Second and more importantly, the lidocaine-induced increase in 2,32,33 delivered, but also has to include PDs as this part was membrane fluidity is gravity dependent. Compared to the 1g neglected until now. Data from PK and PD experiments have to be ground control, the increase in fluidity is significantly lower in integrated for better understanding the gravity dependence of microgravity. the effect of pharmaceuticals to assess the yet unknown risk for The demonstrated decrease in membrane fluidity (indicated by astronauts. As PD experiments can be conducted with artificial the increase of FP) after the opening of the parachute and the systems and cell cultures, these experiments can easily be consequential onset of gravity (Fig. 1a) was expected, as an repeated with a high number of samples, delivering good increase in gravity should decrease membrane fluidity. However, statistical data. Therefore, data from these basic experiments can as there is a lidocaine-induced increase in fluidity, the FP signal support pharmacological experiments with human test subjects in does not fully recover to the measured value before the mixing. space, as these experiments usually are conducted with a limited Nevertheless, the general gravity dependence of membrane number of subjects, as the pool of possible test subject is limited 13,14 fluidity could be verified. After the initial 24.2g shock during to (mainly) astronauts and volunteers during parabolic flights. parachute deployment, the average g-load was ≥1g. The fast high- In addition, basic research concerning the biophysical and g spikes are not visible in the FP signal, but this is due to the limit molecular principles behind drug–target interactions can be used in sampling rate of the system. to optimize experiment design and analysis of PK experiments From these findings, it is evident that the PD properties of with astronauts. By revealing that the biophysical membrane lidocaine are different in weightlessness compared to 1g on Earth. properties are affected by gravity and that this affects the A general gravity dependence of PDs was postulated before, incorporation of anesthetics, as shown in this manuscript, the 2,4 and the few experiments on board the ISS focusing on PDs statement that “the intrinsic ability of a drug to cross a membrane or strongly suggested that PDs should also be affected by gravity, to be actively transported is unlikely to be changed in microgravity” 2,3 but it was never investigated up to now. could be disproved. The presented sounding rocket experiment is an experiment in A recent review from 2017 revealed that this topic is also of real microgravity, which shows that at least the PDs of high interest for jet fighter pilots, as a risk for a difference hydrophobic and amphiphilic substances indeed is gravity disposition of chemicals in the body during high-g maneuvers was dependent. Up to now, only experiments under so called found, but—similar to astronauts in weightlessness—the risk “simulated gravity” (e.g., bed rest studies) exist. These experi- cannot be assessed yet as there is not enough data. ments can be used to monitor general physiological adaptations Therefore, to further improve the knowledge about the due to changes in, e.g., blood distributions and to investigate interaction of PDs and gravity, a systematic investigation of microgravity-related effects, but as they are still executed in 1g membrane interactions and receptor–ligand interactions of conditions, these types of experiments cannot simulate changes in relevant pharmaceuticals and substances is necessary, not only membrane fluidity, as this is only changed in real microgravity and in microgravity but also in hypergravity. 13,29 hypergravity. Therefore, experiments that investigate the With an optimized hardware, other, easier accessible research molecular and biophysical principles of membrane interaction platforms as parabolic flights or drop towers can be used. The Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2019) 5 F.P.M. Kohn and J. Hauslage used hardware is based on a so called stopped-flow system, which With these two fluorescence intensities, the rotational speed of the fluorophore can be measured and this can be used to investigate is regularly used to investigate PD reactions. Depending on the membrane fluidity and is used to measure the effect of anesthetics such mixing speed, extremely fast reactions can be measured (in the 17,23 as lidocaine on membrane fluidity. The fluorescence signals of the two range of milliseconds for industrial systems). As it was shown in PMTs were used to calculate FP in mP. As the polarization signals are the data, the lidocaine-induced change in membrane fluidity is sensitive to the cuvette position, the signals of the experiments were within the range of seconds and reaches a stable plateau. A fast normalized to 100 mP to measure the relative changes in fluidity in 1g and system with a faster mixing time could be used in parabolic flights microgravity. or drop tower to significantly increase the amount of available data. The number of experiments could also be increased by Measurement of vesicle size performing parallel experiments during the same flight. Then also The size of the flight sample (before and after addition of 16 mM lidocaine the different gravity phases can be investigated in more detail by, in flight) was measured by DLS after landing of the experiment. Twenty e.g., starting control experiments in 1g to monitor the general separate measurements have been made for each condition, with 50 µl of gravity dependence of membrane fluidity throughout the whole sample per measurement. flight. Also, centrifuges can be used to investigate the gravity dependence of PD in increased gravity. 