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62cd82f227b1e463bb36b984 | 34 | Bodipy 576/589 labeling of VCB VCB was labeled with Bodipy 576/589 following protocol reported previously . Briefly, the VCB complex was mixed with Bodipy 576/589 NHS ester in a 20:1 molar ration and incubated at room temperature (protect from light) for 2h in reaction buffer (0.1 M sodium phosphate, 75 mM KOAc, 2 mM DTT, pH 7.4). The reaction was quenched by diluting 10 times with reaction buffer and the unreacted dye was removed with a PD-10 MiniTrap desalting column (GE Healthcare) equilibrated with 100 mM Bis-Tris pH 7.0, 100 mM NaCl, 1 mM DTT, pH 7. The eluted labeled protein solution was collected and concentrated with Pierce Concentrator, 3K MWCO (Thermo scientific). |
62cd82f227b1e463bb36b984 | 35 | HEK 293 cells were transfected with LRRK2-NanoLuc vector (Promega, NV3401) for 24h, harvested, and resuspended into OptiMEM media without phenol red (Life Technologies). The cells were then seeded into white 384well plate (Corning3570) at a density of 6000 cells/well. Digitonin solution (final concentration 50 µg/ml), testing PROTACs at decreasing concentrations (11 concentrations with 2-fold serial dilution starting from 33 µM) or DMSO, and VCB protein labeled with Bodipy 576/589 (final concentration 0.5 µM) were added separately. Each well was added with NanoBRET NanoGlo Substrate (Promega, N2160) before performing the NanoBRET reading on PHERAstar (BMG LABTECH) plate reader equipped with a 450-nm bandpass filter (donor) and a 600-nm long pass filter (acceptor). NanoBRET ratio of each well was expressed in milliBRET according to the equation: mBRET = [(signal at 610 nM/signal at 450 nM) -(signal at 610 nMno tracer control/signal at 450 nMno tracer control)]×1000. The background signal as shown in the DMSO control samples was subtracted from each sample. |
62cd82f227b1e463bb36b984 | 36 | Caco-2 cells with Transepithelial electrical resistance (TEER) (TEER= (Resistance sample -Resistance blank)×Effective Membrane Area) = 450 ± 19 Ω•cm2 Ω•cm 2 were used for the experiment. Compounds were dissolved in appropriate buffer (10 mM DMSO stock solutions were diluted with HBSS buffer to a final concentration of 10 µM testing compound and 0.4% DMSO, Lucifer Yellow was introduced in the apical side buffer to test the intactness of the mono-cell layer) and was applied to the apical or basolateral donor side for measuring A -B or B -A permeability (two replicates), respectively. The apical and basolateral plates were prewarmed to 37 °C before placing the apical plate onto basolateral plate. After incubating at 37 °C for 90 min, the apical plate and basolateral plate were separated, and the donor or receiver samples were analyzed with UPLC-MS/MS. |
62cd82f227b1e463bb36b984 | 37 | Frozen plasma was thawed at 37 °C and centrifuged at 3000 rpm for 8 min to remove clots and the supernatant was used in the experiment. The pH of the plasma was recorded and only pH range between 7.4 and 8 was used. The plasma and compounds solution was pre-warmed to 37 °C. 10 µl prewarmed testing compound or reference compound (procaine) solution (20 µM in 0.05 mM sodium phosphate buffer (pH7.4) with 0.5% BSA) was mixed with 90 µl of plasma at different time points to allow for 5, 15, 30, 45, and 60 min of incubation time. For 0 min, the plasma was mixed with vehicle only. Acetonitrile was added to the compound and plasma mixture to quench the reaction and the resulting mixture was centrifuged (5594 g for 15 min). The supernatant was taken and diluted before LC-MS analysis. |
62cd82f227b1e463bb36b984 | 38 | Solubility in Phosphate buffer and Fed State Simulated Intestinal Fluid (FeSSIF) 8 µl of reference or test compound stock solution (10 mM in DMSO) was added into 792 µl of 100 mM phosphate buffer (pH 7.4) or FeSSIF (pH 5.8). The resulting mixture was shaken for 1h (1000 rpm) at room temperature, then centrifuged for 10 min (12000 rpm) to remove un-dissolved particles. The supernatant was collected and diluted 10 times and 100 times separately with 100 mM phosphate buffer or FeSSIF. 5 µl of the supernatant samples (no diluted, 10 times diluted, 100 times diluted) were mixed with 95 µl of acetonitrile (containing internal standard) separately before injecting into LC-MS/MS for analysis. |
62cd82f227b1e463bb36b984 | 39 | Mouse liver microsome stability 1.5 µl testing compound or reference compound (500 µM in 5% DMSO and 95% acetonitrile) was mixed with 18.75 µl of 20 mg/mL liver microsome (Corning) and 479.75 µl potassium phosphate buffer (0.1 M potassium phosphate buffer, 1 mM EDTA, pH 7.4). The reaction was started by mixing 30 µL of the above mixture (pre-warmed to 37 °C) with 15 µl of 6 mM NADPH stock solution (pre-warmed to 37 °C). After incubating for 5, 15, 30, or 45 min, 135 µl of acetonitrile containing internal standard was added to stop the reaction. For 0 min, the compound and microsome mixture was mixed with acetonitrile first before adding NADPH. After quench, the reaction mixture was centrifuged, and the supernatant was taken and diluted for LC-MS analysis. |
62cd82f227b1e463bb36b984 | 40 | Mouse Hepatocyte stability 50 µl of pre-warmed hepatocytes (2 × 10 6 cells/ml) in suspension media (Krebs-Henseleit buffer (Sigma) containing 5.6 g/l HEPES) was mixed with 50 µl prewarmed compound dosing solution (2 µM in Krebs-Henseleit buffer with 1% DMSO). After incubating at 37 °C for 15, 30, 60 or 120 min, 100 µl of acetonitrile containing internal standard was added to quench the reaction. For 0 min incubation, acetonitrile was mixed with hepatocytes first before adding compound solution. After quenching, the mixture was shaken at the vibrator for 10 min (600 rpm/min) and then sonicated for 2 min before centrifugation (5594 g for 15 min). The supernatant was taken and diluted for LC-MS analysis. |
62cd82f227b1e463bb36b984 | 41 | PK profiling was outsourced and undertaken by Shanghai ChemPartner Co., Ltd. All animal experiments performed were conducted in compliance with the Institutional Animal Care and Use Committee (IACUC) and the Office of Laboratory Animal Welfare (OLAW) guidelines. Six-to eight-week-old C57BL/6 male mice purchased from Jihui Laboratory Animal Co. LTD were used in the study. XL01126 was formulated in 10%HP-β-CD in 50mM Citrate buffer pH=3.0 at 1 mg/ml for IV injection and at 3 mg/ml for IP and PO injection. For IV injections, 5 mg/kg of XL01126 was administered into the tail vein. For IP and PO injections, 30 mg/kg of XL01126 was administered via intraperitoneal injection or oral gavage, respectively. The animals were restrained manually at the designated time points (0.083, 0.25, 0.5, 1, 2, 4, and 8 h); approximately, 110 μl of blood sample was collected via facial vein into K2EDTA tubes. Three mice per time point were used, resulting in a total of 21 mice for each administration route. The blood sample was put on ice and centrifuged at 2000g for 5 min to obtain the plasma sample within 15 min. The plasma, brain, CSF samples were stored at approximately -70 °C until analysis. |
60c75769bb8c1a04ad3dc828 | 0 | Social media such as Twitter has become an important communication tool for scientists. Twitter is a social networking service on which users post short messages called tweet, interact with each other by sending a reply and share other users' tweets by retweet. Scientists are using hashtags such as #chemtwitter and #ScienceTwitter to create communities on Twitter for networking and information exchange . Twitter activities about a research paper are considered to be one of the altmetrics , which is an alternative to citation counting for measuring scientific impact. Some studies reported that the number of tweets on a research article is positively correlated to the article citations . Attracting attention on Twitter is getting more important to promote scientific works. |
60c75769bb8c1a04ad3dc828 | 1 | A Twitter bot is an automated program on Twitter controlled through Twitter API. Bots are common on Twitter, and they usually deliver tweets like news and blog updates . Twitter bots are also used for scientific purposes, such as posting updates of journal publications or preprints. While most bots automatically post tweets, they can also respond to replies from other users. We can construct an interactive application using Twitter API. In this paper, we report case studies of cheminformatics tools demonstrated on Twitter. |
60c75769bb8c1a04ad3dc828 | 2 | In this section, we introduce a Twitter bot @retrosynthchan [7], which conducts retrosynthetic analysis by AiZynthFinder . The bot responds to a request given as a SMILES string and returns the results of retrosynthetic analysis for the given molecule. Users are expected to send a reply containing only a SMILES string right after the bot's screen name (e.g., @retrosynthchan c1ccccc1Br). |
60c75769bb8c1a04ad3dc828 | 3 | The simplest version of a cheminformatics tool bot has been operating on @souyakuchan account [10] since 2017 in the form of a bot that draws a 2D structural formula image for replies containing a SMILES or an IUPAC name . The retrosynthesis bot was implemented in December 2020 for the blog event called Drug Discovery (dry) Advent Calendar 2020 hosted by @souyakuchan. |
60c75769bb8c1a04ad3dc828 | 4 | The workflow of the bot program is as follows: (1) retrieve a reply to the bot via Twitter API, (2) check whether the given string is valid as SMILES using RDKit and send a request confirmation, (3) run retrosynthesis analysis by AiZynthFinder , (4) send the retrosynthesis result images via Twitter API. The details of each step will be discussed. |
60c75769bb8c1a04ad3dc828 | 5 | Retrieve reply A reply in Twitter is a message toward a specific account, and it usually starts with the screen name of the receiver (e.g., @jack How are you today?). At the time of this writing, the Twitter API provides a method to retrieve tweets that contain a specific keyword in real-time (POST statuses/filter ). The bot receives a reply to the bot by retrieving tweets that contain the bot's screen name with this API. A Python library Tweepy is used to handle Twitter API. |
60c75769bb8c1a04ad3dc828 | 6 | Confirm request After receiving a reply, the string is separated into parts by a space. The second part is assumed to be a SMILES string since the first part is the bot's screen name. The SMILES string is processed by RDKit to check the validity. The bot returns the input molecule's chemical structure if the input is valid, otherwise, send back an error message to the sender and finish. The confirmation is sent as a reply to the users by a Twitter API POST statuses/update using Tweepy. |
60c75769bb8c1a04ad3dc828 | 7 | Run retrosynthetic analysis Retrosynthetic analysis of the molecule represented by the given SMILES is conducted by AiZynthFinder , open-source software for retrosynthetic planning. The input molecule is broken down into purchasable precursors with the algorithm based on a Monte Carlo tree search guided by a neural network. The synthesis route images are generated for individual routes. |
60c75769bb8c1a04ad3dc828 | 8 | Send synthetic pathway images The generated synthetic route images are sent to the user as a reply with images. Twitter allows to attach only up to four images in one post, so three images are connected into one image using Pillow to send a maximum of twelve synthesis routes. The generated images are sent back to the users with the Twitter API using Tweepy. |
60c75769bb8c1a04ad3dc828 | 9 | The bot attracted attention from Twitter users interested in chemistry, and it gained more than one hundred followers in the first two weeks. The followers include chemists in academia, researchers in the drug industry, undergraduate or graduate chemistry students, etc. The Twitter bot helped the retrosynthesis software outreach to a broader range of people. |
60c75769bb8c1a04ad3dc828 | 10 | Next, we introduce a 3D molecule viewer which can be played on a Twitter timeline inlinely via our web server chemical.space . This feature is based on the Twitter Player Card system . The Player Card system is used as a video/audio player in the Twitter timeline, wherein the 3D molecule viewer works as interactive video content. This feature is currently available on web browsers, while the Twitter mobile application can not load Player Card directly in the timeline due to the limitation of Twitter's specifications. Users can view any Protein Data Bank (PDB) structures interactively without leaving their timeline on the browser. To embed the viewer in a tweet, users are expected to post a tweet containing a PDB ID string right after the bot's screen name with a #pdb hashtag (e.g., @souyakuchan 1ema #pdb). |
60c75769bb8c1a04ad3dc828 | 11 | As a 3D molecule viewer, we adopted LiteMol , which is a JavaScript application written in TypeScript. The way of receiving requests is the same as in Case 1. After receiving the PDB ID via users' tweets, the bot makes a reply in which the URL of our hosting server of LiteMol is indicated. Also, to provide Twitter Player Card, our viewer web page serves meta information in HTML header named twitter:card. |
60c75769bb8c1a04ad3dc828 | 12 | A screenshot of the viewer played on a Twitter timeline is shown in Figure . After receiving a PDB code by a mention tweet, the bot immediately replies with an URL in which the 3D viewer Player Card can be expanded. Users can view the 3D structure interactively as it is in the Twitter timeline. |
60c75769bb8c1a04ad3dc828 | 13 | The 2D chemical structural formula editor integrated with tweet posting function (available at chemical.space/editor) is one of the easiest ways to share one's own idea of chemical structures on Twitter. Users can share their manually edited structural formula as a SMILES or Chemical Markup Language (CML) format. By using CML, users can include the information of coordinate and rotation of each object. This is a better way to share complex structures, whereas SMILES can only represent the molecular graph information, which has the advantage of the smallest data size. This editor has one more useful feature, in which users can send their manually drawn chemical structure to @retrosynthchan bot (shown in Case Study 1) by just clicking the RETROSYNTHESIZE button. |
60c75769bb8c1a04ad3dc828 | 14 | Among the existing well-designed traditional browser-based 2D molecule editors such as JSME and MarvinSketch , we chose Kekule.js for its ease of integration as a web application and its sufficient functionality. Its JavaScript-based editor can input and output SMILES strings and CML strings of manually drawn structural formulas. While SMILES only stores simple characters to express atoms, bonds, rings, and their orders, CML has coordinate and rotation information of each atom and bond. Furthermore, CML contains many XML markup tags in addition to that information. Meanwhile, CML strings become too long to tweet because one tweet accepts only 280 characters. However, URL strings are not included in this limitation. To embed CML strings into an URL, chemical.space/editor compresses CML strings by gzip using pako library, then encodes it into Base64url using brianloveswords/base64url . |
60c75769bb8c1a04ad3dc828 | 15 | The 2D molecule editor hosted on chemical.space/editor is shown in Figure . Users can utilize it to draw structural formulas from scratch in their browser and post them to Twitter in SMILES or CML format using the button at the bottom of the editor. We have also prepared a button that allows users to submit their structural formula to the retrosynthetic analysis bot introduced in Case Study 1. Structural formulas posted in CML format can be re-edited by opening the URL in the tweet, which is useful for open discussion on compounds on the SNS. |
60c75769bb8c1a04ad3dc828 | 16 | As presented in these case studies, it is technically possible to make any cheminformatics tool accessible and noticeable from social networking services in the form of interfacing with Twitter bots. However, to make effective impressions on the scientific community and stimulate discussion, it is necessary to use appropriate hashtags that are widely prevailing and be tweeted by accounts with a large number of followers and strong influence. It would be helpful for scientists to follow accounts of mutual interest regularly and grow their follow/follower count appropriately. Tweets with high-impact and sensational content are likely to generate more impressions, but the information described in the tweet must have a certain level of accuracy. As with the sanitization of structural formulas , it is also beneficial for peers of scientists to ensure each other's scientific soundness on the SNS timeline. An attempt to socially share ideas for compounds has also emerged very recently in the fight against the pandemic coronaviral disease COVID-19 (e.g., the COVID Moonshot project ), which is an example of the trend toward open science in drug discovery. Looking ahead to applications in drug discovery, we also need widgets in which users can interactively view and discuss the interaction and activity of compounds with target proteins, which is a future challenge. As a semi-permanently maintained database infrastructure, Twitter has the potential to store an unlimited amount of chemical information. |
