Hydrophilic polymeric fluorescent probes based on benzothiadiazole constructed for real- time monitoring of lipid droplet levels in cells

Hydrophilic polymeric fluorescent probes based on benzothiadiazole constructed for real- time monitoring of lipid droplet levels in cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hydrophilic polymeric fluorescent probes based on benzothiadiazole constructed for real- time monitoring of lipid droplet levels in cells Jin-Hua Jiang, Wei-Long Cui, Yun-Hao Yang, Jian-Yong Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6800301/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Photochemical & Photobiological Sciences → Version 1 posted 13 You are reading this latest preprint version Abstract A water-soluble polymer probe NBMA- co -PEGMA 400 was constructed by selecting benzothiazole as a fluorescent parent and modifying it with hydrophilic PEGMA 400 moiety by reversible addition-breakage chain transfer polymerization (RAFT). Furthermore, the probe was sensitive to environmental polarity and had some solvent discoloration effect. More significantly, the polymer probe had some membrane permeability, which enabled the localization of intracellular LDs and real-time assessment of lipid droplets levels in dissimilar cells. The results demonstrated the potential of polymeric probes for applications in areas such as biomonitoring and early disease diagnosis. RAFT polymerization Fluorescent copolymers Lipid droplets Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The lipid droplets, also described as a liposome, is a dynamic subcellular organelle within the cell[ 1 , 2 ]. It is involved in the regulation of neutral lipid storage and metabolism, cell membrane architecture, and intermembrane regulation and transport, and thus this signaling molecule plays an important role in various physiological activities[ 3 – 6 ]. Studies have shown that the lipid droplet is a dynamic and complex multifunctional organelle. It interacts with other organelles and plays an important role in processes such as lipid protein degradation and regulation of gene expression[ 7 , 8 ]. In addition, the number and polarity of lipid droplets vary from cell to cell, and even from one physiological state to another within the same cell[ 9 – 13 ]. By studying and monitoring these differential changes, it helps to understand the determinants of LDs formation and the involvement of LDs in the pathogenesis of metabolic diseases, even cancer[ 14 , 15 ]. It has potential applications in areas such as bio-clinical monitoring and early prevention[ 16 – 18 ]. Recently years, fluorescence detection technology has been widely used in the fields of protein labeling[ 19 – 21 ], analytical monitoring[ 22 – 24 ] and bioimaging[ 25 , 26 ]. Compared with traditional detection methods, fluorescent probes have been preferred by experts because of their high sensitivity, high specificity and good signal stability[ 27 – 32 ]. Previously, a large number of fluorescent probes had been widely reported for the specific detection of lipid droplets, including some for the diagnosis and monitoring of lipid droplet pathogenesis[ 33 – 37 ]. However, most of these fluorescent probes for lipid droplets detection were small molecule probes, which inevitably had the limitations of poor water solubility and biocompatibility. In contrast, polymeric probes have been modified to improve overall water solubility and biocompatibility. Polymer probes for the detection of metal ions [ 38 – 40 ], pH [ 41 ], biothiols [ 42 ], and drug delivery [ 43 ] have been widely reported. Among the many polymerization strategies, reversible addition-fragmentation chain transfer polymerization (RAFT) has attracted increasing attention from researchers due to its broad monomer selectivity, easy molecular modification and narrow molecular weight distribution[ 44 – 46 ]. Therefore, it is necessary to combine the advantages of both small-molecule fluorescent probes and polymeric macromolecules to design and construct a novel fluorescent polymer for real-time monitoring of lipid droplet status in various cells. Herein, we designed and developed a novel lipid droplet-targeted polymer fluorescent molecule (NBMA-co-PEGMA400) by combining the advantages of high quantum yield, good photostability and large Stokes shift of benzothiadiazole[ 47 ]. As anticipated, the polymer molecule exhibited good pH stability, water solubility and biocompatibility. Simultaneously, this polymer responded to solvent polarity changes and exhibited certain solvent discoloration effects. Furthermore, NBMA- co -PEGMA 400 was successfully applied to the imaging detection of lipid droplets in various cancer cells. It was shown that NBMA- co -PEGMA 400 could differentiate the levels of different intracellular lipid droplets. 