1g reference experiment. The ground reference experiment was per- As vesicle preparations are stable up to several weeks, this type formed on the same day as the microgravity experiment in the recovered of experiment could be a good candidate for an automated ISS experiment hardware. Ambient conditions (pressure and temperature) were identical to the flight conditions and the same vesicle and asolectin experiment, as no cell culture system is needed. preparation as used for the rocket mission was used to ensure identical experiment conditions. METHODS Experiment hardware Sounding rocket The experiment was part of the Mapheus program of the German The experiment hardware was designed and built to be usable during a sounding rocket flight, and all required tests (e.g., on g-load and vibrations) Aerospace Center (DLR). It was carried out during the Mapheus 6 flight at were passed successfully. The hardware consists of three units: (1) the Esrange Space Center, Sweden. The rocket was launched on 14 May 2017. This sounding rocket system was an unguided two-stage rocket and rocket structure, (2) the late access unit (LAU), and (3) the experiment consisted of a VSB-30 vehicle and the scientific payload on top. During module (Fig. 4). the flight, a microgravity time of up to 6 min can be achieved with a −3 −4 microgravity quality of about 10 –10 g. The total flight time of Mapheus Experiment module 6 (from launch to landing of the payload) was 858 s. The experiment module contained the fluidic and the optical system. Two standard syringes (Luer taper; 10 ml) were operated by a motor-driven Vesicle preparation syringe pump. They were connected to a coin-shaped quartz glass cuvette (volume approximately 50 µl) by silicone tubes. The content of both The vesicles were made from asolectin (from soybean) as the preparation of these vesicles is easy and can be established in a laboratory quite fast. syringes was mixed in a t-connector that was connected to the inlet port of Asolectin is a mixture of unsaturated phospholipids whose main the cuvette. To avoid overpressure in the system, a waste container was components are soybean phosphatidylcholine (25%), phosphatidyletha- connected to the outlet port of the cuvette. Fluorescence was excited by a ultraviolet light-emitting diode (UV-LED) nolamine, and phosphatidylinositol. It is regularly used in pharmacolo- with an additional bandpass filter (365 nm ± 10 nm). By using a long pass gical experiments as it is easy to obtain lipid extract of a biological membrane. filter (430 nm), the excitation wavelength was shielded from the two digital Asolectin was dissolved in chloroform (10 mg/ml). Afterwards 1 ml of PMTs (Hamamatsu H7155) and thus only emission wavelength was this solution was filled in a round-bottomed flask and the solvent was recorded. FP was measured by using three polarizers: one in front of the UV-LED, and one in front of each PMT (parallel and perpendicular to the removed in a rotary evaporator (300 mbar, 1 h) until the lipid remained as a excitation polarizer). Before each experiment, the experiment unit was dry thin film. To remove the last residue of solvent, the flask was stored in a fume hood overnight. After these steps, the lipid film was rehydrated with placed inside the pressure-tight chamber of the LAU and was connected to 2+ 2+ 20 ml of phosphate buffered saline (PBS; without Ca and Mg ) and the the power and control system. A basic schematic of the fluidic and the optical system is shown in Fig. 5. fluorescent dye DPH (1,6-diphenyl-1,3,5-hexatriene; dissolved in dimethyl sulfoxide (DMSO); end concentration 10 µM) for 1 h at 30 °C. To protect the dye from light, the flask was completely covered with aluminum foil. The rehydrated lipid was sonicated (1 h; temperature was kept below 40 °C) and afterwards centrifuged for (30 min; 3000 × g; room temperature). The supernatant containing the DPH-doped vesicles was carefully removed and stored in the fridge in an opaque tube (4 °C). The dyed vesicles were stable for 14 days. Nevertheless, during the sounding rocket mission, fresh vesicles were made 24 h before each launch attempt. Lidocaine solution 2+ 2+ Lidocaine hydrochloride was dissolved in PBS (without Ca and Mg ). A stock solution of 32 mM was used to obtain a final concentration of 16 mM after mixing it with the asolectin vesicle solution. This concentration was determined in previous calibration experiments during the design of the experiment hardware. Measurement of membrane fluidity Changes in membrane fluidity were measured by t-format FP. In short, a fluorescent probe, DPH in this experiment, was excited by polarized light. The emission wavelength was measured by two photomultipliers (PMT), Fig. 4 Picture of the experiment hardware. (1) Rocket structure, (2) again with polarizers parallel (0°) and perpendicular (90°) to the excitation late access unit (LAU), and (3) experiment module. The experiment light. For this experiment, an excitation wavelength of 365 nm was used. module fits into the pressure chamber of the LAU. The LAU is The PMTs recorded emission wavelengths >430 nm. integrated into the structure housing shortly before launch npj Microgravity (2019) 5 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA F.P.M. Kohn and J. Hauslage The polarizers and optical filters were purchased from Edmund Optics Germany. The PMTs are from Hamamatsu Photonics (Hamamatsu, Japan). The electronics were purchased from R+S components Germany and Reichelt Elektronik (Sande, Germany). The metal components and the cuvette were manufactured at the workshops of DLR (Cologne, Germany) and the University of Hohenheim (Stuttgart, Germany). Data and statistical analysis Major goal of this pathfinder experiment was to assess the question whether the incorporation of hydrophobic and amphiphilic substances is gravity dependent as previously proposed. Owing to volume and mass limitations of a sounding rocket mission, the flight data represent only one replicate. Statistical analysis was performed using SPSS 25 and GraphPad Prism 6. Data are expressed as mean ± SD of “n” of the 525 time points of the complete experiment. The mean of each group was calculated with a minimum of 94 time points (number of time Fig. 5 Schematic of the experiment optical set-up and fluidic points: 1g before: 98; 1g+lidocaine: 287; 0g before: 98; 0g lidocaine: 287; system. Vesicles and test substances are mixed and transferred to reentry+lidocaine: 94; presented in Fig. 2). Data were tested for normality the sample cuvette. Fluorescence polarization is measured con- using D’Agostino–Pearson omnibus normality test. Normally distributed tinuously for 525 s data were analyzed for statistical significance using two-tailed Student's t test For non-normally distributed data, statistical significance among the