60c75769bb8c1a04ad3dc828 | 17 | We presented three examples of SNS integrated cheminformatics tools in this study. Twitter integration can offer a convenient interface to run cheminformatics software without a laborious environment setup, and users can easily share the result by tweet and retweet. Providing a demonstration on social media will be a new approach to present one's research and stimulate discussion in a more accessible manner. If you would like to add your own tool to our bot functions, please contact the authors for support. |
61ba768b7f367e19f4550e22 | 0 | A class of emergent phenomenon , known as cooperativity, grants the bypassing of large energy barriers in a wide variety of systems, ranging from spin flipping in magnetic materials to protein folding . In solid state phase transitions, cooperative mechanisms occur by a concerted displacement of molecules with each molecule retaining its nearest neighbors. This results in ultrafast kinetics and lower energy barriers, relative to typical nucleation and growth type mechanisms, which occur molecule by molecule. The displacive nature of cooperativity, well documented in inorganic martensitic transitions, results in unique behaviors, such as reversible shape changes in the crystal leading to thermosalient motion and shape memory . A key distinction from nucleation and growth rises from interactions with defects within the crystal, which results in avalanche behavior due to pinning of the phase boundary . While cooperative behavior has been well documented in metal alloy based martensitic transitions, it has rarely been reported in molecular crystals. As such, the fundamental origin of cooperativity in organic crystals is not well understood and the molecular design inducing this behavior is still unknown. |
61ba768b7f367e19f4550e22 | 1 | Recently, cooperative transitions have been observed in key organic semiconductor molecules such as 6,13-bis(triisopropylsilylethnyl) pentacene (TIPS-P), among other p-type semiconductors . Cooperativity in TIPS-P led to fast switching of the electronics coupled with large recoverable crystal shape change , resulting in super-and ferro-elastic mechanical effects . In the case of TIPS-P, the rotation of the TIPS groups played a key role in the observed cooperative phenomena . Besides dynamic rotation, other systems have shown the importance of molecular tilting and order-disorder mechanisms to resolve cooperative behaviors . The switching of electronic properties coupled with the rapid mechanical effects of cooperative phase transitions presents a wide design space for novel organic electronics such as thermally activated actuators and highly flexible devices . |
61ba768b7f367e19f4550e22 | 2 | However, studies involving structural transitions in n-type counterparts (necessary for full logic design) have severely lagged behind. N-type semiconductors require relatively low lying LUMO levels for efficient electron transport, which are difficult to stabilize under ambient conditions . One route to design stable n-type molecules involves forming a quinoidal structure using strong electron withdrawing groups such as cyano groups and attaching long alkyl chains to the core. The cyano groups break the aromaticity of the conjugated core and planarize the molecule by stabilizing the quinoidal resonance structure that exhibits double bonds between the rings. On the other hand, alkyl chains are widely used to impart solution processability and helps prevent reactions with water and oxygen in the solid state . At the same time, these quinoidal cores stabilize an exotic biradical ground state that exists in equilibrium with the quinoidal form . The presence of biradicals results in interesting spin-spin interactions between the conjugated cores and, in extreme cases, results in formation of "pancake" bonding . These unique interactions are also tunable through slight changes in environment (concentration, temperature etc.). Along with intermolecular interactions, biradical formation appears to have self-doping effects capable of tuning the charge carrier densities and the resulting electronic properties . While both the quinoidal core and alkyl chains are key to engineering n-type semiconductor molecules, the effect on structural transitions is still unexplored. |
61ba768b7f367e19f4550e22 | 3 | In this work, we report an intriguing coexistence of both cooperative and nucleation and growth type mechanisms during polymorphic transitions in single crystals of 2DQTT-o-B, one of the highest performing n-type organic semiconductors to date . The I-II phase transition, which exhibits cooperativity, is accompanied by a significant conformational change of the alkyl side chains, showing the importance of side chain flexibility in accessing cooperative phenomena. In stark contrast, the II-III transition shows a nucleation and growth mechanism driven by an increase in core interactions based on biradical formation in combination with melting of the alkyl side chains. This is the first time that both cooperative and noncooperative transitions are reported in a single system. Further, the phenomenon of biradical interactions directly inducing a polymorphic phase transition has not been reported before. Finally, we demonstrate the use of cooperative behavior through a thermally actuated switching device, a functionality unavailable to a nucleation and growth type mechanisms. |
61ba768b7f367e19f4550e22 | 4 | Polymorph Transition Behaviors. Crystals of 2DQTT-o-B (Figure ) were either grown via drop casting from a 1:1 para-dichlorobenzene and decane mixture or slow evaporation from a 1:1 dichloromethane and ethyl acetate mixture to form both small (50-300µm) and large (1-3mm) crystals, respectively (Methods Section). Single crystal X-ray diffraction showed crystals of 2DQTT-o-B packed in a C 2/c unit cell with 1-D π-stacks which was designated polymorph I . Based on the BFDH morphology (Figure ), we observe the π-π stacking occurs along the long axis of the crystal (Figure ). These π-stacks then pack into layers separated by the alkyl side chains (Figure ), which results in the thin needle like crystals observed in polarized optical microscopy (POM). We set to investigate two thermally induced structural transitions in single crystals of 2DQTT-o-B using polarized optical microscopy, where we heated and cooled crystals at a constant 5 °C min -1 . We observed two reversible phase transitions designated the I-II transition at 164 °C and II-III at 223 °C. The phase transitions were accompanied by modulation of the brightness and color, reflecting changes in the refractive index of the material (Figure ,e, Movies 1-2,3-4). We observed distinct transition behavior suggesting a cooperative transition mechanism for the I-II transition as opposed to a nucleation and growth mechanism during the II-III transition. Below we discuss evidence from in situ POM in detail and this inference is further supported by in situ GIXD, Raman spectroscopy and EPR spectroscopy discussed later. |
61ba768b7f367e19f4550e22 | 5 | Using python-based image analysis, we were able to select individual crystals and track the progression of the phase transition by calculating an average intensity for each frame. The I-II transition proceeded remarkably fast upon heating (Figure , Movie S1,2). Across many crystals, the heating transition typically occurred within one frame, less than 0.1 second, providing a lower bound for the propagation speed of at least 2000 µm s -1 , implying a cooperative-type mechanism. While difficult to capture under normal circumstances due to the speed, this rapid transition is accompanied by a well-defined phase front propagating through the crystal, distinct from typical diffuse phase boundaries under nucleation and growth mechanisms. Initiation of such phase front typically occurred at the crystal tips, or at cracks within the crystal. Even when the transition initiated at both tips simultaneously, the resulting crystal was a single domain after transition, suggesting a close orientational relationship between the two phases. This is consistent with our earlier work by Chung and Diao et. al. which showed defects facilitated phase boundary initiation in similar cooperative transitions of ditBu-BTBT followed by cooperative propagation. |
61ba768b7f367e19f4550e22 | 6 | During cooling the sharp phase boundary becomes much more evident due to avalanche behavior, where the phase boundary became pinned during transition (Figure , Movie S2). These avalanches follow behavior seen in inorganic martensitic transitions, where defects in the lattice, such as dislocations, create a local free energy minimum and corresponding energy barrier . The resulting rough free energy landscape causes the boundary to move in a series of jerks and requires thermal fluctuations to jump over the defect barriers. As mentioned, the avalanches were found during cooling, which is clearly observed as plateaus in the crystal intensity during the temperature changes (Figure ). This suggests the initial heating transition may be resulting in an increasing number of defect sites within the crystal that cause avalanches in subsequent cycles. However, despite taking several seconds, up to minutes, to become unpinned, this typically does not change the speed of the transition as each avalanche still occurs within less than one frame of the video (Figure ). |
61ba768b7f367e19f4550e22 | 7 | Moreover, the direction of the phase boundary is only observable during these pinning events. In these cases, we observe two main crystallographic directions along which the boundary forms: the (010) and (025) planes both of which are connected to the π-stacking direction. The (010) direction lies perpendicular to the 1-D π-stacks, suggesting the cooperative behavior is transmitted down the π-stacks, similar to a domino effect (Figure ). On the other hand, the (025) plane parallels the conjugated core of the molecule and was indexed to the π-π stacking peak in grazing incidence X-ray diffraction . The crosshatch structure of the π-stacking results in two mirrored directions within the crystal, following the two-phase boundary directions (Figure ). This would suggest a cooperative behavior is transmitted along the 1-D π-stack and is inhibited by dislocations present in those stacks. |
61ba768b7f367e19f4550e22 | 8 | In contrast, the II-III phase transition (Figure , Movies S3,4) showed nucleation at several points within the crystal and transformed over several minutes, with polymorph III increasing in brightness under polarized microscopy. The II-III transition exhibited no clear phase boundary, instead we observed a diffuse spreading of the intensely bright polymorph III. The most common nucleation points are near the edges of the crystal, though cracks and other defects will act as nucleation spots (Figure ) as well. By tracking the intensity change over temperature, we observe a quite smooth change, with no avalanche like behavior (Figure ). Upon cooling, the reverse transition began at a temperature below that of the I-II transition, exhibiting a hysteresis of 100 °C, an order of magnitude greater than the I-II transition which showed a hysteresis of only 17 ± 5 °C, confirming a much larger energy barrier for nucleation and growth mechanism. This matches with observations from DSC using powder samples, showing a hysteresis for the I-II and II-III transitions of 6°C and 75 °C, respectively . In single crystals, the hysteresis for the II-III transition varied substantially and, in some cases, the III-I transition did not occur during constant cooling, resulting in the kinetic trapping of polymorph III at room temperature. This suggested a nucleation and growth behavior, shown in Figure , where nucleation occurred at edges or defects and diffusively spread through the crystal. Thermosalient behavior. Upon undergoing the I-II transition, we found the crystal decreased in length by 3.6 ± 1.3% on average (Figure , Table ). This change in length was matched quite closely with the 2.9% decrease in the b-direction of the unit cells obtained through fitting the thin film GIXD diffraction patterns (Figure ), as we previously reported . This shape change led to cracking of pinned small crystals of ~100 µm in size (Figure , Movie S5), which occurs due to the buildup of strain at the phase front caused by the change in shape coupled with crystal-substrate interactions that act to prevent the shape change. In an extreme case, we observed a crystal embedded in a surrounding film that formed during the drying process, which cracked upon transition. This case also resulted in the formation of "bright bands" along the phase boundary, visible in Figure , which would be consistent with strain induced birefringence we expect from a crystal attempting the I-II transition while trapped in a solid film. On the other hand, for unpinned large crystals (1-3mm), we observed a pronounced thermosalient behavior during the I-II transition (Figure , Movie S6). While the crystal will bend during the I-II transition, reversing the transition will not recover original shape, implying a plastic deformation. This may be a result of strain buildup at the phase boundary causing the π-stacks within the crystal to irreversibly slip along the greasy alkyl chain layers. |
61ba768b7f367e19f4550e22 | 9 | In the II-III transition, however, we observed a slight expansion to the crystal without thermosalient behavior (Figure ). The change did not conform to the unit cell changes, as polymorph III exhibits a vastly different symmetry and unit cell relative to polymorph II (Figure ) and results from thermal expansion during heating. This reconstructive transition also leads to the formation of multiple grain boundaries within the crystal, breaking single crystallinity (Figure ). Formation of these domains mitigate strain build up preventing any thermosalient motion from occurring due to the crystal expansion. These observations are consistent with the understanding that the nucleation and growth mechanism does not incur shape change owing to a single-to-polycrystalline transition . This stands in contrast to the cooperative martensitic transition that preserves single crystallinity and thus exhibits shape reversibility corresponding to the unit cell change. |
61ba768b7f367e19f4550e22 | 10 | Uncovering the origins of the transition behaviors requires understanding the molecular and structural modifications during the phase transitions. To that end, we performed in situ grazing incidence X-ray diffraction (GIXD) during thermally triggered polymorph transitions. A GIXD video of the I-II transition was obtained by continuous heating at 5 °C min -1 and capturing the diffraction pattern every 5 seconds (Movie S7). The unit cells for polymorphs I and II were extracted from the GIXD patterns via a regression fitting the diffraction pattern as discussed in previous work . This fitting for polymorph I also matched the single crystal unit cell obtained at room temperature . During the I-II transition, we observed minor changes to the unit cell, showing peak movements in the q z direction while keeping the Bragg rods in place and maintaining the monoclinic symmetry (Figure ). The largest extent of peak shift in the q z position occurred right before the I-II polymorph transition, similar to pretransition behavior observed by Panda and Naumov et. al. (Figure ). This pretransition behavior is typical of pretransition regions of critical phenomena , such as in the lattice fluctuations necessary to drive the avalanche behavior . Comparing the fitted unit cells for polymorphs I and II (Figure ), there is a significant increase in the β-angle during the I-II transition, consistent with the observed out of plane shift. By comparing single crystal X-ray diffraction with GIXD for polymorph I, we showed this angle reflects the molecular tilt with respect to the substrate, measured to be 66° (Figure ), commensurate with the complement of the β-angle, measured to be 67°3 . During the I-II transition, the β-angle changed from 113° to nearly 90°, suggesting the molecules standing straight up from a more tilted position, like reverse dominos. |