2. Experimental 2.1. Materials and Instruments Unless otherwise noted, all reagents and drugs are commercially available. They can be used directly without secondary purification. PEGMA 400 was purchased from Shanghai McLean Biochemical Technology Co. and has an average molecular weight of 400. Ultrapure water was used in the experiments. A detailed description of the test equipment and experimental conditions can be found in the ESI. 2.2. Synthesis of polymeric fluorescent probe NBMA- co -PEGMA 400 The synthetic scheme and detailed experimental steps for the synthesis of benzothiadiazole fluorescent monomers are described in ESI† (Scheme S1, ESI†). The synthesis and characterization of chain transfer agents (CTAs) have been described in detail in previous work [ 48 ], and the specific synthesis is shown in scheme 1 . The starting monomer compound NBMA ( 2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]ethyl-2-methyl-2-propenoate ) ( 0.05 mmol, 15.4 mg ) was combined with the chemical PEGMA 400 ( poly(ethylene glycol) methacrylate with a molecular weight of 400 ) ( 1 mmol, 400 mg ), AIBN ( azodyisobutyronitrile ) ( 0. 005 mmol, 0.8 mg), CDP (2-[( dodecylthiocarbothio ) thio]propionic acid) (0.025 mmol, 10.1 mg) in THF (tetrahydrofuran) (3 mL) and added to a 25 mL Schlenk tube. The reaction system was continuously bubbled with N 2 to exclude O 2 , and the reaction system was frozen with liquid nitrogen, then thawed with a vacuum pump where it was charged with nitrogen, and the freeze-thaw cycle was repeated three times to achieve the effect of oxygen removal. Then the reaction was carried out under N 2 at 75℃ for 24 h to obtain the polymer probe NBMA- co -PEGMA 400 , which was purified by dialysis with a 1000 g mol-1 molecular weight cut-off membrane for 72 h. 3. Results and discussion 3.1. Design of the polymeric fluorescent probe NBMA- co -PEGMA 400 . The benzothiadiazole with excellent photophysical properties and a highly water-soluble poly(ethylene glycol) chain were selected for copolymerization. Secondary ammonia and nitro on the NBMA monomer formed a typical D-π-A structural model through the benzene ring, which greatly enhanced the ICT process of the system. The structure of the probe NBMA- co -PEGMA 400 was characterized by 1 H NMR and UV absorption spectroscopy. As shown in Fig. 1 and Figure S6 (ESI†), the characteristic peaks of the benzene ring and amino group on the benzothiadiazole monomer were present in the NMR spectrum of the polymer probe NBMA- co -PEGMA 400 . In addition, the disappearance of the characteristic peak of olefin on NBMA was another evidence of the successful polymerization of NBMA with other monomers (Fig. 1 a). Meanwhile, the characteristic peaks of other monomers PEGMA and CTA could be found in the H NMR spectrum of the polymer NBMA- co -PEGMA 400 . In addition to the 1H NMR studies, the UV absorption and fluorescence emission behaviors of NBMA- co -PEGMA 400 were investigated, as shown in Fig. 1 b, S2. The polymer and the monomer NBMA had an identical absorption peak around 460 nm, and the fluorescence spectra also showed some agreement. This indicated that NBMA- co -PEGMA 400 retained the luminescent properties of benzothiadiazole, which proved the successful preparation of the fluorescent polymer. In addition, the polymerization of this fluorescent polymer was tested by GPC, and the test analysis revealed a single peak, compound normal distribution for the NBMA- co -PEGMA 400 polymer probe, indicating a homogeneous polymer with a single structure. PDI = 1.53, M w =7800, M n =5100 (Figure S1 , Table S1 ). 3.2. Photophysical properties of polymer probes NBMA- co -PEGMA 400 . First, we investigated the absorption and emission behavior of NBMA- co -PEGMA 400 in different solvent environments (Fig. 2 , S2). The maximal absorption peaks of the polymer probes in different solvents were around 460 nm and red-shifted with increased solvent polarity (Fig. 2 a). Meanwhile, the relative fluorescence intensity of NBMA- co -PEGMA 400 was weak in anionic solvents such as PBS buffer, methanol and DMSO, while the relative fluorescence intensity was significantly higher in lipid-soluble solvents such as THF, 1,4-dioxane and ethyl acetate (Figure S2). During the test, the polymer probe NBMA- co -PEGMA 400 was found to be solvent-chromatic because its maximum fluorescence emission wavelength was polar (Fig. 2 b), which shifted to red with increasing solvent polarity. Such observation could be explained by the intramolecular charge transfer (ICT) mechanism upon irradiation due to the dipole moment caused by the electron donating secondary amino group and the electron withdrawing chromonitro group (Fig. 2 b) [ 49 ]. The above experimental results showed that NBMA- co -PEGMA 400 is sensitive to the solvent polar environment and can analyze and detect the polarity of the microenvironment. 