Late access unit different experiment conditions was assessed by Welch–analysis of variance and Games–Howell post hoc test. The LAU had three separate functions. (1) A vacuum-tight chamber allowed experiments to be carried out under normal pressure (approxi- mately 1000 mbar) during the flight of the rocket. (2) It served as power Reporting summary supply for the complete experiment. It was powered by accumulators Further information on experimental design is available in the Nature (12 V, 4800 mAh, NiMH), which were located outside the vacuum chamber. Research Reporting Summary linked to this article. (3) The electronics to control the experiment and to handle the data were located on the aluminum base plate in front of vacuum chamber. Via a vacuum tight plug, data and power lines were connected to the DATA AVAILABILITY experiment unit inside the chamber. The data that support the findings of this study are available from the corresponding The fluorescence signal from each PMT was handled by a dedicated author. microcontroller (Arduino Nano 3.0) and was stored on two memory cards. A third Arduino served as a master controller. It was connected to the main rocket computer and operated the complete experiment after the proper ACKNOWLEDGEMENTS start of experiment (SOE) signal was given by the rocket system. It also The authors thank the Institute of Materials Physics in Space at the DLR (Cologne, synchronized the real-time clock and controlled the subordinate Arduinos Germany) for the flight opportunity. The crew of DLR-MORABA is acknowledged for and the experiment module (pumping and switching of the UV-LED). their support during the certification of the experiment hardware, the preparation of the sounding rocket, and for providing the acceleration data, as well as the team Ambient data from Swedish Space Corporation for the on-site support at Esrange Space Center To be able to execute the 1g reference experiments with the same during the mission. We thank Volkan Cevic for his support during the Mapheus 6 mission. Martin Nawrath from the Academy of Media Arts Cologne is acknowledged temperature profile as given during the rocket flight, temperature was for the Arduino library FreqCounter. This work was funded by University continuously measured at two locations inside the pressure chamber of of Hohenheim and German Aerospace Center (DLR) Institute of Aerospace the LAU (directly at the cuvette and near the inner side of the wall) by two Medicine. battery-powered data loggers (Arexx BS-30). Experiment procedures AUTHOR CONTRIBUTIONS Three hours before liftoff, two syringes were filled with (1) 3.5 ml of F.P.M.K. was the scientific supervisor. He designed the experiment, prepared it during asolectin vesicle preparation and (2) 3.5 ml 32 mM lidocaine solution. The the Mapheus 6 mission, analyzed the data, and wrote the manuscript. J.H. was the silicone tubes and the quartz cuvette were pre-filled with the same vesicle technical supervisor. He designed and constructed the hardware, programmed the preparation to measure the FP baseline signal of the vesicle preparation software, and was responsible for the technical preparation of the experiment on site. without lidocaine after the experiment was started and to remove air He supported manuscript preparation. bubbles from the system. After these steps, the experiment unit was mounted into the pressure chamber of the LAU and it was tightly sealed. Two hours before the end of the countdown, the LAU was integrated ADDITIONAL INFORMATION into the rocket structure, the experiment was connected to rocket service Supplementary information accompanies the paper on the npj Microgravity website module, and the experiment was set to standby. (https://doi.org/10.1038/s41526-019-0064-5). With the onset of microgravity (approximately 70 s after liftoff), the SOE signal was given and the experiment was switched to active: First, the Competing interests: The authors declare no competing interests. baseline FP of the unmodified DPH-doped asolectin vesicles was measured for 100 s. Subsequently, the syringe pump was activated and the content Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims of the two syringes were mixed and the mixture was pumped into the in published maps and institutional affiliations. sample cuvette (18-s pump duration; final lidocaine concentration 16 mM). Excess liquid was pumped into the waste syringe. The FP measurement was continued for additional 425 s to include the gravitational pull after reentry into the atmosphere and opening of the parachute. REFERENCES 1. Musk, E. Making life multi-planetary. New Space 6,2–11 (2018). Materials 2. Wotring, V. E. Space Pharmacology (Springer, New York, 2012). 3. 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Högberg, C.-J. & Lyubartsev, A. P. Effect of local anesthetic lidocaine on elec- trostatic properties of a lipid bilayer. Biophys. J. 94, 525–531 (2008). Open Access This article is licensed under a Creative Commons 20. Kopeć, W., Telenius, J. & Khandelia, H. Molecular dynamics simulations of the Attribution 4.0 International License, which permits use, sharing, interactions of medicinal plant extracts and drugs with lipid bilayer membranes. adaptation, distribution and reproduction in any medium or format, as long as you give FEBS J. 280, 2785–2805 (2013). appropriate credit to the original author(s) and the source, provide a link to the Creative 21. Pleuvry, B. J. Mechanism of action of general anaesthetic drugs. Anaesth. Intensive Commons license, and indicate if changes were made. The images or other third party Care Med. 9, 152–153 (2008). material in this article are included in the article’s Creative Commons license, unless 22. Sanchez, V., Arthur, G. R. & Strichartz, G. R. Fundamental properties of local indicated otherwise in a credit line to the material. If material is not included in the anesthetics. I. The dependence of lidocaine’s ionization and octanol:buffer par- article’s Creative Commons license and your intended use is not permitted by statutory titioning on solvent and temperature. Anesth. Analg. 66, 159–165 (1987). regulation or exceeds the permitted use, you will need to obtain permission directly 23. Yun, I. et al. Amphiphilic effects of local anesthetics on rotational mobility in from the copyright holder. To view a copy of this license, visit http://creativecommons. neuronal and model membranes. Biochim. Biophys. Acta 1564, 123–132 (2002). org/licenses/by/4.0/. 24. Tsuchiya, H., Mizogami, M. & Takakura, K. Reversed-phase liquid chromatographic retention and membrane activity relationships of local anesthetics. J. 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The gravity dependence of pharmacodynamics: the integration of lidocaine into membranes in microgravity