61ba768b7f367e19f4550e22 | 11 | To elucidate the driving force behind this change in tilt, we turned to in situ Raman spectroscopy to provide insight into the molecular changes occurring in the I-II transition (Figure ). Raman has shown to be a powerful tool for tracking polymorphic transitions and understanding the molecular changes that occur as a result of these transitions . Under Raman spectroscopy crystals exhibit both low frequency peaks related to the intermolecular phonon vibrations, sensitive to the crystal structure (< 500 cm -1 ), as well as intramolecular vibrations related to the conformation and chemical environment of the molecule (900-1800 cm -1 ). In the phonon region, we observe two key peaks which are associated with lattice vibrational modes, indicated with black arrows in Figure . We observe a ratio change between these two peaks, as the lowest peak increases in intensity during the I-II transition (Figure ). This is a small effect, indicating modest changes to the lattice packing consistent with our observation of changing molecular tilt from GIXD. Phonon intensity modulation, as opposed to peak shifts or splitting has been shown to be typical in well studied martensitic type cooperative transitions . On the other hand, in the intramolecular vibration region (900-1800 cm -1 ), we observed significant changes to the intensity of the peak at 1409 cm -1 relative to the most intense peak at 1387 cm -1 indicated by the red and blue arrows in Figure , respectively and illustrated in Figure . Based on a combination of DFT calculations (Figure ) and literature, we assigned the peak at 1387 cm -1 to C=C stretching along the backbone, corresponding to the effective conjugation coordinate (ECC) mode for the quinoidal form which we denote as ν(C=C) ECC,Q (Figure , Movie S8,9); the peak at 1522 cm -1 was assigned to other C=C stretching modes in the backbone (Figure , Movie s10); the peak at 1772 cm -1 was assigned to C=O stretching (Figure , Movie S11). While the peak at 1409 cm -1 may correspond to core stretching (Figure , Movie S12), this was assigned to CH 2 deformation in the alkyl chains denoted as δ(CH 2 ) based on wellestablished literature on alkyl chain vibrations. Along with these peaks, we also tracked the peaks at 1025 and 1063 cm -1 which were assigned to C-C stretching of the trans ν(C-C) T and gauche ν(C-C) G isomers in the alkyl chains. The alkyl chain peak assignments (δ(CH 2 ), ν(C-C) T , ν(C-C) G ) were confirmed based on Raman spectroscopy measured on a similar molecule with shorter side chains (Figure ). A complete explanation for these assignments can be found in the supplementary information. |
61ba768b7f367e19f4550e22 | 12 | To investigate the changes in these vibrational modes during the phase transitions, peaks were fit using OriginPro in both the 1000-1100 cm -1 region and 1350-1600 cm -1 region (Figure ). Peak line shape was selected as either Gaussian or Lorentzian based on whether the vibrational mode was associated with the quinoidal core or alkyl chains, respectively. Normally, the line shape in solid crystals is expected to follow a Gaussian curve; however peaks associated with the alkyl chains were fit as Lorentzian due to the disorder, causing more liquid like behavior . In the 1000-1600 cm -1 range, which captures the core and alkyl chain vibrations, we note a significant intensity reduction and redshift of the δ(CH 2 ) peak at 1409 cm - 1 during the I-II transition. We plotted the intensity ratio with respect to ν(C=C) ECC,Q and the peak position to show this sudden drop in intensity and redshift of 7 cm -1 in the δ(CH 2 ) at the I-II transition temperature (Figure ). This trend was repeated across 3 crystals to ensure this change is representative of the I-II transition (Figure ), though the exact transition temperature varied by 5 °C for each crystal due to avalanche behavior. The redshift is indicative of reduced steric hinderance or an increase in the attractive forces between alkyl chains. The intensity decrease, on the other hand, implies alterations to either the orientation or distribution of vibrational states in the alkyl chains. Taken together, these changes show a substantial reorientation of the alkyl chains occurs during the I-II transition. For the II-III polymorph transition, we see large changes in the unit cell, as polymorph III appears to form a likely hexagonal crystal system (Figure ), obtained from the GIXD pattern as discussed in previous work . This is consistent with other previous observations which have shown crystals have a propensity to increase in symmetry at higher temperatures . GIXD video of the II-III transition (Figure , Movie S13) showed the significant reduction in observable peaks, as a result of increased symmetry of the hexagonal lattice as well as reduced long-range order as higher q peaks are no longer measurable (to be discussed later). Despite the reduced diffraction of other Bragg rods at higher q values, the π-stacking peak remains intense and well defined in polymorph III, in line with a continued dominant π-stacking motif. The reconstructive transition is further highlighted by observing the complete disappearance of the polymorph II diffraction peaks at the expense of polymorph III peaks (Figure ), with no shift in position prior to transition, which is consistent with a nucleation and growth type mechanism. |
61ba768b7f367e19f4550e22 | 13 | Upon investigation of the II-III transition mechanism via Raman spectroscopy, we observed something quite unexpected: we see the marked weakening of the core stretching peak ν(C=C) ECC,Q at 1387 cm -1 , while 2 new peaks appear at higher wavenumbers, 1442 and 1497 cm -1 (Figure ). This indicated a modification to the conjugated core itself. We initially suspected this may be a degradation of the molecule at such high temperatures. However, the temperature at which the peaks appeared were highly correlated with the II-III transition and, moreover, this peak formation was found to be reversible (Figure ). Along with this, we also observed a change in the intensity ratio between the peaks located at 1025 and 1063 cm -1 , previously assigned to the trans (ν(C-C) T ) and gauche (ν(C-C) G ) isomers, respectively. Based on these observations, we hypothesize a transformation from a quinoidal structure to aromatic structure accompanied by decreased order of the alkyl side chains; such quinoidal to aromatic transitions have been observed in similar systems . |
61ba768b7f367e19f4550e22 | 14 | Previous work on similar quinoidal systems has revealed the presence of a singlet biradical ground state which affects the molecular structure and, by extension, the intermolecular interactions. Moreover, previous work on these biradical forms showed a sensitivity to temperature offering the intriguing possibility that the II-III transition occurred alongside a quinoidal to aromatic transition . The key change in molecular structure between a quinoidal and aromatic form is the alternation between the single and double bonds along the conjugated backbone, which is described by the bond length alternation (BLA) parameter, the average difference between consecutive single and double C-C bonds. A BLA < 0 defines the quinoidal structure whereas a BLA > 0 defines the aromatic form. Because of this change in bond length, Raman spectroscopy is quite sensitive to these biradical forms, due to the difference in vibrational frequency of the ECC mode in the quinoidal and aromatic systems . We simulated the Raman spectrum for this scenario by forcing the BLA into the aromatic form via substituting the cyano groups for methyl groups on the ends of the molecules and compared this to the spectra obtained from polymorph III (Figure ). This allowed us to assign the two new peaks at 1442 and 1497 cm -1 to the ECC of the aromatic form ν(C=C) ECC,A (Figure , Movie S14) and asymmetric stretching modes along the conjugated backbone (ν(C=C) asym ) (Figure , Movie S15) respectively. The complete explanation for these assignments is found in the supplementary information. |
61ba768b7f367e19f4550e22 | 15 | Based on this assignment, we plotted the intensity ratio of the 1442 cm -1 to the 1387 cm -1 peaks, representing the ratio of aromatic to quinoidal forms, as we increased temperature (Figure , top). We observe a significant increase in the biradical form starting at the II-III transition. Moreover, the intensity ratio of trans to gauche C-C stretching also changed during the transition (Figure , bottom). As polymorph III formed, this ratio decreases drastically, suggesting significant changes in the alkyl chain dynamics. In previous works, the intensity ratio of these two conformers provide an indication of disorder in the alkyl side chains . As more gauche isomers form in the system, the alkyl chains become bent and twisted within the occupied volume. A complete melting of the alkyl side chains is represented by a ratio of 1, as the chains become free to bend in any direction. In the 2DQTT-o-B system, the intensity ratio of trans to gauche decreases from 8 to 1 across the II-to-III transition. These results strongly suggest that the formation of biradical species and alkyl chain melting are the molecular origins of the II-III phase transition. To confirm the formation of biradicals, we also simulated and compared the absorption spectroscopy as well as measured the spin states in the crystals through in situ EPR spectroscopy to directly observe the formation of these biradical states. UV-Vis was simulated for both the quinoidal and aromatic forms via setting the BLA value for each of the systems (see methods). The simulated UV-Vis spectra for the quinoidal molecule showed good agreement with the experimental spectra (Figure ) though we expect the redshift and vibronic fine features resulting from crystalline packing not to be captured in the simulation. For the aromatic molecule, the simulation showed a significantly red shifted and broadened peak compared to the quinoidal form, similar to the redshift observed in the experimental spectra (Figure ). Based on Raman, we expect a mixture of quinoidal and aromatic forms, which may account for a smaller extent of red shift observed than predicted by simulation (Figure ). Nonetheless, the good qualitative match between simulated and experimental spectra suggests that the biradical aromatic form provides a good model for describing the molecular system in polymorph III. Finally, as expected from our hypothesis, in situ EPR showed an increasing presence of spins in our system as we increased temperature (Figure ). EPR directly measures the increasing unpaired spins associated with the biradical state (Figure ) and indeed, we see a dramatic increase in the concentration of spins within the material (40x increase) upon reaching the II-III transition (Figure ). This confirms that the II-III transition not only exhibits a large structural change, but also that a key part of this transition involves accommodating the abruptly increased concentration of biradical species through a reconstructive structural transition (Figure ). ). Much like stacks of dominos tilting all at once, molecules along the b-axis of the crystal constituting the 1-D π stacks stand up during the I-II transition. In this case, the tipping of the molecule is caused by a conformation change in the alkyl side chains, which results in the molecule standing upright during heating and allowing the molecule to slip back into a tilted position during cooling. We suggest engineering of these alkyl side chains may be crucial in facilitating the I-II cooperative behavior. While recently rotation of bulky side chains have been shown to be critical for expressing cooperative behavior in molecular crystals , cooperative polymorph transition has rarely been studied in molecules with alkyl side chain motifs -a dominant side chain design in organic crystals . Until now, their role in facilitating cooperative transitions has not been well understood. |
61ba768b7f367e19f4550e22 | 16 | The II-III phase transition, however, was shown to be driven by the formation of biradical species and is facilitated by increased mobility of the alkyl side chains during a melting process. At room temperature, the concentration of biradical cores is quite low, meaning the potential for spin-spin interaction is quite low. However, as the temperature increases through the II-III transition, the biradical concentration abruptly increases conducive to forming new spin-spin interactions . Typically, in molecules where the biradical ground state is dominant, the major packing motifs are either face-on dimer pairs when unhindered or ladder-like stacking when steric hinderance prevents the former. In both cases, the high spin centers (where the radical is most localized) will line up and, in some circumstances, form a pseudo σ-bond . |
61ba768b7f367e19f4550e22 | 17 | One possibility of molecular packing change during II-III transition is shown in Figure , where molecules form dimers with the nearest neighbor in the original polymorph II structure. Since the crystal packs in these 1-D π-stacks with dimer-like pairs already, this would be a natural progression. While the dimer formation may be plausible, we cannot say with certainty how those dimers then pack to form polymorph III. Regardless of the new core packing motif, to facilitate the II-III also necessitates alkyl chain melting, indicated by increased gauche conformers. Moreover, this side chain melting is consistent with the kinetic trapping observed (Figure and Movie S4). As we observe upon cooling to 37 °C, the alkyl chains return to a frozen state however there appears to be residual biradicals not yet quenched by cooling, owing to a residual lifetime of the biradical state (Figure ). As a result, polymorph III is trapped and would not return to polymorph I due to freezing the alkyl chains before the conjugated cores rearrange back into polymorph I that may require quenching of biradical species. However, without the full structure of polymorph III, we are left only to postulate possible molecular packings. Nevertheless, we can suggest the nucleation and growth polymorph transition is the result of increased core-core interactions triggered by the biradical formation, and the large packing changes are necessarily facilitated by increased molecular mobility from the melted alkyl side chains. Electronic and device properties. We worked to demonstrate the control and design of organic electronics utilizing these transition mechanisms. We fabricated single crystal devices to measure the twopoint-probe conductivity of each polymorph (Figure ). The crystals were pre-bent fixed at two ends by PEDOT:PSS electrodes. When heated to trigger I-II transition, the crystal would straighten from the prebent state due to shape change shown in Figure . We observed a 6-fold decrease in conductivity at the I-II transition, with good recoverability during cooling albeit with a hysteresis. Moreover, we see a moderate increase in conductivity upon polymorph II-III transition. We suspect this recovery of conductivity may be a result of charge carrier doping through the presence of biradicals, similar to Wudl et. al., which showed a self-doping effect . This self-doping process may compete with the reduction in charge transfer pathways due to structural transitions. |
61ba768b7f367e19f4550e22 | 18 | We then were able to harness the shape change discovered in the I-II transition for reliable thermal actuation (Figure ). For the device, we attached a crystal at both ends using PEDOT:PSS and upon heating, the crystal became dislodged on one side due to the cooperative shape change. This allowed for switching on and off the device by cycling between polymorphs I and II, exhibiting an on/off ratio of 500 and effectively reaching the noise floor for device measurement in the off state. Because the crystal became detached in the polymorph II state, we observed no conductivity and cooling back to polymorph I reforms the contact between the crystal and the PEDOT:PSS, turning the device back on. This provided quite reliable cycling for several cycles until out of plane bending prevented stable contact between the PEDOT:PSS contacts. |