npj Microgravity , Volume 5 (1) – Mar 6, 2019

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Life Sciences; Life Sciences, general; Classical and Continuum Physics; Biotechnology; Immunology; Space Sciences (including Extraterrestrial Physics, Space Exploration and Astronautics) ; Applied Microbiology
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www.nature.com/npjmgrav ARTICLE OPEN The gravity dependence of pharmacodynamics: the integration of lidocaine into membranes in microgravity 1 2 Florian P. M. Kohn and Jens Hauslage To realize long-term manned space missions, e.g. to Mars, some important questions about pharmacology under conditions of different gravity will have to be answered to ensure safe usage of pharmaceuticals. Experiments on the International Space Station showed that the pharmacokinetics of drugs are changed in microgravity. On Earth, it is well known that the incorporation of substances into cellular membranes depends on membrane fluidity, therefore the finding that membrane fluidity is gravity dependent possibly has effects on pharmacodynamics of hydrophobic and amphiphilic substances in microgravity. To validate a possible effect of gravity on pharmacodynamics, experiments have been carried out to investigate the incorporation of lidocaine into plain lipid membranes under microgravity conditions. In microgravity, the induced increase in membrane fluidity associated with lidocaine incorporation is smaller compared to 1g controls. This experiment concerning the gravity dependence of pharmacodynamics in real microgravity clearly shows that the incorporation of amphipathic drugs into membranes is changed in microgravity. This might have significant impact on the pharmacology of drugs during long-term space missions and has to be investigated in more detail to be able to assess possible risks. npj Microgravity (2019) 5:5 ; https://doi.org/10.1038/s41526-019-0064-5 INTRODUCTION is surface pressure dependent. Furthermore, it was demon- strated that ligand–receptor interactions are membrane fluidity Long-lasting manned space missions beyond Earth's orbit, e.g., to dependent, using the nicotinic acetylcholine receptor as a model Mars are already planned by space agencies and private system. corporations. Then humans will have to live in closed environ- From experiments in clinorotation and in real microgravity ments under space conditions including microgravity for long 13,14 and hypergravity it is known that membrane fluidity itself is periods without immediate access to medical or pharmacological affected by gravity. In microgravity membrane fluidity is increased; support from Earth. To minimize the risk for astronauts, the effects in hypergravity it is decreased. of relevant drugs under space conditions will have to be Following that the incorporation of hydrophobic (and other) investigated in detail before such manned mission will start. To substances and ligand–receptor interactions depends on mem- assess whether the action of pharmaceuticals is different in brane fluidity, which in turn is gravity dependent, it can be weightlessness compared to Earth, pharmacokinetics (PKs) and concluded that a major part of pharmacological relevant pharmacodynamics (PDs) have to be investigated in more detail to 2,3 substances might have changed PDs in microgravity compared establish a “space pharmacology”. According to National to 1g conditions. Aeronautics and Space Administration, there is evidence for A major class of drugs in the above stated field of space inadequate treatment of astronauts during and after a mission. pharmacology are anesthetics, which partially act via membrane Limited data are available for PKs in real weightlessness, incorporation. They are of special interest as a change in their PD showing that, e.g., the peak concentration of acetaminophen in properties could have severe consequences for astronauts. the saliva is significantly smaller, and the time to reach it is 4,5 To assess whether PDs of hydrophobic anesthetics is indeed significantly longer compared to Earth. Unfortunately, no data affected by gravity, we have used lidocaine as a model substance, exist for PDs in real weightlessness yet. Up to now, only ground- as it is well known that it incorporates into the membrane, based experiments (“simulated microgravity”), e.g., from bed rest increasing membrane fluidity. The lidocaine-induced fluidization studies have been performed in 1g. Lidocaine is a local anesthetic of membranes was measured by fluorescence polarization (FP) in and antiarrhythmic drug included in the International Space 1g laboratory conditions and in the microgravity phase during a Station (ISS) pharmacy, with potential variability in its PKs due to sounding rocket flight. metabolism by polymorphic enzymes. For this drug, PK changes have been detected in subjects exposed to 4-day head down tilt tests. RESULTS On Earth, it is well known that the incorporation of hydrophobic, amphiphilic, and other similar drugs into mem- During the microgravity phase of a rocket flight, the incorporation branes depends on membrane fluidity, as well as it has been of 16 mM lidocaine into plain lipid vesicle membranes was shown that the incorporation of Alamethicin into lipid monolayers measured by FP as described in the Methods section. 1 2 Department of Membrane Physiology (230b), Institute of Physiology, University of Hohenheim, Stuttgart, Germany and German Aerospace Center (DLR), Institute of Aerospace Medicine, Gravitational Biology, Linder Hoehe Cologne, Germany Correspondence: Florian P. M. Kohn (florian.p.m.kohn@uni-hohenheim.de) Received: 18 September 2018 Accepted: 5 February 2019 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA F.P.M. Kohn and J. Hauslage The data from the microgravity experiment was compared with membrane fluidity was significantly decreased compared to the 1g ground reference experiment. In both experiments, 16 mM microgravity conditions. lidocaine decreased FP, which means that the fluidity of the To assess whether lidocaine incorporated into the lipid bilayer, membrane is increased (Fig. 1a). Compared to the 1g reference, an additional 1g experiment was executed after the rocket flight. the lidocaine-induced increase in membrane fluidity was smaller The vesicle size of the untreated vesicles (not flown but same in microgravity. vesicle preparation) and of the returned flight sample was After the deployment of the parachute (big spike in Fig. 1a), the measured by dynamic light scattering (DLS) in the laboratory. FP slightly increased, which means that membrane fluidity was The vesicle size of the returned flight sample (treated with 16 mM decreased. This was due to the increased gravity conditions lidocaine during the flight; no additional treatment on Earth) caused by the reentry into the atmosphere and the deployment of increased by 10.6%. No time-dependent variation of vesicle size the parachute (Fig. 1b). was observed (Fig. 3). A statistical analysis of the flight and 1g data (Fig. 2) revealed that the increase in membrane fluidity of asolectin vesicles after DISCUSSION the addition of 16 mM lidocaine is significant, but more importantly, it showed that the increase was significantly less in There is experimental evidence that lidocaine affects neuronal microgravity compared to 1g. After the onset of gravity, sodium channels directly by binding to the D4-S6 region of the α- Fig. 1 a Normalized data of the changes in fluorescence polarization (FP) of the microgravity experiment and 1g ground reference. In both cases, upon addition of 16 mM lidocaine (start of mixing is indicated by arrow; mixing indicated by # ; artifacts caused by moving air bubbles are marked by # ), FP decreased. A reduced FP indicates a decrease in membrane viscosity or an increase in membrane fluidity. It is visible that, during microgravity, the increase membrane fluidity is less compared to 1g. The big spike was caused by the high g-load (24.2g) during the deployment of parachute. One datapoint (second 428) during the deployment was removed as an artifact due to the mechanical shock. b FP flight data compared to the acceleration profile (vector addition of all three axes x, y, and z). At the end of the flight, the average gravity increases again, the big spike indicates the opening of the parachute. With onset of gravity, FP slightly increased again. The dotted lines indicate 0g and 1g Fig. 2 Statistical analysis of the data from the flight and 1g experiment. In 1g and in microgravity, 16 mM lidocaine significantly reduced fluorescence polarization (FP), so membrane fluidity is increased. A comparison between the lidocaine-induced changes of FP reveals a significant smaller decrease in microgravity compared Fig. 3 The change in vesicle size after the addition of 16 mM to 1g. During the reentry phase, FP significantly increases again due lidocaine measured in 1g after the flight. The untreated vesicle to the general gravity dependence of membrane fluidity itself, but it preparation of the rocket mission (not flown) was compared to the does not recover to the values before the addition of lidocaine returned flight sample. The mean vesicle radius increased from as this decreases FP. n (from left to right) = 98, 287, 98, 287, 94. 72.51 nm before the addition to 80.18 nm afterwards. n = 20. Mean ± SD, Welch–analysis of variance and Games–Howell post hoc Boxplot: median, 25 and 75 percentile, whiskers min to max. test. *p < .0005 Unpaired t test, *p < .0001 npj Microgravity (2019) 5 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; F.P.M. Kohn and J. Hauslage subunit and indirectly by modification of membrane proper- (which are parts of PDs) or use hydrophobic substances have to be 19,20 ties. How and to what extent these direct and indirect effects conducted in real microgravity (and hypergravity). on ion channels are intertwined for the pharmacological effect is It is known that the properties of the nicotinic acetylcholine 15,17,21 still under discussion. receptor are affected by the biophysical properties of the The direct effect is preferred by many researchers, but it is well membrane. Following this, it can be postulated that also known that ion channel parameters, as the closed-state prob- receptor–ligand interactions and other signal cascades should be affected by gravity as most of the receptor proteins are ability of the nicotinic acetylcholine receptor, for example, clearly membrane proteins. From experiments during parabolic flight depend on membrane fluidity. Dissolved lidocaine exist in missions (generating real microgravity and hypergravity), electro- uncharged and positively charged form, the uncharged mole- physiological data are available, indicating that synaptic transmis- cules can diffuse through the membrane and interact with ion sion at the motor end plate is also affected. Of course, it has to channels directly, and the charged lidocaine accumulates in the be investigated in more detail to be able to make a definitive hydrophobic part of the lipid membrane. statement. By incorporation into the membrane, lidocaine increases 23,24 25 Coming back to the ascertained gravity dependence of membrane fluidity and decreases membrane thickness. This 23,26,27 lidocaine PDs, the question arises about the mechanism. One can be measured by optical methods as FP, for instance. plausible explanation is that the number of incorporated However, no absolute values of membrane viscosity can be molecules is reduced in microgravity due to the general gravity obtained with this technique. Nevertheless, it can be used for dependence of membrane fluidity. From Earth-bound experi- relative assessments, e.g., if the fluidity of a sample decreases or ments, we know that the increase in membrane fluidity depends increases or if ≥2 samples differ after treatment with different on lidocaine concentration. It is increased with rising lidocaine lidocaine concentrations. concentration. With these prerequisites, the presented experiment now clearly If the result of this experiment is interpreted to state that less