61ba768b7f367e19f4550e22 | 19 | We observed both cooperative and nucleation and growth behavior within the 2DQTT-o-B system, allowing for direct comparison of molecular origins of these transitions in a single material. We discovered the I-II transition exhibited cooperative behavior, resulting in a rapid, shape changing transition dominated by avalanches as defects were introduced. The transition was found to be driven by changes in the alkyl side chain confirmation, as revealed by Raman spectroscopy. This was in striking contrast with the slow, smooth II-III transition showing clear nucleation and growth behaviors. Unlike the I-II transition, the II-III transition was driven by changes in spin-spin interactions through biradical formation which is the observation of biradical interactions triggering a polymorphic phase transition. |
61ba768b7f367e19f4550e22 | 20 | We then harnessed cooperative behaviors for the design and application of novel organic electronic switching behavior. Not only were we able to tune the conductivity by orders of magnitude via the structural changes, but we could also use the mechanical effects to sever the connection with one of the PEDOT:PSS contacts. By taking advantage of the mechanical effects of the cooperative shape change, we could switch on and off a single crystal device through rapid temperature actuation. This opens the door for fusing the novel mechanical effects of cooperative transitions with device architecture for new functionality. Ultimately, understanding the origin of these transition mechanisms offers pathways to rationally designing organic semiconductors to access cooperative transitions with these exciting properties. |
61ba768b7f367e19f4550e22 | 21 | Single crystal fabrication. Single crystals of 2DQTT-o-B were fabricated through a dropcasting method. Solutions with concentrations between 10-15 mg mL -1 of 2DQTT-o-B dissolved in a 1:1 mixture of dichlorobenzene and decane were heated to 100 °C. 5-15 µL were dropped onto PTS-treated silicon wafers (SiO 2 /Si) and allowed to dry overnight. For large crystals (SCXRD and Raman experiments), 2DQTTo-B was dissolved in a 1:1 mixture of dichloromethane and ethyl acetate at 1 mg mL -1 in a 30 mL vial (up to about 15 mL of solution was used). The solution was then capped with parafilm and 1-5 holes were created in the film using a needle. The vial was then placed in a glove bag under nitrogen atmosphere for slow evaporation over a few weeks. The nitrogen in the glove bag was refilled every other day to prevent oxygen from reacting with the molecule and ensure saturation of solvent vapors did not occur. |
61ba768b7f367e19f4550e22 | 22 | Polarized optical microscope. Single crystals grown under the dropcasting procedure were placed on a Nikon H550S with a high-speed camera (Infinity 1) and heating stage (Linkam 402). The chamber was sealed with a magnetic lid and O-ring during heating. The temperature ramp was kept at a constant 5 °C min -1 . Videos were recorded at framerates ranging from 1 to 7.5 fps. Video analysis was performed using a python program to obtain an average intensity value of a given crystal at each frame. |
61ba768b7f367e19f4550e22 | 23 | Grazing incidence X-ray Diffraction. GIXD was performed at beamline 8-ID-E of the Advanced Photon Source at Argonne National Laboratory . The data were collected at 10.91 keV on a 2D Pilatus 1M detector. Films of polymorph I were obtained by solution coating from a solution of 2DQTT-o-B dissolved in chloroform and chlorobenzene at 6 mg mL -1 onto SiO 2 treated with trichloro(phenyl)silane (PTS). Solution coating was performed at 85 °C at a blade speed of 0.3 mm s -1 and a blade gap of 100 μm. These films were annealed under nitrogen atmosphere for 30 minutes at 100 °C to convert the films to polymorph I. In situ thermal annealing measurements were conducted in a He environment with the sample on a commercial thermal stage (Linkam HFSX350-GI), with the temperature ramped at 10 °C min - 1 and the exposure taken after equilibration at the target temperature for approximately 5 min. GIXD videos (Movies S7,13) were obtained via taking continuous exposures at a constant heating of 5 °C min -1 . GIXSGUI software was used to correct for detector nonuniformity, beam polarization and to reshape the 2D data into the representation q z vs q r 65 . The incident angle was set at 0.14° right above the critical angle for total reflectance of the organic thin film. Unit cells were extracted based on the discussion in the previous paper by Davies and Diao et. al., 2021. Raman Spectroscopy. A Raman confocal imaging microscope with a 532 nm laser (Laser Quantum Ventus 532 with max power 50 mW) and 50× long working distance objective lens (HORIBA LabRAM HR 3D) equipped with HORIBA Synapse back-illuminated deep-depletion CCD camera was used to collect spectra. Using a 300 g mm -1 grating, we used a scan exposure time of between 20-60 s. An optical density filter of OD = 0.2 was used (OD = log(power transmission factor)), and no beam damage was observed only at the highest temperatures after prolonged periods of laser exposure (>5min). To eliminate this effect, each spectrum was recorded in a new position on the crystal to prevent overexposure of any particular area. For variable-temperature experiments, the samples were collected using a Linkam THMS600 heating stage with a closed chamber. The heating and cooling rate was kept at 10 °C min -1 . Each temperature was equilibrated until the temperature reading stabilized and the Raman laser was refocused to account for substrate thermal expansion (approximately 5 minutes). |
61ba768b7f367e19f4550e22 | 24 | Electron Paramagnetic Resonance Spectroscopy. Crystals of Polymorph I were grown via the slow evaporation method from DCM and EA described in the crystal fabrication above, were dried out and then placed in an EPR tube. The crystal sample was then measured on an EMXplus running the Xenon software with variable temperature (part no. E0000071) for in situ EPR measurements. The temperature was stepped by 25 °C up through the II-III phase transition. The Intensity at each temperature was then doubly integrated and plotted vs temperature. Electronic properties. For single crystal devices, large needle like crystals were grown from a 1 mg mL -1 solution of 1:1 mixture of dichloromethane and ethyl acetate. These crystals were pre-bent and attached to a glass substrate, using conductive PEDOT:PSS. Pre-bending allowed for the devices to be heated through the I-II transition without cracking or breaking the device. The device was measured in air using a Keysight B1500A semiconductor parameter analyzer. The current through the crystal was measured by contacting the PEDOT:PSS with 2 probes and current was obtained as a function of source-drain voltage. |
61ba768b7f367e19f4550e22 | 25 | Actuator devices were fabricated by attaching the crystal with PEDOT:PSS without pre-bending the crystal. During the first cycle of the I-II transition, one end becomes detached from the PEDOT:PSS due to the crystal shape change. This device can then be cycled multiple times as once cooled down, the crystal reconnects with the PEDOT:PSS contact. |
61ba768b7f367e19f4550e22 | 26 | DFT calculations. Ground-state geometries of isolated molecules were optimized at the DFT level using the ωB97X-D functional and def2TZVPP basis set. IR and Raman frequencies were calculated at the same level of theory. For the quinoidal form, the original molecule was used, however for the aromatic form, the cyano-groups were cleaved. This allowed for fully relaxed structures to be used in both simulations while capturing the correct bond length alternation observed in each conjugated core. The UV-Vis absorption spectra were likewise calculated at the same level of theory, using the 10 lowest singlet and triplet states in the TD-DFT calculation. All calculations were performed using Gaussian16 Rev. B. Side chains were removed for computational efficiency. In this case, the quinoidal geometry optimization was allowed to relax fully, while for the aromatic geometry the carbon atoms along the backbone were fixed to enforce the proper aromatic single-double bond alternation pattern (i.e., that opposite the quinoidal bonding pattern) and the result of the structure was allowed to relax. |
63db7b2be136d9bc552935d3 | 0 | Cell-free expression (CFE) is a highly versatile tool used to achieve rapid RNA/protein synthesis through in vitro transcription and translation (IVTT) of natural or synthetic DNA. Avoiding cell-based synthetic biology limitations, such as laborious genetic encoding, gene delivery and slow design-build-test cycles, 2 CFE is ideal for high-throughput drug screening, the study of biological processes, gene circuits, and the purification of proteins otherwise challenging to express (e.g., containing non-natural amino acids). As a result, CFE technologies have recently gained great interest from the biomanufacturing field and industry. Moreover, CFE mixtures can be encapsulated in lipid bilayers to form synthetic cells for applications in drug delivery and studying cellular communication. The ability to control the function of these cell-free systems will be a key step to advance this technology towards more complex gene circuits or in situ formation and release of therapeutics. Control can be achieved with molecular inputs for RNA switches and transcription factor-based biosensors, with the analyte being the trigger for CFE activation. However, these signal molecules can be difficult to apply as, when added at different timepoints, they will change the concentrations of the components and are mostly not applicable for encapsulated systems such as synthetic cells. Furthermore, most of these molecular systems have been optimised for use in living cells and do not show optimal activity in cell-free systems. Light, on the other hand, is an ideal stimulus for controlling cell-free system systems as it can be applied remotely to closed systems in a spatiotemporal manner, has low toxicity, and is bioorthogonal to most biological processes. Furthermore, unlike other remote stimuli, it is possible to use multiple, orthogonal wavelengths of light to control different biological processes in the same system, opening up the prospect of precise, remotecontrol of cell-free systems. |
63db7b2be136d9bc552935d3 | 1 | Gene expression in cell-free systems has previously been controlled with light, by either chemically introducing light-activated photocages into the DNA templates for transcription, or by using light-sensitive proteins. The majority of these systems have leaky 'off-states', where expression still occurs without light, and in the case of light-sensitive proteins require the expression of additional genes, both of which limit their application. We have previously developed an approach for the light-activation of cell-free protein synthesis (CFPS) by attaching photocaged biotin/monovalent streptavidin (mSA) to a T7 promoter sequence to sterically block transcription, prior to illumination . Photocleavable biotin linkers were attached to amino-modified oligonucleotides and used as primers to amplify any gene of interest for control using light. The advantage of this approach is that it is made from commercially available modified nucleic acids and photocleavable biotin, and has a tight 'offstate', with negligible expression without illumination. We have used this light-activated DNA (LA-DNA) to spatiotemporally activate protein synthesis in synthetic tissues, activate communication between synthetic cells and living cells, and, by employing orthogonal photocages, to engineer a dual wavelength cell-free AND-gate. The main disadvantage of our current approach is that it is an irreversible ON switch. Azobenzene-modified DNA promoters have been used as photoreversible switches; however, they are leaky in the 'off-state' and do not degrade the RNA already generated. In this project, we have applied our simple photocleavable biotin/streptavidin approach to develop lightcontrolled CFPS OFF switches from antisense oligonucleotides (ASOs) (Fig. ). ASOs are short DNA sequences that can selectively degrade a target mRNA in the presence of Ribonuclease H (RNase H), a common endonuclease. While light-activated ASOs have previously been generated, they are difficult to synthesise and orthogonally controlled versions have never been realised. The use of ASOs in cell-free systems has been greatly underexplored; however, 'transfection-style' methods have been developed to explore their use in synthetic cells. Here, we attached orthogonal UV (nitrobenzyl) and blue (coumarin) photocaged biotins, and then monovalent streptavidins, to amino-C6-dTs positioned across ASOs to tightly control gene knockdown in cell-free systems using light (Fig. ). We demonstrated the orthogonal degradation of two different mRNAs, depending on the wavelength used, and combined UV-controlled ASOs with our previously generated blue lightactivated DNA templates to precisely remote-control CFPS (Fig. ). |
63db7b2be136d9bc552935d3 | 2 | Initially, we tested three variants of an ASO sequence we had previously identified that targets the mRNA for mVenus (mV), a yellow fluorescent protein. Each variant contained three thymines (dTs) replaced with amino-C6-dTs at different positions. The three amino-ASOs were reacted with a biotinylated 2-nitrobenzyl N-hydroxysuccinimide (NHS)-ester photocleavable group (uvLA-biotin, Fig. ) and purified by HPLC (see Supplementay Information), prior to binding either monovalent streptavidin (mSA) or wild-type streptavidin (tetSA) to sterically block RNase H from binding the hybrid DNA:RNA duplex. These UV light-activatable (uvLA) ASOs were tested against mV-mRNA with or without illumination (Supplementary Fig. ) and analysed by agarose gel electrophoresis. A better 'off-state' (least mRNA degradation) was observed when the modified dTs were distributed throughout the entire sequence in ASO uvLA-V1, rather than concentrated at the terminus. Moreover, when bound to mSA rather than tetSA, the light-sensitive moieties appeared to photocleave more efficiently upon UV irradiation, resulting in a better ON/OFF ratio. Thus, we continued using mSA in the subsequent experiments. |
63db7b2be136d9bc552935d3 | 3 | To produce a better ON/OFF ratio, we proceeded to substitute one further dT with amino-C6-dT, for a total of four modifications (Fig. ). In addition, we screened for a new ASO targeting the same mRNA region, but with a higher potency and thymidines denser and more symmetrically distributed in the sequence (Fig. [mV220 vs mV229] and Supplementary Fig. ). Both the previous and the newly designed sequences containing four amino-C6-dTs (V2 and V3 respectively) were then modified with uvLA-biotin and caged with mSA (Fig. ). As expected, the addition of a fourth amino-dT resulted in a negligible decrease in ASO activity, while a fourth photocleavable streptavidin moiety visibly improved the 'on/off-state' (Fig. ). As modified ASO uvLA-V3 showed the best ON/OFF ratio, this was selected for further experiments. To control in vitro transcription (IVT) a linear DNA template encoding mVenus was transcribed in the presence of no ASO, V3, or uvLA-V3, and RNase H (Supplementary Fig. ). Without illumination, similar levels of mRNA were transcribed when using uvLA-V3 as if no ASO was present. Whereas when IVT took place following illumination, mRNA degradation was observed to similar levels of V3 amino-ASO (representing 100% photocleavage). We then moved to control cell-free protein synthesis with the uvLA-V3 ASO, using a commercial CFPS kit (PURExpress). V3 or uvLA-V3, and the mV DNA template were added to the CFPS system (supplemented with RNase H), and the yield of intact mVenus was measured by fluorescence after 4 hours (Fig. ). UV light irradiation in the absence of an ASO only resulted in a minor, non-significant decrease (p-value = 0.23) in protein synthesis levels, showing little/no UV-damage. The amino-ASO V3 decreased mV synthesis by 89% compared to the no ASO control, with no significant difference following illumination (p-value = 0.265). In the absence of UV light, uvLA-V3 only decreases protein synthesis by ~15% (nonsignificant, p-value = 0.084), whereas when irradiated with UV for 5 minutes, protein synthesis was reduced by ~75% compared to the no ASO control, and 84% of ASO activity was recovered when compared to the amino-ASO V3. These results demonstrate we were able to tightly control gene knockdown of CFPS using our light-activated ASOs. |
63db7b2be136d9bc552935d3 | 4 | We recently reported a blue light-activatable photocaging group to control CFPS, which acts orthogonally to UV light. By combining a blue light-activatable (bLA) mV DNA template with the uvLA-V3 ASO, we envisaged a system where transcription could be selectively turned ON with blue light and translation turned OFF with UV light (Fig. ). We initially tested this in an IVT reaction using the bLA-mV DNA template in the presence of RNase H and uvLA-V3 (Supplementary Fig. ). Following blue light irradiation, we observed an increasing amount of mV-mRNA produced over three hours, as measured on agarose gel electrophoresis, similar to the amount produced from a non-modified DNA template (Supplementary Fig. ). The IVT reactions were then incubated for a further hour with or without UV illumination. When irradiated with UV light the mRNA was almost completely degraded in one hour, due to activation of uvLA-V3. Without UV illumination, no degradation was observed, demonstrating uvLA-V3 was not activated. |
63db7b2be136d9bc552935d3 | 5 | After the successful proof of concept with an IVT system, we moved on to apply this blue ON and UV OFF system in CFPS (Fig. ). We incubated the bLA-mV DNA template in the CFPS system at 37 °C in the presence of uvLA-V3 and RNase H for 4 hours under different illumination conditions and measured intact protein yield by fluorescence (Fig. ). Illumination with only blue light showed an equal protein output compared to the no ASO control (p-value = 0.196), due to uvLA-V3 not activating under blue light. Irradiation with UV light alone did not activate the bLA-mV DNA template and no protein was produced after 4 hours, as only the ASO was activated. UV light was then applied at different timepoints, following blue light-activation of the template, to halt cell-free protein synthesis upon demand by degrading the mRNA already generated. When irradiated with UV at the same time as blue light, prior to incubation, a high degree of gene knockdown (74%) was observed, as any mRNA formed would be quickly degraded by RNase H and the uncaged ASO. When irradiating with UV following blue light-activation and 30 minutes incubation, protein synthesis had already initiated but was halted at 51%, compared to when no ASO was present. This is in line with previous data, showing that CFPS systems produce a substantial amount of mRNA and protein in the first stages of the reaction. Lastly, when irradiated with UV following blue lightactivation and 3 hours incubation, no reduction in protein output was observed (p-value = 0.519), as expected, because mVenus production already reached a plateau. This demonstrated a two wavelength ON and OFF switch for CFPS, with the ability to temporally activate and halt protein synthesis. |
63db7b2be136d9bc552935d3 | 6 | As we had orthogonal UV and blue photocages that could be attached to amino-C6-dT modifications, we next aimed to generate two ASOs that would bind different mRNA and selectively degrade their target with each wavelength (Fig. ). To allow the fluorescent measurement of a different target in CFPS, we aimed to identify a new ASO sequence that could target the mRNA encoding for the red fluorescent protein mCherry (mC), without binding mV-mRNA. Being derived from fluorescent proteins of different organisms (dsRed vs. avGFP), mC has an orthogonal excitation/emission spectrum to mV and a large difference in the DNA sequence. After screening several ASOs to identify a good target point in mC-mRNA, with sequences containing multiple Ts across the length of the ASO (Supplementary Fig. ), a second screening process was carried out to find a sequence with orthogonality to mVenus (Supplementary Fig. ). The same test was also performed with the mVenus ASOs, in which we identified that V2 was orthogonal to mC, whereas V3 showed some crosstalk (Supplementary Fig. ). |
63db7b2be136d9bc552935d3 | 7 | The two ASOs chosen from these screens for orthogonal targeting of mC (C1) and mV (V2), both contained four amino-C6-dT modifications. They were modified with bLA-biotin and uvLA-biotin (Fig. ) respectively, purified by HPLC (see Supplementary Information) and bound to mSA (forming bLA-C1 and uvLA-V2). mV-and mC-mRNA were incubated with the two photocaged ASOs and RNase H, to test light-controlled mRNA degradation (Fig. ). In the absence of irradiation, both mRNAs remained intact. Strikingly, upon illumination with blue light, mC-mRNA was degraded while mV-mRNA stayed intact. Similarly, mV-mRNA was degraded selectively after irradiation with UV light, with no degradation being observed for the mC-mRNA. We then used these orthogonal LA-ASOs to selectively control the cellfree protein synthesis of the two different proteins (Fig. ). The DNA templates of mV and mC, bLA-C1, uvLA-V2, and RNase H were added to the CFPS system, incubated for 4 hours following different patterns of illumination, and the fluorescence of both proteins was measured. Without illumination, both proteins were expressed to similar levels observed in the absence of ASOs (17% increase for mC and 23% decrease for mV, both non-significant). Upon UV irradiation, uvLA-V2 selectively knocked down mV fluorescence by 56%, whereas mC was only reduced by 16% (non-significant, p-value = 0.129). Vice versa, upon blue light irradiation, bLA-C1 selectively knocked down mC fluorescence by 62%, with a 30% reduction observed for mV fluorescence (significant against positive control, p-value = 0.032). Excitingly, this demonstrated we were able to control gene knockdown in CFPS using two orthogonally light-controlled ASOs. |
6797e88a81d2151a02309d46 | 0 | A plethora of lipids exist in living organisms that perform various functions ranging from cell membrane formation, to energy storage, and signaling. To rationalize lipid functions and characterize the dynamic adaption of cellular lipid structures to external stimuli, the field of lipidomics thrives to detect as many lipids as possible and decipher their molecular architecture in one sample. This endeavor, however, is a demanding bioanalytical task as subtle changes in the molecular makeup of lipids often hinder unambiguous annotation of lipid structures and reliable predictions of lipid isomers are complicated by the intertwined nature of enzymatic processes involved in lipid metabolism. For example, cells adapt their phospholipid fatty acid (FA) composition to their needs in a biochemical cascade termed Lands cylce. In this cycle, attachment of FAs to lysolipids occurs with the help of lysophospholipid acyltransferase enzymes. Whereas the involved enzymes in the Lands cycle are mostly known, their specificity and selectivity have not been studied in detailed. Zhao et al. recently showed that upregulation of phosphatidylcholine (PC) 18:1/16:0 in tumor tissue is indeed connected with the increased lysophosphatidyl acyltransferases-1 (LPCAT1) expression compared to surrounding healthy material showcasing the need of structurally resolved lipidomics workflows. For this reason, multiple new methods have been developed that that track lipid structure changes, mainly relying on mass spectrometry (MS). Sensitive detection with direct infusion methods, or shotgun MS, of lipids has been implemented on multiple state-of-the-art mass spectrometric platforms. By electrospray ionization (ESI) detection of hundreds of lipids is routinely feasible by making use of high mass resolution and mass accuracy capabilities of modern MS instruments. To resolve the structural complexity of lipid mixtures, hyphenation of MS methods with separation systems such as liquid chromatography (LC) or ion mobility spectrometry (IMS) can facilitate disentangling complex samples. LC-MS and shotgun MS lipid annotations additionally make use of tandem mass spectrometric methods to reveal lipid class and FA composition. |
6797e88a81d2151a02309d46 | 1 | To gain further insights into lipid structures and reveal FA C=C (DB) positions, DB geometries, and stereospecific numbering (sn) isomers, specialized derivatization, separation, and/or tandem mass spectrometry methods are required. One of the first lipid structure selective methods, termed OzID, was introduced by Blanksby and co-workers utilizing ozone to cleave DB bonds. OzID has been used to investigate DB positions as well as sn-isomers of lipids. Most recently it was used in a large-scale shotgun MS study to investigate sn-isomer abundances in different cell lines. In this study Michael et al. revealed that sn-isomers compositions can differ between cell lines and they identified very long chain FAs attached to phospholipids. Structure selective fragments are also obtained by ultraviolet photodissociation (UVPD), a method pioneered for lipids by Brodbelt and co-workers. By exciting lipid ions with UV light, DB positions, sn-isomers and cyclopropanation sites are identified. But also other methods that use laser light can yield information about lipid structures. The selection of ions attached to lipids can regularly alter fragmentation pathways facilitating formation of fragments that are indicative for DB or methyl branching sites. Lipid fragmentation can also be influenced by introducing new functional groups into lipids by derivatization methods prior to ionization. Whereas a large number of derivatization strategies have been developed, epoxidation and Paternò-Büchi (PB) workflows are most regularly used. All these methodologies can provide some additional information for the overall lipid structure such as DB position, sn-isomers, or FA modifications. However only electron impact excitation of ions from organics has provided direct evidence that E/Z DB isomers of lipids can be distinguished based on fragment ion abundances. To further improve the annotation capabilities for isomers in lipidomics workflows, IMS and LC have been used in combination with these advanced tandem mass spectrometry methods. For example, OzID has been combined with reversed-phase LC (RPLC) for the analysis of the structural diversity of FAs in human plasma. RPLC was also employed to separate PB mixtures and epoxidized lipids . |
6797e88a81d2151a02309d46 | 2 | Here, we report a new epoxidation workflow that contributes to these ongoing efforts to fully characterize lipid structures in lipidomics studies. The epoxidation method relies on the photochemical reaction of unsaturated lipids in a flow reactor to form epoxides with 60 -80 % apparent reaction yield. Whereas the formed epoxides can reveal DB position, we describe the capabilities to also identify sn-isomers based on MS n experiments. In addition to DB position and sn-isomer discrimination capabilities, the reported workflows allow improved separation of epoxidized lipids by RPLC compared to non-reacted lipids as well as the identification of E/Z isomers of DBs by comparing RPLC profiles of non-reacted lipids before and after the epoxidation procedure. To demonstrate the capabilities of the method for shotgun and LC-MS lipidomics workflows, we identified DB positions of PCs, PEs, PSs, DGs, TGs in complex polar lipid extracts and revealed 56 sn-isomers in HeLa and H9c2 cell extracts. |
6797e88a81d2151a02309d46 | 3 | The human cervical HeLa and rat H9c2 myoblast cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, PAN Biotech) with added 10 % fetal bovine serum (Sigma Aldrich) as well as 1% 10,000 U/mL penicillin and streptomycin. The medium of incubated cells was removed and cells were resuspended in Dulbecco's Phosphate Buffered Saline (DPBS, PAN Biotech). Next DPBS was removed, and adherent cells were detached with trypsin/EDTA (PAN Biotech) by incubating for 2 min at 37°C and 5% CO2. The cell suspension was diluted with DMEM and spun down (300 g, 5 min). Cell pellets were resuspended in DPBS counted in a hemocytometer to obtain 10 6 cell aliquots and were transferred into 2 mL homogenization tubes. After removing DPBS, cell pellets snap frozen in liquid nitrogen and kept at -80 °C till use. |
6797e88a81d2151a02309d46 | 4 | For preparing working solutions for epoxidation, lipid standards (FAs, PCs, PE, PG, PS, PI, PA) were diluted in ACN to a final concentration of 0.2 mg/mL. Benzil was dissolved in ACN:water (1:1, v/v) and sonicated for 6 min to achieve a final concentration of 0.2 mg/mL. The working solution was prepared by adding 50 µL benzil solution and 425 µL acetonitrile:water (1:1, v/v) to 25 µL of solution containing lipid standards. For liver/heart and cell extracts, 50 µL benzil was added into 15 µL liver/heart extract (25 mg/mL) stock solution and dried cell extract sample respectively, and add ACN:water (1:1, v/v) to get to the final volume at 500 µL. The working solution was vortexed for 20 s and stored at 4 °C until further use. |
6797e88a81d2151a02309d46 | 5 | Briefly, after adding 375 µL of methanol to one million HeLa or H9c2 cells, the mixture was incubated at RT for 1 h while vortexing at 600 rpm. Next, 1.25 mL of MTBE and 313 µL of deionized water were added, followed by centrifugation at 10,000 rpm for 10 min. The upper MTBE phase was carefully collected, dried in a SpeedVac, and stored at -80 °C for subsequent use. |
6797e88a81d2151a02309d46 | 6 | Photochemical epoxidation. For the reaction of standards, a volume of 500 µL working solution was injected in flow reactor (Figure ), and the outlet was either connected to a HESI ion source for reaction monitoring or collected in a vial for further processing after observing epoxidized lipids in the mass spectrometer. The injection pump flow rate, controlled by the instrument Tune software, was set to a 40 µL/min. For extracts, the same reaction protocol was applied but the reacted solution was collected, dried, and stored at -80 °C. Before LC-MS or shotgun MS measurements of extracts the reaction solutions was reconstituted in isopropanol:methanol(1:1,v/v) without or after addition of 200 µM NaOAc, respectively. The flow reactor was cleaned with, 500 µL dichloromethane, 500 µL acetonitrile, and 500 µL water after every reaction. |
6797e88a81d2151a02309d46 | 7 | Mass Spectrometry. MS 1 and MS n measurements of oxidation products were conducted using a Velos Pro or an Eclipse mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The instruments were equipped with either a heated electrospray ionization (HESI-II) probe (Thermo Fisher Scientific) or a nanoESI source. HESI settings included a voltage of ± 3.5 kV, an inlet temperature of 300 °C, a sheath gas flow of 10, and an aux gas flow of 8. nanoESI settings included a voltage of 1 kV. In all experiments, the isolation window was ±0.5 m/z, the collision setting (HCD/CID) was 30 NCE, and the MS n resolution was 15,000 at m/z 400. Detailed information about RPLC experiments and MS settings are included in the Supporting Information. |
6797e88a81d2151a02309d46 | 8 | To investigate photochemical lipid derivatization, a simple flow reactor was designed illustrated in Figure . Multiple potentially photoreactive aldehydes and ketones were tested with the aim to derivatize unsaturated lipids in a 254 nm-triggered PB reaction. Whereas most compounds reported in the literature, for instance 3-acetyl pyridine, yielded the desired oxetanes, the mass spectrum of PC 16:0/18:1(9Z) spiked with benzil (Figure ) did not contain corresponding PB products. Instead, a pronounced mass spectrometric signal shifted +15.99 Da from the precursor m/z at 776.58 was present (Figure ). An additional, signal was also observed at +31.98 Da compared to the protonated precursor m/z but in lower intensity. These results are consistent with the epoxidation, ketone formation, or hydroxylation of PC 16:0/18:1(9Z) that is initiated by 254 nm light and requires benzil. |
6797e88a81d2151a02309d46 | 9 | The absence of similar results in experiments with PC 32:0 suggests that DBs are the primary reactive sites (Figure ). Additionally, the MS 2 -CID spectrum of [PC 16:0/18:1(9Z)+O+H] + resulted in the formation of DB position specific fragments (Figure ), consistent with literature reports for epoxidized PCs. Because we did not observe addition of benzil to unsaturated lipids or any lipidbenzil-associated fragments and no other oxygen source was added to the reaction solution, we hypothesized that dissolved molecular oxygen could be involved in the reaction. To confirm the photoactivation of molecular oxygen by benzil, we purged the reaction solution with N2 for 4 h prior to initiating the photochemical reaction of PC 16:0:/20:4(5Z,8Z,11Z,14Z). This resulted in the reduction of the +15.99 Da and 31.98 Da signals by a factor of ~20 after UV irradiation in the flow reactor (Figure ) compared to purging the reaction solution with air (Figure ). These results are consistent with the involvement of dissolved O2 in the reaction. Our findings are in line with prior reports in the organic chemical literature that showed the photoactivation of O2 by benzil. These results indicate that the use of benzil in a water/ACN solution can effectively epoxidize unsaturated lipids. |