shows two things. First, the incorporation of lidocaine into the lidocaine molecules are integrated into the membrane, this would membrane increases membrane fluidity (FP is decreased). The have a huge impact on space pharmacology. It would mean that increase in vesicle size after the addition of lidocaine (Fig. 3)is a in microgravity the dose of hydrophobic medication (as many strong support that lidocaine molecules really incorporate into the anesthetics are) must be increased. This could—in worst case— membrane. More molecules per membrane area could correlate negatively influence the therapeutic index. The same would be with an increase in vesicle size. true for many other drugs as outlined already shortly. Of course, There are also light scatter experiments indicating that the size 28 this has to be verified with additional experiments not only on the of spheroid cells is increased in microgravity. As a lidocaine- 25 pharmacokinetic but also on the pharmacodynamic level as well induced decrease in membrane thickness was found, an as with efficacy studies. interesting experiment would be to monitor membrane thickness Consequently, on long-lasting human space missions, this has in microgravity and hypergravity to evaluate whether this has an to be taken into account, and sufficient data have to be provided effect on the found general gravity dependence of membrane by setting up a research field of space pharmacology, which not fluidity. only focuses on PKs, where first results already have been Second and more importantly, the lidocaine-induced increase in 2,32,33 delivered, but also has to include PDs as this part was membrane fluidity is gravity dependent. Compared to the 1g neglected until now. Data from PK and PD experiments have to be ground control, the increase in fluidity is significantly lower in integrated for better understanding the gravity dependence of microgravity. the effect of pharmaceuticals to assess the yet unknown risk for The demonstrated decrease in membrane fluidity (indicated by astronauts. As PD experiments can be conducted with artificial the increase of FP) after the opening of the parachute and the systems and cell cultures, these experiments can easily be consequential onset of gravity (Fig. 1a) was expected, as an repeated with a high number of samples, delivering good increase in gravity should decrease membrane fluidity. However, statistical data. Therefore, data from these basic experiments can as there is a lidocaine-induced increase in fluidity, the FP signal support pharmacological experiments with human test subjects in does not fully recover to the measured value before the mixing. space, as these experiments usually are conducted with a limited Nevertheless, the general gravity dependence of membrane number of subjects, as the pool of possible test subject is limited 13,14 fluidity could be verified. After the initial 24.2g shock during to (mainly) astronauts and volunteers during parabolic flights. parachute deployment, the average g-load was ≥1g. The fast high- In addition, basic research concerning the biophysical and g spikes are not visible in the FP signal, but this is due to the limit molecular principles behind drug–target interactions can be used in sampling rate of the system. to optimize experiment design and analysis of PK experiments From these findings, it is evident that the PD properties of with astronauts. By revealing that the biophysical membrane lidocaine are different in weightlessness compared to 1g on Earth. properties are affected by gravity and that this affects the A general gravity dependence of PDs was postulated before, incorporation of anesthetics, as shown in this manuscript, the 2,4 and the few experiments on board the ISS focusing on PDs statement that “the intrinsic ability of a drug to cross a membrane or strongly suggested that PDs should also be affected by gravity, to be actively transported is unlikely to be changed in microgravity” 2,3 but it was never investigated up to now. could be disproved. The presented sounding rocket experiment is an experiment in A recent review from 2017 revealed that this topic is also of real microgravity, which shows that at least the PDs of high interest for jet fighter pilots, as a risk for a difference hydrophobic and amphiphilic substances indeed is gravity disposition of chemicals in the body during high-g maneuvers was dependent. Up to now, only experiments under so called found, but—similar to astronauts in weightlessness—the risk “simulated gravity” (e.g., bed rest studies) exist. These experi- cannot be assessed yet as there is not enough data. ments can be used to monitor general physiological adaptations Therefore, to further improve the knowledge about the due to changes in, e.g., blood distributions and to investigate interaction of PDs and gravity, a systematic investigation of microgravity-related effects, but as they are still executed in 1g membrane interactions and receptor–ligand interactions of conditions, these types of experiments cannot simulate changes in relevant pharmaceuticals and substances is necessary, not only membrane fluidity, as this is only changed in real microgravity and in microgravity but also in hypergravity. 13,29 hypergravity. Therefore, experiments that investigate the With an optimized hardware, other, easier accessible research molecular and biophysical principles of membrane interaction platforms as parabolic flights or drop towers can be used. The Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2019) 5 F.P.M. Kohn and J. Hauslage used hardware is based on a so called stopped-flow system, which With these two fluorescence intensities, the rotational speed of the fluorophore can be measured and this can be used to investigate is regularly used to investigate PD reactions. Depending on the membrane fluidity and is used to measure the effect of anesthetics such mixing speed, extremely fast reactions can be measured (in the 17,23 as lidocaine on membrane fluidity. The fluorescence signals of the two range of milliseconds for industrial systems). As it was shown in PMTs were used to calculate FP in mP. As the polarization signals are the data, the lidocaine-induced change in membrane fluidity is sensitive to the cuvette position, the signals of the