6797e88a81d2151a02309d46 | 10 | Consequently, we varied the reaction time, flow rate, solvent composition, and benzil concentration to optimize the epoxidation yield and minimize byproducts (Figure -S5 and Table ). For authentic standards, the apparent reaction yield after parameter optimization based on intensities is 60-80 % (Figure ) in line with reports with other epoxidation reactions. To validate that the reaction products are primarily epoxides, LC-MS 2 of FA standards after lightinduced benzil reaction were compared to FA epoxide standards as shown in Figure . These results confirm that the major reaction product of the described reaction are epoxides. Intriguingly, the experiments with FAs (Figure ) reveal that E/Z isomerization leads to the formation of cis and trans epoxides that are separated by LC-MS (Figure ). These findings are in line with the proposed reaction mechanism of this epoxidation reaction and in analogy to recent reports of E/Z identification of lipids in PB workflows. Figure : Benzil-induced oxidation of unsaturated lipids via 254 nm light. A) Mass spectrum of reaction products after oxidation of PC 16:0/18:1(9Z) and B) MS 2 of the base peak at m/z 776.58 with DB-selective ions highlighted. Mass spectra of reaction products after oxidation of PC 16:0/20:4(5Z,8Z,11Z,14Z) were obtained following 4 h of purging the reaction solution with nitrogen C) and synthetic air D). Some signals are labelled with corresponding m/z values and highlighted in red. |
6797e88a81d2151a02309d46 | 11 | After establishing the relevant aspects of the mechanism of benzil-mediated photochemical epoxidation for lipid analysis, epoxidized authentic standards were activated via CID to identify structure-selective fragments. The results for FAs are discussed in Figure and results for GPLs are shown in Figure for PE 16:0/18:1(9Z) and Figure for other PEs and other lipid classes. First, we investigated the MS 2 of protonated compounds. Consistent with previous reports for other lipid classes, CID of epoxidized PE 16:0/18:1;9Ep results in pronounced head group loss ions at m/z 593.51 as well as additional fragments at around m/z 460 (Figure ). Two of these signals, i.e., m/z 451.38 and m/z 467.37, are consistent with epoxide fragmentation enabling DB position assignment. But also fragments stemming from FA loss are observed (Figure ) revealing the FA composition. Similar results are obtained in an MS 3 assay for negative ions (Figure ) as reported before. Next, we fragmented sodiated GPLs after epoxidation and results are shown in Figure is attributed to the loss of the sodiated epoxidized FA 18:1 moiety from the five-membered ring intermediate formed upon neutral head group loss (Figure ). Because this fragment can only be formed after formation of the five-membered ring intermediate (Figure ) and multiple groups have shown that this dominantly occurs with the involvement of sn-2 FAs of the precursor ion, this fragment is sn-isomer selective. This is also consistent with its absence in the MS 3 of sodiated PE 18:1;9Ep/16:0 (Figure ). Because the unsaturated FA in PE 18:1;9Ep/16:0 is located at sn-1, the corresponding MS 3 contains a fragment at m/z 361.27 indicating the loss of a sodiated FA 18:1;9Ep moiety from the sn-1 site (Figure ). Plausible fragmentation pathways consistent with MS 2 and MS results are given in Figure . Similar results are obtained for MS experiments of sodiated GPLs with improved signal-to-noise for PCs compared to MS 3 (Figure ). Taken together, these results demonstrate that an MS n assay of benzil-mediated epoxidation of sodiated GPLs can yield sn-position, DB position, head group, and FA composition. |
6797e88a81d2151a02309d46 | 12 | The extracted ion chromatogram (EIC) of non-epoxidized PC 34:1 reveals two signals that are separated in the mixture of all three isomers but do not significantly differ. The first signal at around 22 min RT (Figure ) overlaps with the PC 34:1 mixture prior to reaction (Figure ). The coelution of snand DB position GPL isomers is also consistent with literature reports that demonstrated the inability to separate most GPL snand DB position isomers when using a RP column, unless the attached FA residues on sn-1 and sn-2 significantly differ in length or DB number. This is in contrast to E/Z DB stereoisomers that are regularly separated. Therefore, the second signal in Figure |
6797e88a81d2151a02309d46 | 13 | Next, we used the developed workflow to investigate lipid isomer abundances in bovine liver and heart extracts by RPLC-MS 2 . After performing untargeted lipidomics of the unreacted extracts, we selected a target list of lipids of different classes (PE, PC, PS, LPE, LPC, DG, TG) at the sum composition level to investigate their lipid composition after photochemical benzil-epoxidation. Lipids for which full assignment of head group, FA composition, and DB positions in positive-ion mode as [M+H] + ions after photochemical epoxidation was possible are listed in Table (heart extract). Comparison between the identified lipid composition and their relative isomeric abundances for some of these lipids in liver and heart extracts is show in Figure and additional examples are provided in Figure . |
6797e88a81d2151a02309d46 | 14 | The comparison between the EIC of PE 34:1;Ep in heart and liver extract is shown in Figure and ), the highest intensity feature in the EIC is assigned to PE 16:0_18:1;9Ep. These spectra are highlighted by blue circles in Figure . With the additional information from the LC trace of the lipid without epoxide after reaction, the lipid PE 16:0_18:1(9Z) can be assigned. All investigated monounsaturated lipids for these two extracts contained only Z DB geometries as expected (see Figure for examples). The shoulder at 19.2 min in heart extract contains fragment ions at m/z 479.41 and m/z 495.40 revealing that PE 16:0_18:1;11Ep is responsible for the broadening of the chromatographic peak (MS 2 ; orange circles). For liver extracts the broadening of the EIC is not present and the majority of MS 2 spectra are assigned to PE 16:0/18:1;9Ep with the exception that two MS 2 spectra contain low intensity signals at m/z 465.39 and m/z 481.39 (Figure ). These signals are consistent with low levels of PE 16:0_18:1;10Ep. Therefore, these results in combination with LC traces from unreacted lipids after epoxidation reveal that heart extract and liver extract contains PE 16:0_18:1(9Z) as well as PE 16:0_18:1(11Z) and PE 16:0_18:1(9Z) as well as low levels of PE 16:0_18:1(10Z), respectively. |
6797e88a81d2151a02309d46 | 15 | Results for other lipid compositions are shown in Figure . Because E/Z assignment was not possible for PUFAs, only DB position and FA composition are reported. Photo-epoxidation of unsaturated lipids and RPLC-MS 2 enables the assignment of the FA composition as well as DB positions for DG, TG, PC, PE, PS, LPC, and LPE enabling to differentiate the isomer composition between the two extracts. For example, DG 36:1 in liver only contains DG 18:0_18:1(9), whereas the same lipid is composed of DG 18:0_18:1(9) and DG 18:0_18:1 (11) in heart (Figure ). Beside revealing DB positional isomers, FA compositional isomers can be resolved. This is the case for TG 52:2 for which TG 16:0_18:1_18:1(9), TG 16:0_18:1_18:1 (11), and TG 16:0_18:0_18:2 (9,12) are observed in both extracts but their relative abundance differs (Figure ). Beside the well-known DB positions for FA 18:1 at Δ9 and Δ11, our method identifies DBs at Δ10 and Δ12 in low relative abundance. These results are consistent with PB assignments of these DB isomers from bovine extracts and were recently also observed in low abundance for FAs. All other DB positions are in line with canonical DB positions and have the highest abundance (above 85 %). That the determination of DB positions is not limited to lipids with only a few DBs is showcased for PEs and PCs. For example, PE 38:4 consists only of PE 16:0_22:4 (5,8,11,14) in heart extract but liver is composed of PE 16:0_22:4 (5,8,11,14), PE 18:0_20:4 (7,10,13,16), and low abundant PE 18:1(12)_20:3 (Figure ). These results demonstrate that the coupling of photo-chemical epoxidation with chromatographic separation enables assignment of DB positions, FA composition, and for lipids with monounsaturated FA moieties the DB geometry in complex mixtures supporting RPLC-MS workflows for global lipidomics. |
6797e88a81d2151a02309d46 | 16 | The sn-isomer composition of GPLs for different cell lines can differ and polyunsaturated FA moieties not necessarily only populate sn-2 positions. This highlights the tight level of control cells exerts to build individual lipidomes as recently reported by Michael et al. for four different cell lines. To demonstrate the availability of sn-isomer information from photo-epoxidized unsaturated GPLs, we used the developed methodology to chart the sn-isomer abundance of PCs in shotgun MS experiments for HeLa and H9c2 cells (Figure ). For example, Figure and 5B shows the MS 4 spectra of sodiated PC 36:1;Ep with highlighted sn-isomer diagnostic ions. Whereas the tandem mass spectrometric result for H9c2 cells, rat cardiomyocyte cells, signals for the sn-isomer pair PC 18:0/18:1 and PC 18:1/18:0 as well as PC 16:0/20:1, fragmentation of epoxidized and sodiated PC 36:1 in the immortalized cervical cancer HeLa cell line yields the same fragments as H9c2 but additionally reveals the presence of PC 20:1/18:0. In addition to the four sn-isomers for PC 36:1 in Figure photochemical epoxidation via benzil allowed to reveal 56 sn-isomers present in at least two of the three biological replicates and 34 sn-compositions that were present in all three replicates in at least one of the two cell lines. The relative sn-isomer intensity (intensity of one sn-isomer divided by the sum of all signals of the sn-isomer pair) is shown in Figure . In agreement with the recent study by Michael et al., PCs with a small difference between the lengths or DB number of the FA moieties in sn-1 and sn-2 exhibit subtle differences in sn-isomer intensities. In both cell lines for example, fragments for the sn-pairs PC 16:0_18:2 and PC 16:0_16:1 have close to the same intensity ratios. This contrasts with the pairs for PC 16:0_20:4 and PC 18:0_20:4 for which the sn-2 location of the PUFA is in large excess to the other isomer in agreement with reported selectivity of LPCATs to preferably attach PUFAs to sn-2. But also differences between the cell lines and occupation preferences of snsites exist. HeLa cells, for example, have higher PUFA intensities in sn-1 position compared to H9c2 cells. One example is the sn-pair PC 18:1_20:3 for which HeLa cells have about the same relative intensity for the two isomers but the isomer PC 18:1/20:3 is preferred for H9c2 cells (Figure ). |
6797e88a81d2151a02309d46 | 17 | Photochemical epoxidation of unsaturated lipids by benzil and dissolved oxygen in a flow reactor revealed new possibilities for lipid structure analysis in RPLC-MS n and shotgun MS n experiments. By epoxidizing unsaturated FAs, glycerolipids (GLs), and GPLs, DB positions are pinpointed in positive-or negative ions mode across these experimental platforms. This increases the number of lipid classes for which DB positions are accessible in RPLC-MS n of epoxides. An additional benefit of RPLC of epoxidized lipid products compared to non-reacted counterparts is the improved separation capabilities for DB position and sn-isomers (Figure ). The herein newly described photochemical reaction workflow for lipidomics leads to the isomerization of DB bonds in non-reacted and reacted unsaturated lipids. As DB E/Z isomers are readily resolved in RPLC of lipids, the benzil-mediated photochemical epoxidation allows annotation of DB geometries. Furthermore, addition of sodium ions to epoxidized unsaturated lipids yielded sn-specific fragments in MS n experiments. This opens the possibility to study snisomerism as showcased for two lipid cell extracts. Therefore, we describe a single derivatization strategy that facilities assignments of all major aspects of GL and GPL structures, i.e., head group, FA composition, DB position, DB geometry, and sn-isomerism, in eukaryotes if appropriate sample preparation or separation methods are employed. |
6797e88a81d2151a02309d46 | 18 | Despite the described benefits, there are areas for improvement. These include the development of custom-made software solutions to identify corresponding fragments ions, increase sodiated lipid ion intensities for RPLC-MS n , and improvement of E/Z separation for assignment of DB geometries in lipids with multiple DBs. These shortcomings will be addressed in future studies aiming to overcome these challenges. With the presented methodology and potential future improvements, we envision to study the intricate effects of heart diseases on structural features of lipids to link these structural changes to altered metabolic processes after myocardial infarction or heart transplantation. |
660d320691aefa6ce1b04362 | 0 | Photodynamic therapy (PDT) as a burgeoning noninvasive therapeutic method has been utilized in preclinical treatment thanks to its easy accessibility, low toxicity, high spatiotemporal precision, and less incidence to evolve drug resistance. Photosensitizer (PS) is the primary element of PDT, which can generate two kinds of reactive oxygen species (ROS) upon photoexcitation to ablate cancer cells. One is type I ROS depending less on oxygen, such as hydroxyl radical (OH • ), superoxide radical (O2 •-) and hydrogen peroxide (H2O2), which shows great potential for antitumor treatment under hypoxia. The other is type II ROS including singlet oxygen ( 1 O2). Besides the efficient therapy capability, fluorescence photosensitizers can visualize the tumor locations and image organelles in cancer cells as well. Thus, imageguided PDT is a versatile theranostic technique. While, the "always on" mode of photosensitizers contributes to a low target-to-background ratio on account of a high background interference. Therefore, development of "off-on" photosensitizer system with high sensitivity to tumor microenvironment change is extremely desirable to detect cancer in the initial phase. |
660d320691aefa6ce1b04362 | 1 | Hypoxia is a significant feature of most solid tumors, which is stemmed from the imbalance between the enhanced oxygen depletion caused by the fast proliferation of tumor cells and an insufficient oxygen supply arisen arising from limited neovascularization. Hypoxia is also associated with tumor invasion, metastasis, increased resistance to radiotherapy and chemotherapy. Moreover, hypoxic tumors generally display much stronger reducibility than those normoxic cells due to the overexpression of reductases, such as nitro-reductase, azo-reductase, glutathione, NAD(P)H: quinone oxidoreductase 1 and so on. By virtue of the intense reductive ability, numerous hypoxia responsive probes have been created. Among these, azobenzene derivatives are a kind of classical hypoxia-sensitive probes, which can be reduced to amine derivatives by breaking the azo group using azo-reductase in hypoxic microenvironment. Usually, azobased luminophores emit weak fluorescence upon excitation owing to the excited energy consumption by rapid intramolecular rotation around the N=N double bond. After reduction of the azo group, intense fluorescence is attained that is the design principle for hypoxia-responsive probes. To now, many hypoxia-sensitive probes have been constructed by azo reduction strategy through using a single molecule system. For example, Nagano and co-workers reported a series of azo-containing probes composed of a NIR cyanine fluorophore and a fluorescence quencher moiety at two ends of azo group showing high sensitivity to hypoxia. However, these traditional fluorescence probes after dissociation always encounter aggregation-caused quenching (ACQ) effect in the aggregated state or physiological environment, which largely reduces the ROS generation efficiency and fluorescence quantum yield (QY). Moreover, the direct covalent linking strategy also confronts complicated synthesis and limited activity adjustment. |
660d320691aefa6ce1b04362 | 2 | The emerging aggregation-induced emission luminogens (AIEgens) with high QY, good photostability and efficient ROS generation in the aggregated state are ideal candidates to construct chemo/bioprobes. While, AIEgens emitting bright fluorescence upon aggregation can cause large background noise during bio-imaging. To fulfill the whole potential of AIE photosensitizers and smartly tune their photophysical properties, a noncovalent strategy based on the host-guest interaction is proposed. In contrast to single molecule systems, supramolecular interactions can be facilely operated and extensively used for high throughput screening. Organic macrocycles as the main host compounds are essential ingredients in supramolecular assembly, which can selectively include guest molecules in their inherent cavities to form complexes. Among them, calixarene affording a cone conformation is well-known for easy alteration, good compatibility, and multiple responses to stimuli. Now, calixarene derivatives have been widely utilized for biomedical materials in bio-imaging, drug delivery, and therapeutic agents. For example, Guo reported several calixarene-based supramolecular fluorescence probes for hypoxia imaging but without deeply exploring their antitumor capability. Herein, we develop a supramolecular assembly strategy based on sulfonate-functionalized azocalix [4]arene (SAC4A) and red-emitting AIEgen for hypoxia-responsive bioimaging and photodynamic therapy. In this system, cationic AIEgen composed of triphenylamine and quaternary ammonium salt (namely TPA-H) can be encapsulated in the cavity of azocalix [4]arene through electrostatic interaction (Scheme 1A). The as-prepared supramolecular complexes result in a quenched fluorescence. When exposing the complexes under hypoxia, the azo group is reduced to aniline derivative and released the included AIEgen, recovering its pristine fluorescence for cell imaging. Interestingly, the dissociative TPA-H undergoes cell membrane-to-mitochondria translocation during fluorescence imaging, constructing a realtime self-reporting system to monitor the PDT process in situ (Scheme 1B). Moreover, TPA-H can generate efficient singlet oxygen ( 1 O2) and superoxide radical anions (O2 •-) under light excitation for PDT with good performance. In vivo hypoxiaresponsive tumor imaging and therapy manifest that our supramolecular complexes have good biosafety and efficient antitumor efficacy. It is predicted that such host-guest strategy avoids complicated molecular synthesis and paves a way for efficient image-guided PDT. |
660d320691aefa6ce1b04362 | 3 | During supramolecular assembly, the positive photosensitizer should be pre-included in the cavity of SAC4A through charge-aided hydrogen-bonding interaction. Then, upon encountering hypoxic microenvironment, the as-obtained supramolecular complexes dissociated by reduction of azo group are essential to release the photosensitizer. Negative sulfonate groups decorated at the para-position of azobenzene are aimed to give a water-soluble calixarene and provide electrostatic interaction with positive AIEgen. The coneshaped SAC4A has a big 3D cavity that is large enough to encapsulate TPA-H. Therefore, our hypoxia-responsive hostguest assembly strategy is feasible theoretically. The detailed synthetic routes of TPA-H and SAC4A are presented in Scheme S1-S2. Their molecular structures were characterized by 1 H and C NMR and high-resolution mass spectrometry (HRMS) (Figure -S8, Supporting Information). |
660d320691aefa6ce1b04362 | 4 | The photophysical properties of TPA-H and its supramolecular assemblies were first evaluated by ultravioletvisible (UV-vis) absorption and photoluminescence (PL) spectra. As illustrated in Figure , a main absorption profile of TPA-H is observed at 465 nm and its maximum PL band centers at 654 nm in pure dimethylsulfoxide (DMSO) solution. AIE behaviors of TPA-H were investigated in DMSO and dichloromethane (DCM) mixtures. TPA-H emitted weak red fluorescence in DMSO. When adding poor solvent DCM to the solution gradually, increased fluorescence was observed accompanied by some hypsochromic shift in PL spectra due to twisted intramolecular charge transfer (Figure ). Above results demonstrated TPA-H is AIE-active. Then, theoretical calculations were carried out to optimize its molecular structure and orbital by using density functional theory (DFT) methods (Figure ). The whole molecule adopted a twisted conformation due to introduction of triphenylamine unit, which is favorable for efficient emission in the aggregated state. The highest occupied molecular orbital (HOMO) of TPA-H mainly located on the triphenylamine moiety, while the lowest unoccupied molecular orbital (LUMO) concentrated on the diazosulfide and phenyl rings. The energy gap was computed to be 1.90 eV. In addition, the lowest energy gap of singlet and triplet states (ΔES1-T2) of TPE-H was computed to be 0.06 eV, which is favorable to promote intersystem crossing (ISC) to triplet state. TPA-H fluoresced brightly with a QY of 9.0% in aqueous medium. Upon addition of 2.0 equivalents (eq.) of SAC4A, the fluorescence intensity dramatically decreased with a QY of 0.85%, implying strong electrostatic interactions (Figure ). Therefore, it is anticipated that TPA-H and SAC4A formed supramolecular complexes (TPA-H⊂SAC4A). PL titration was carried out to further verify the host-guest assembly. With enhancing the concentration of SAC4A, the fluorescence intensity of TPA-H gradually reduced and achieved equilibrium until 2.0 equivalents of SAC4A was added (Figure ). After supramolecular assembly of SAC4A and TPA-H, the zeta potential of the resultant nanoparticles changed from ∼15.0 mV for TPA-H to ∼-26.4 mV for TPA-H⊂SAC4A due to the negative charges of sulfonate motif in SAC4A (Figure ). Additionally, the time-resolved fluorescence decay curves of TPA-H and its supramolecular complexes were tested. TPA-H showed a lifetime of 1.78 and 1.90 ns in DMSO solution and powders, respectively. After selfassembly with SAC4A in aqueous solution, the lifetime of TPA-H⊂SAC4A decreased to 1.22 ns (Figure and). Dynamic light scattering (DLS) data showed that TPA-H formed micro-assemblies with a mean diameter of about 1554 nm in water solution (Figure ). After complexation with SAC4A, the aggregate size decreased to 243 nm owing to dissolution enhancement by water soluble of SAC4A (Figure ). Then, the photostability of supramolecular nanomaterials was evaluated under different times, different pH values and various species like HPO4 -, CO3 -, Mg 2+ , Na + , Cu 2+ , K + , Ca 2+ , they all showed little influence on their PL properties (Figure ). To explore the potential self-assembly mechanism, 2D COSY NMR, 1 H NMR titrations, and 2D NOESY NMR of TPA-H with SAC4A were implemented in d6-DMSO. Through analysis of 2D COSY NMR, their specific protons are clearly characterized (Figure ). When adding 0 to 2.0 equivalents of SAC4A to TPA-H, the signal of the methyl proton (Ha), phenyl ring protons (Hb and Hc) and vinyl double bonds (Hd and He) of TPE-H presented evident upfield-shifts by -0.036, -0.035, -0.031, -0.019 and -0.018 ppm (Figure and Table ) showing effective shielding effect by the aromatic rings of SAC4A. It was properly inferred that the quaternary ammonium moiety was inserted in the cavity of SAC4A. The protons of triphenylamine and diazosulfide unit showed minor change, implying these segments kept away from the cavity. Due to the inclusion of TPA-H, the phenyl ring proton Hf and Hg of SAC4A gave an obvious downfield shift by 0.015 and 0.011 ppm (Figure ). The 2D NOESY spectrum of mixture of TPA-H and SAC4A showed an obvious NOE signal between vinyl proton (He) of TPA-H and phenyl proton (Hf) of SAC4A, indicating that TPA-H is partially inserted in the cavity of SAC4A (Figure ). Sodium dithionite (Na2S2O4) as a reducing reagent was selected to verify the reduction of azo group in SAC4A by using HRMS spectroscopy. Upon addition of Na2S2O4 to SAC4A solution, the N=N double bond rapidly decomposed to form p-aminocalix [4]arene (Figure and). Then, the reaction between TPA-H⊂SAC4A and sodium dithionite was studied by using UV and PL spectroscopy. As shown in Figure , the UV intensity was evidently increased after complexation with SAC4A in aqueous medium. When sodium dithionite was added in the complex of TPA-H⊂SAC4A, the absorption band of the mixture showed a similar profile with TPA-H. Meanwhile, the red fluorescence at 654 nm was turned on gradually over time (Figure ). Above data supported our hypothesis that azo group of SAC4A was broken to release the included TPA-H and emit bright fluorescence, which further stimulates us to study their hypoxia-responsive bioimaging. The HeLa cells were incubated in normoxic (20% O2) and hypoxic (less than 0.1% O2) conditions with the same concentration of TPA-H⊂SAC4A nanomaterials. Figure and Figure revealed that the fluorescence intensity enhanced more than 2-fold in hypoxia than that in normal oxygen concentration. This indicated that SAC4A is decomposed in hypoxic microenvironment and released TPA-H from its cavity. ROS generation efficiency is a key factor to evaluate the PDT ability. The whole ROS capability was assessed by indicator 2,7-dichlorodihydrofluorescein (DCFH-DA) under white light. DCFH-DA was weakly emissive under light irradiation (Figure and Figure ). While, its fluorescence intensity was drastically increased with a 54-fold enhancement after illuminating for 6 s, proving a rapid and high ROS generation efficiency. After complexation with SAC4A, the ROS generation efficiency was largely suppressed. To determine the species of ROS, 9,10-anthracenediylbis(methylene)-dimalonic acid (ABDA) was utilized to detect type II ROS 1 O2 through using absorption spectra. As shown in Figure and Figure , ABDA was almost no change upon exposure to white light. While, upon addition of TPA-H, a large decrease in the absorption profile was observed with a 91% of ABDA consumption. Interestingly, the supramolecular complexes TPA-H⊂SAC4A totally inhibited the 1 O2 generation. Then, we verified that if TPA-H can produce type I ROS, dihydrorhodamine 123 (DHR123) was first selected as radical ROS indicator to monitor O2 •-. From the PL spectra we can see that DHR123 was almost unchanged under illumination (Figure and Figure ). However, the mixture of DHR123 and TPA-H showed a sharp enhancement in PL intensity. After host-guest interaction with SAC4A, the O2 •-generation efficiency largely decreased. In addition, type I ROS species were also explored by electron spin resonance (ESR) spectra using 4-amino-2,2,6,6-tetramethylpiperidine (TEMP) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin-trap probes for 1 O2 and free radicals, respectively. Compared with the dark state, TPA-H presented strong 1 O2 generation (Figure ), and no ESR signals were attained in TPA-H⊂SAC4A system, which is coincident with absorption data. Moreover, TPA-H and its supramolecular assemblies showed evident O2 •-signals after interaction with DMPO in methanol (Figure ) and no obvious OH• radical response was observed in water (Figure ). Thus, such results indicated that TPE-H can efficiently generate 1 O2 and O2 •- ROS. While, SAC4A can largely restrain 1 O2 generation after interaction with TPA-H. Then, DCFH-DA was used to detect intracellular ROS generation (Figure ). After treating with DCFH-DA and TPA-H, bright green fluorescence was observed in HeLa cells under light excitation, while no fluorescence was detected in the dark state and DCFH-DA group, implying that TPA-H can produce ROS under light illumination in living cells. TPA-H exhibited remarkable 1 O2 radical signals after interaction with TEMP in water (D), exhibits a small amount of O2 •-radical signaling after interaction with DMPO in methanol (E) and no OH• radical signaling after interaction with DMPO in water (F). TPA-H⊂SAC4A exhibits obvious O2 •-radical signaling (E) after interaction with DMPO in methanol and no 1 O2 (D) and OH• (F) radical response in water. Light power: 100 mW cm -2 . |
660d320691aefa6ce1b04362 | 5 | Then, the cytotoxicity of TPA-H was assessed by CCK-8 assay in HeLa cells. As illustrated in Figure , the viability of HeLa cells is higher than 80% after incubation in 10 μM of TPA-H for 24 h under dark state, implying their good biocompatibility. Upon the white light illuminating for 10 min, only 8.6% of cell viability remained in the presence of 10 μM of TPA-H. The photostability of TPA-H and Mito-Tracker Green were evaluated in HeLa cells (Figures 4B and Figure ). Through continuous laser irradiation, the PL intensity of the HeLa cells stained by TPA-H presented minor change under confocal microscope after illuminating for 3 min, revealing the good photostabilities. As a control, the PL intensity of Mito-Tracker Green distinctly reduced under the same condition (Figure ). As depicted in Figure , TPA-H was used to stain HeLa cells under different incubation time. After staining with TPA-H (2 μM) for 10 min, bright red fluorescence (λem = 600-700 nm) was observed on cell membrane. Surprisingly, when extending the illumination time, TPA-H gradually migrated to other organelle in cell (Figure ). To confirm the specific location, co-localization experiments were conducted with TPA-H and commercial Mito-tracker green (mitochondria staining dye). The red fluorescence of TPA-H was mainly located on cell membrane at the initial stage with a low Pearson's correlation coefficient (PC). Upon prolonging the incubation time to 2, 6 and 9 h, the co-localization with Mitotracker green has an increased PC from 0.31 to 0.52 to 0.74 to 0.86, demonstrating TPA-H gradually translocated from cell membrane to mitochondria. This in situ real-time visualizing fluorescence migration in cells is beneficial to understand PDT process. Moreover, the live/dead cell co-staining assay further supported the PDT properties of TPA-H in the presence of Calcein-AM and propidium iodide (PI). Calcein-AM emits green fluorescence for live cells detection and PI shows red fluorescence in dead cells. When the HeLa cells were incubated with 2.5 µM of TPA-H, bright green fluorescence and slight red emission was were observed under light irradiation (Figure ). Upon increasing the concentration to 10 µM, the green fluorescence totally disappeared and all cells exhibited strong red fluorescence, indicating the high PDT activity of TPA-H. We next demonstrated the PDT ability of TPA-H⊂SAC4A nanoparticles in hypoxic microenvironment. SAC4A showed good biocompatibility no matter in hypoxia or normoxia condition with/without light illumination (Figure ). The supramolecular assemblies also afforded high cell viability in the dark state with or without oxygen supply (Figure ), meaning no PDT was triggered off. When TPA-H⊂SAC4A nanoparticles were incubated in HeLa cells with normal O2 condition under light irradiation, obvious cell killing was obtained with increasing the nanoparticle concentrations. As above-mentioned, TPA-H⊂SAC4A assemblies can drastically inhibit 1 O2 generation, but still produce a small quantity of O2 •-. Therefore, it can kill HeLa cells through PDT. In contrast, when they were incubated in hypoxia under illumination, the cell viability largely decreased, indicating TPA-H can be released from the cavity by decomposition of SAC4A to generate efficient ROS to ablate HeLa cells. Moreover, the live/dead cell co-staining assay was carried out to explore the PDT properties of TPA-H⊂SAC4A under hypoxia or normoxia in the presence of Calcein-AM and PI. As depicted in Figure , when HeLa cells were incubated with TPA-H⊂SAC4A in normoxia condition, bright green fluorescence and small red emission were observed under light irradiation. Upon reducing O2 concentration to 0.1%, strong red fluorescence was attained, indicating many cells were killed by PDT of TPA-H. Thus, we successfully constructed a hypoxia-responsive supramolecular phototheranostic system. In consideration of the efficient cell killing effect of TPA-H, we further explored whether TPA-H treatment will destroy the cell cycle. In this experiment, HeLa cells were treated with TPA-H for 30 min under light irradiation and then determined by flow cytometry. It was worth noting that there occurred S phase accumulation with the increasing concentration of TPA-H (Figure ). In addition, CDK-2, Cyclin A2, P21 and P53 are important modulators for the cell cycle S phase. Western blot analysis manifested that TPA-H treatment resulted in CDK-2 and Cyclin A2 protein downregulation, while P21 and P53 expressions were significantly improved (Figure ). Of note, P21 is cyclin-dependent kinase inhibitor and P53 is tumor suppressor protein. Above results demonstrated that TPA-H may prevent HeLa cell propagation arising from cell cycle S arrest. The good performance of TPA-H and TPA-H⊂SAC4A nanoparticles in cell experiments motivated us to further study in vivo hypoxia-responsive behaviors involving HeLa tumorbearing BALB/c nude mice model. After intratumoral injection of TPA-H (100 μL, 1 mg mL -1 ), the tumor site can be evidently observed strong fluorescence signals at 1 to 48 h, demonstrating its excellent tumor retention property (Figure ). In contrast, no fluorescence signals were detected at the initial stage after injection of TPA-H⊂SAC4A nanoparticles (Figure ). When prolonging the time to 12 h, obvious fluorescence was observed in the tumor site. This is because SAC4A was gradually decomposed in hypoxic tumors and released the included TPA-H to image the tumor tissues. Then, tumors and heart, liver, spleen, lung, kidney were collected to test their fluorescence intensities (Figure ). Intense fluorescence signals were detected in tumor site, but almost no fluorescence signals were obtained in the main organs, which validated the prominent specificity and sustained tumor imaging ability. |