experiments were within the range of seconds and reaches a stable plateau. A fast normalized to 100 mP to measure the relative changes in fluidity in 1g and system with a faster mixing time could be used in parabolic flights microgravity. or drop tower to significantly increase the amount of available data. The number of experiments could also be increased by Measurement of vesicle size performing parallel experiments during the same flight. Then also The size of the flight sample (before and after addition of 16 mM lidocaine the different gravity phases can be investigated in more detail by, in flight) was measured by DLS after landing of the experiment. Twenty e.g., starting control experiments in 1g to monitor the general separate measurements have been made for each condition, with 50 µl of gravity dependence of membrane fluidity throughout the whole sample per measurement. flight. Also, centrifuges can be used to investigate the gravity dependence of PD in increased gravity. 1g reference experiment. The ground reference experiment was per- As vesicle preparations are stable up to several weeks, this type formed on the same day as the microgravity experiment in the recovered of experiment could be a good candidate for an automated ISS experiment hardware. Ambient conditions (pressure and temperature) were identical to the flight conditions and the same vesicle and asolectin experiment, as no cell culture system is needed. preparation as used for the rocket mission was used to ensure identical experiment conditions. METHODS Experiment hardware Sounding rocket The experiment was part of the Mapheus program of the German The experiment hardware was designed and built to be usable during a sounding rocket flight, and all required tests (e.g., on g-load and vibrations) Aerospace Center (DLR). It was carried out during the Mapheus 6 flight at were passed successfully. The hardware consists of three units: (1) the Esrange Space Center, Sweden. The rocket was launched on 14 May 2017. This sounding rocket system was an unguided two-stage rocket and rocket structure, (2) the late access unit (LAU), and (3) the experiment consisted of a VSB-30 vehicle and the scientific payload on top. During module (Fig. 4). the flight, a microgravity time of up to 6 min can be achieved with a −3 −4 microgravity quality of about 10 –10 g. The total flight time of Mapheus Experiment module 6 (from launch to landing of the payload) was 858 s. The experiment module contained the fluidic and the optical system. Two standard syringes (Luer taper; 10 ml) were operated by a motor-driven Vesicle preparation syringe pump. They were connected to a coin-shaped quartz glass cuvette (volume approximately 50 µl) by silicone tubes. The content of both The vesicles were made from asolectin (from soybean) as the preparation of these vesicles is easy and can be established in a laboratory quite fast. syringes was mixed in a t-connector that was connected to the inlet port of Asolectin is a mixture of unsaturated phospholipids whose main the cuvette. To avoid overpressure in the system, a waste container was components are soybean phosphatidylcholine (25%), phosphatidyletha- connected to the outlet port of the cuvette. Fluorescence was excited by a ultraviolet light-emitting diode (UV-LED) nolamine, and phosphatidylinositol. It is regularly used in pharmacolo- with an additional bandpass filter (365 nm ± 10 nm). By using a long pass gical experiments as it is easy to obtain lipid extract of a biological membrane. filter (430 nm), the excitation wavelength was shielded from the two digital Asolectin was dissolved in chloroform (10 mg/ml). Afterwards 1 ml of PMTs (Hamamatsu H7155) and thus only emission wavelength was this solution was filled in a round-bottomed flask and the solvent was recorded. FP was measured by using three polarizers: one in front of the UV-LED, and one in front of each PMT (parallel and perpendicular to the removed in a rotary evaporator (300 mbar, 1 h) until the lipid remained as a excitation polarizer). Before each experiment, the experiment unit was dry thin film. To remove the last residue of solvent, the flask was stored in a fume hood overnight. After these steps, the lipid film was rehydrated with placed inside the pressure-tight chamber of the LAU and was connected to 2+ 2+ 20 ml of phosphate buffered saline (PBS; without Ca and Mg ) and the the power and control system. A basic schematic of the fluidic and the optical system is shown in Fig. 5. fluorescent dye DPH (1,6-diphenyl-1,3,5-hexatriene; dissolved in dimethyl sulfoxide (DMSO); end concentration 10 µM) for 1 h at 30 °C. To protect the dye from light, the flask was completely covered with aluminum foil. The rehydrated lipid was sonicated (1 h; temperature was kept below 40 °C) and afterwards centrifuged for (30 min; 3000 × g; room temperature). The supernatant containing the DPH-doped vesicles was carefully removed and stored in the fridge in an opaque tube (4 °C). The dyed vesicles were stable for 14 days. Nevertheless, during the sounding rocket mission, fresh vesicles were made 24 h before each launch attempt. Lidocaine solution 2+ 2+ Lidocaine hydrochloride was dissolved in PBS (without Ca and Mg ). A stock solution of 32 mM was used to obtain a final concentration of 16 mM after mixing it with the asolectin vesicle solution. This concentration was determined in previous calibration experiments during the design of the experiment hardware. Measurement of membrane fluidity Changes in membrane fluidity were measured by t-format FP. In short, a fluorescent probe, DPH in this experiment, was excited by polarized light. The emission wavelength was measured by two photomultipliers (PMT), Fig. 4 Picture of the experiment hardware. (1) Rocket structure, (2) again with polarizers parallel (0°) and perpendicular (90°) to the excitation late access unit (LAU), and (3) experiment module. The experiment light. For this experiment, an excitation wavelength of 365 nm was used. module fits into the pressure chamber of the LAU. The LAU is The PMTs recorded emission wavelengths >430 nm. integrated into the structure housing shortly before launch npj Microgravity (2019) 5 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA F.P.M. Kohn and J. Hauslage The polarizers and