660d320691aefa6ce1b04362 | 6 | The PDT treatment in vivo was carried out on 30 tumorbearing BALB/c mice, and divided into 6 groups: PBS + light, SAC4A + light, TPA-H, TPA-H + light, TPA-H⊂SAC4A, TPA-H⊂SAC4A + light groups. After intratumoral injection of PBS, TPA-H and TPA-H⊂SAC4A, the tumor sites of PBS + light, TPA-H + light, TPA-H⊂SAC4A + light groups were exposed under white light for 30 min, and other groups without light irradiation were served as a control. The tumor size in volume and body weight were monitored every 2 days during the treatment. As illustrated in Figure and 6D, the mice in the group PBS + light, SAC4A + light, TPA-H, TPA-H⊂SAC4A, showed similar tumor growth trend, implying that PBS and SAC4A with light irradiation, TPA-H and TPA-H⊂SAC4A without light irradiation, had no antitumor activity. However, the group of TPA-H + light showed outstanding PDT efficacy. As a hypoxia-sensitive PDT model, TPA-H⊂SAC4A + light group also afforded good antitumor activity. The stable body weights of all mice indicated good biocompatibility of TPA-H and TPA-H⊂SAC4A (Figure ). Hematoxylin and eosin (H&E) staining of tumors and major organs were conducted in each group to explore systemic toxicity. As shown in Figure , no obvious damage and inflammatory lesion were obtained in the heart, liver, spleen, lung and kidney, but TPA-H + light and TPA-H⊂SAC4A + light groups showed remarkable necrosis of the cell nucleus. Additionally, blood routine assays of above all groups were implemented after 14 days' treatment, and few variations of the hematological indexes were detected in Table , further illustrating excellent antitumor activity and low systematic toxicity. |
660d320691aefa6ce1b04362 | 7 | In summary, a hypoxia-responsive supramolecular photodynamic therapy system was constructed based on a cationic AIEgen (TPA-H) and water-soluble azocalixarene (SAC4A). Upon supramolecular assembly of TPA-H and SAC4A through electrostatic interaction, the red fluorescence of TPA-H was quenched and its ROS generation was largely inhibited. In hypoxic tumors, the azo group of SAC4A can be reduced to aniline derivative and released the included TPA-H to recover pristine fluorescence and ROS. Cell imaging revealed that free TPA-H translocated from cell membrane to mitochondria, achieving dual-organelle targeting and a realtime self-reporting system to monitor dynamic molecule migration in situ. In vivo hypoxia-responsive tumor imaging and therapy were performed to find this supramolecular complexes have good biosafety and efficient antitumor activity. This work presented a promising platform for the construction of hypoxia-responsive supramolecular photosensitizer system and it will enrich the biomedical study based on supramolecular strategy. |
6490738ba2c387fa9a92d863 | 0 | our own experiments, as well as with other experiments and simulations in the literature. In the sequel, we use the model to study the partitioning of a weak diprotic acid at various pH of the supernatant. Our results show that the ionization of the acid is enhanced in the PEC phase, resulting in its preferential accumulation in this phase, which monotonically increases with the pH. Currently, this effect is still waiting to be confirmed experimentally. We explore how the model parameters (particle size, charge density, permittivity and solvent quality) affect the measured partition coefficients, showing that fine-tuning of these parameters can make the agreement with the experiments almost quantitative. Nevertheless, our results show that charge regulation in multivalent solutes can potentially be exploited in engineering the partitioning of charged molecules in PEC-based systems at various pH values. |
6490738ba2c387fa9a92d863 | 1 | Mixing of oppositely charged macromolecules can result in the formation of polyelectrolyte complexes. This associative process is often accompanied by a phase separation, in which the system demixes into two phases: the polymer-rich polyelectrolyte complex (PEC) phase, co-existing with a dilute supernatant phase which is almost free of the polymers. When the PEC phase is liquid, it is termed a complex coacervate. Complex coacervates have found use in hygiene products, food industry, and they are prospective candidates for medical applications, water purification or development of programmable materials. Furthermore, the mechanism of coacervation inspired a plethora of derived soft materials based on the assembly of polyelectrolytes with other charged objects, such as multivalent macroions, colloids, co-polymers yielding coacervate core micelles or proteins. Naturally, the use of polyelectrolyte complexes in such delicate arrangements requires a high level of fundamental understanding of the underlying physics and chemistry which govern the properties of these materials. |
6490738ba2c387fa9a92d863 | 2 | Recently, it has been recognized that cells utilize coacervation and liquid-liquid phase separation to compartmentalize and regulate their matter, and to create microenvironments akin to bionanoreactors for catalyzing chemical reactions or protein folding. This recognition sparked a new interest in the old phenomenon of complex coacervation. Furthermore, it has been demonstrated that coacervates made of synthetic polyelectrolytes can be used to sequestrate proteins, similar to the membraneless organelles which can selectively encapsulate proteins from cytoplasm. |
6490738ba2c387fa9a92d863 | 3 | Such an uptake can be not only selective, but also can preserve the activity and secondary structure of proteins, which is necessary for potential applications in drug delivery or peptide therapy. In addition, sequestration of proteins by coacervates can be regulated by change of pH or mixing ratio of polyelectrolytes. Generally, it seems to be clear that electrostatic interactions play a key role in the partitioning whereas hydrophobicity or other specific interactions fine-tune the behavior while the liquid nature of both phases ensures that the whole system remains close to thermodynamic equilibrium. |
6490738ba2c387fa9a92d863 | 4 | Systematic exploration of thermodynamic stability of coacervates established phase diagrams as a function of chain length, mixing ratio, pH or salt concentration. The general idea seems to be to use experimental setups with minimal number of welldefined components, such that their properties can be varied systematically. Other works, focusing for instance on the thermodynamics of the complex formation, visco-elasticity or interfacial properties, show that the coacervate phase behavior is universal to a high degree. Concurrently, theoretical approaches such as scaling (blob) arguments, mean-field and liquid-state theories, field-theoretic calculations or transfer-matrix formalism, have identified the key actors: charge correlations, counterion condensation or chain connectivity. Additionally, it has been shown, that specific chemistry of the polymer, counterions or solvent can strongly affect the stability and composition of the complexes. Nevertheless, the agreement between the experiment and theory remained mostly qualitative. While the experimental results reflect on both generic physical and specific chemical effects, the theories can systematically describe typically only the former effect, whereas the latter often enters the calculations in the form of phenomenological material constants, which are hard to evaluate or predict from theory. To enable further progress, it seems necessary to disentangle the universal and system-specific effects and to understand how they both contribute to the net result. |
6490738ba2c387fa9a92d863 | 5 | The partitioning of solutes between the coacervate and supernatant remains a challenge for theory and modeling. It is still poorly understood even for the simple ions, let alone more complex organic molecules or proteins. In thermodynamic equilibrium the chemical potential of each species i, which can be exchanged between the two phases (PEC and supernatant), must be the the same, |
6490738ba2c387fa9a92d863 | 6 | Clearly, this partition coefficient is controlled by the excess chemical potentials and the main challenge for theory and simulations is to correctly predict this term. In general, the excess chemical potential includes steric, short-range (Van der Waals) and electrostatic contributions and a Donnan contribution. If the coacervate is charge-balanced, then the Donnan contribution is zero. In such case, the partition coefficient is predominantly controlled by the subtle balance between the electrostatic interactions and steric effects whereas the system-specific short-range interactions further modulate the result. |
6490738ba2c387fa9a92d863 | 7 | None of these contributions can be neglected in a dense multi-component system, such as the coacervate phase, therefore, their estimation remains the main theoretical challenge. For example, many theoretical models and simulations predict that monovalent ions should preferentially accumulate in the coacervate phase whereas experiments show that simple salts, such as NaCl, slightly prefer the supernatant phase. It took several decades to establish a qualitative explanation of this observation. While many theories and experiments investigated the partitioning of monovalent salts, much less is known about the partitioning of multivalent ions or small organic molecules. In brief, these experimental studies have shown that multivalent ions generally tend to accumulate in the polyelectrolyte complex phase. Furthermore, the partitioning of multivalent solutes depends also on their size,hydrophobicity and charge. |
6490738ba2c387fa9a92d863 | 8 | Thus, it is clear that the partitioning of organic and multivalent molecules is strongly afffected by short-range interactions and charge-charge correlations, in addition to the effects which determine the partitioning of monovalent salt ions. Nevertheless, a systematic understanding of how these parameters affect the partitioning has not been fully established yet. Within the mean-field picture, the degree of ionization, α can be described by an augmented Henderson-Hasselbalch equation pH |
6490738ba2c387fa9a92d863 | 9 | where ze is the charge and ψ is the mean electrostatic potential. Thus the partitioning of charge-regulating solutes is affected by the difference in ψ between coacervate and the supernatant in two different ways: (1) at fixed valency of the solute, it determines the electrostatic contribution to the excess chemical potential; (2) at pH which is not too far from pK A of the solute, valency of the solute can be switched as it is exchanged between the phases. While the first effect has been considered in the existing theories, the second one has not yet been analyzed theoretically, although there is clear experimental evidence that a change in pH dramatically affects solute partitioning. To bridge this knowledge gap between theory and experiment, various molecular models have been designed for use in molecular simulations, which have become a strong tool for the polymer physics. The prevalent approaches in particle-based simulations of polyelectrolyte complexes can be classified in three categories. First, simulations exploring only a single pair of oppositely charged chains in dilute conditions. These studies correctly identified the main thermodynamic driving forces of polyelectrolyte complexation but they provided very little information about the behaviour of the complexes in the bulk. Second, simulations using many chains in a large simulation cell, directly emulating the phase separation. In these simulations, a nanodroplet of polyelectrolyte complex is formed inside an otherwise empty box. Such simulations suffer from massive finite-size effects, caused by the explicit interface of the coacervate nanodroplet. Extrapolation of these results to the macroscopic systems is problematic not only because the interface contributes much less to the bulk properties but also because the bulk of the coacervate phase is poorly defined in such simulation. Third group of the simulations is based on the Gibbs ensemble, where one simulates coexistence of two bulk phases (PEC and supernatant) using two simulation cells, avoiding the need for simulating their interface. 102 Such a simulation typically assumes that there is no polymer in the supernatant phase, while simultaneously small ions and other solutes can be exchanged between both phases. This third group of simulations is well suited for predicting the phase stability of coacervates and partitioining of solutes between the phases. It avoids the drawbacks of the previous two, therefore, the only limitation of this group consists in the quality of the molecular model. Indeed, the simulations of the third group qualitatively reproduced the experimentally observed phase diagrams as a function of salt concentration and also the partitioning of monovalent ions. In addition, by tuning the model parameters, a quantitative agreement between such simulations and specific experiments could be achieved. Our method falls within this third category, however in contrast to the previous studies, it also includes the effects of pH and charge regulation. These effects have not yet been addressed in molecular simulations although they have been addressed by phenomenological thermodynamic models. Unlike preceding simulation studies, we have not designed our model to reproduce the properties of a specific experimental system. Instead, our model of polymers and solutes are constructed as a generic beadspring model, possessing only a small number of parameters which can be linked with solvent permittivity, size and valency of the monomers and of the small ions. Such a setting allows us to systematically explore the effect of these parameters on the phase stability of the polyelectrolyte complex and on the partitioning of small ions. Only in simulations these parameters can be varied independently, while keeping all other interactions constant, thereby allowing us to distinguish generic features of the phase separation and partitioning from those which are determined by the specific chemistry of the polymers and small ions. |
6490738ba2c387fa9a92d863 | 10 | We use the Grand-reaction Monte Carlo method, 105,106 which allows us to simulate chemical equilibrium within and co-existence between the supernatant and coacervate phases in a broad range of salt concentrations, pH values and in the presence of small molecular or ionic solutes. The Grand-reaction method guarantees the equality of chemical potentials of the partitioning species between the phases, equality of osmotic pressures between the phases, and it also provides an option to assess its self-consistency. Similar to the preceding studies, our model assumes that there is no polymer in the supernatant phase. This approximation works well far from the critical point whereas it fails in the critical region, which defines the range of applicability of our approach. In the current work, we use only conventional trial Monte Carlo insertion moves, which can become inefficient when simulating the partitioning of bulky solutes. This deficiency can be suppressed by coupling our method to the continuous fractional component ansatz 107,108 or to thermodynamic integration, which we plan to do in the future. |
6490738ba2c387fa9a92d863 | 11 | Although our model has not been fine-tuned to reproduce a specific experimental system, we first validate our simulation results against published experimental data on the partitioning of NaCl. Second, we describe the partitioning of CoCl 2 comparing our simulations with our experiments using complexes of poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH), again reaching a nearly quantitative agreement. |
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