optical filters were purchased from Edmund Optics Germany. The PMTs are from Hamamatsu Photonics (Hamamatsu, Japan). The electronics were purchased from R+S components Germany and Reichelt Elektronik (Sande, Germany). The metal components and the cuvette were manufactured at the workshops of DLR (Cologne, Germany) and the University of Hohenheim (Stuttgart, Germany). Data and statistical analysis Major goal of this pathfinder experiment was to assess the question whether the incorporation of hydrophobic and amphiphilic substances is gravity dependent as previously proposed. Owing to volume and mass limitations of a sounding rocket mission, the flight data represent only one replicate. Statistical analysis was performed using SPSS 25 and GraphPad Prism 6. Data are expressed as mean ± SD of “n” of the 525 time points of the complete experiment. The mean of each group was calculated with a minimum of 94 time points (number of time Fig. 5 Schematic of the experiment optical set-up and fluidic points: 1g before: 98; 1g+lidocaine: 287; 0g before: 98; 0g lidocaine: 287; system. Vesicles and test substances are mixed and transferred to reentry+lidocaine: 94; presented in Fig. 2). Data were tested for normality the sample cuvette. Fluorescence polarization is measured con- using D’Agostino–Pearson omnibus normality test. Normally distributed tinuously for 525 s data were analyzed for statistical significance using two-tailed Student's t test For non-normally distributed data, statistical significance among the Late access unit different experiment conditions was assessed by Welch–analysis of variance and Games–Howell post hoc test. The LAU had three separate functions. (1) A vacuum-tight chamber allowed experiments to be carried out under normal pressure (approxi- mately 1000 mbar) during the flight of the rocket. (2) It served as power Reporting summary supply for the complete experiment. It was powered by accumulators Further information on experimental design is available in the Nature (12 V, 4800 mAh, NiMH), which were located outside the vacuum chamber. Research Reporting Summary linked to this article. (3) The electronics to control the experiment and to handle the data were located on the aluminum base plate in front of vacuum chamber. Via a vacuum tight plug, data and power lines were connected to the DATA AVAILABILITY experiment unit inside the chamber. The data that support the findings of this study are available from the corresponding The fluorescence signal from each PMT was handled by a dedicated author. microcontroller (Arduino Nano 3.0) and was stored on two memory cards. A third Arduino served as a master controller. It was connected to the main rocket computer and operated the complete experiment after the proper ACKNOWLEDGEMENTS start of experiment (SOE) signal was given by the rocket system. It also The authors thank the Institute of Materials Physics in Space at the DLR (Cologne, synchronized the real-time clock and controlled the subordinate Arduinos Germany) for the flight opportunity. The crew of DLR-MORABA is acknowledged for and the experiment module (pumping and switching of the UV-LED). their support during the certification of the experiment hardware, the preparation of the sounding rocket, and for providing the acceleration data, as well as the team Ambient data from Swedish Space Corporation for the on-site support at Esrange Space Center To be able to execute the 1g reference experiments with the same during the mission. We thank Volkan Cevic for his support during the Mapheus 6 mission. Martin Nawrath from the Academy of Media Arts Cologne is acknowledged temperature profile as given during the rocket flight, temperature was for the Arduino library FreqCounter. This work was funded by University continuously measured at two locations inside the pressure chamber of of Hohenheim and German Aerospace Center (DLR) Institute of Aerospace the LAU (directly at the cuvette and near the inner side of the wall) by two Medicine. battery-powered data loggers (Arexx BS-30). Experiment procedures AUTHOR CONTRIBUTIONS Three hours before liftoff, two syringes were filled with (1) 3.5 ml of F.P.M.K. was the scientific supervisor. He designed the experiment, prepared it during asolectin vesicle preparation and (2) 3.5 ml 32 mM lidocaine solution. The the Mapheus 6 mission, analyzed the data, and wrote the manuscript. J.H. was the silicone tubes and the quartz cuvette were pre-filled with the same vesicle technical supervisor. He designed and constructed the hardware, programmed the preparation to measure the FP baseline signal of the vesicle preparation software, and was responsible for the technical preparation of the experiment on site. without lidocaine after the experiment was started and to remove air He supported manuscript preparation. bubbles from the system. After these steps, the experiment unit was mounted into the pressure chamber of the LAU and it was tightly sealed. Two hours before the end of the countdown, the LAU was integrated ADDITIONAL INFORMATION into the rocket structure, the experiment was connected to rocket service Supplementary information accompanies the paper on the npj Microgravity website module, and the experiment was set to standby. (https://doi.org/10.1038/s41526-019-0064-5). With the onset of microgravity (approximately 70 s after liftoff), the SOE signal was given and the experiment was switched to active: First, the Competing interests: The authors declare no competing interests. baseline FP of the unmodified DPH-doped asolectin vesicles was measured for 100 s. Subsequently, the syringe pump was activated and the content Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims of the two syringes were mixed and the mixture was pumped into the in published maps and institutional affiliations. sample cuvette (18-s pump duration; final lidocaine concentration 16 mM). Excess liquid was pumped into the waste syringe. The FP measurement was continued for additional 425 s to include the gravitational pull after reentry into the atmosphere and opening of the parachute. REFERENCES 1. Musk, E. Making life multi-planetary. New Space 6,2–11 (2018). Materials 2. Wotring, V. E. Space Pharmacology (Springer, New York, 2012). 3. 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