3.3. Study of the water solubility of probe NBMA- co -PEGMA 400 . Water is an indispensable component of living organisms. It is an important factor in maintaining normal physiological functions of cells. To compensate for the poor water solubility of the fluorescent monomer, the introduction of the hydrophilic chain segment PEGMA improved the overall water solubility of the polymer. The water solubility of NBMA- co -PEGMA 400 was then characterized for different concentrations. As shown in Fig. 3 a, the absorbance of the polymer probe increased with the concentration of the probe in water, and the absorbance was linear with the probe concentration within 300 µg/mL, and the R 2 of the fitted curve was 0.996 (Fig. 3 b). This indicated that the polymer probe NBMA- co -PEGMA 400 had better water solubility in the in vitro environment, which laid the foundation for the probe to cope with the complex intracellular environment. 3.4. Study of the water solubility of probe NBMA- co -PEGMA 400 . The optical properties of the probe NBMA- co -PEGMA 400 in the THF/PBS binary mixture system were next investigated. In addition to the large amount of cytoplasm within the cells of living organisms, there was also a large amount of lipid media. The ability to label LDs in these complexed environments became an important indicator of polymeric probes. The emission of NBMA- co -PEGMA 400 showed an exponential increase with increasing THF content in the system (Fig. 4 b), accompanied by a slight blue shift (Fig. 4 a). The affinity of the polymer NBMA- co -PEGMA 400 for lipid-soluble solvents was further demonstrated, providing theoretical support for experiments using this polymer to label intracellular lipid droplets. 3.5. Investigation of the stability and selectivity of polymer NBMA- co -PEGMA 400 . Next, we explored the emission behavior of the polymer probe under different pH environments in vitro. As showed in Fig. 5 a, the fluorescence emission of NBMA- co -PEGMA 400 was almost unaffected by the pH solution environment, which was consistent with the data in Fig. 5 a and showed excellent acid and base resistance. Then, after continuous irradiation of the polymer under Toluene and PBS buffer system for 4500 s under 520 nm excitation, NBMA- co -PEGMA 400 still maintained stable fluorescence emission. In addition, the fluorescence intensity of NBMA- co -PEGMA 400 changed slightly in the configuration of solvents containing competing molecules such as metal ions, reactive oxygen species, and anions. This indicated that the polymer probe was highly resistant to interference. The above results indicated that the polymer probe has good photostability, pH resistance and excellent selectivity, and was suitable for application in intracellular lipid droplet imaging. 3.6. The co-localization fluorescence imaging of the copolymer NBMA- co -PEGMA 400 . The biocompatibility of the polymer NBMA- co -PEGMA 400 was tested using the MTT method prior to cell imaging. As showed in Figure S5, different concentrations of NBMA- co -PEGMA 400 were co-incubated with HeLa cells for 24 hours. The cell survival rate was above 85% in all groups tested, indicating that the polymer had low biotoxicity. This was consistent with our design strategy and allowed NBMA- co -PEGMA 400 to be used for cell imaging. Encouraged by the above test results, we applied NBMA- co -PEGMA 400 and the commercially available targeted lipid droplet dye Nile Red to HeLa and HepG2 cells. NBMA- co -PEGMA 400 showed bright spherical spots in the yellow and green channels after incubation in HeLa and HepG2 cells, respectively (Fig. 6 b, S6b). The combined channels showed good overlap after co-incubation of the polymers with Nile Red dye (Fig. 6 d, S6d). The Pearson's correlation for the two compounds was 0.91 and 0.80 in HeLa and HepG2 cells, respectively. The above results indicate that NBMA- co -PEGMA 400 has the ability to localize LDs in HeLa and HepG2 cells. 3.7. Imaging of LDs levels by polymer probe NBMA- co -PEGMA 400 detection. The number and polarity of LDs vary in different cells. Even when the same cell is in different physiological states, the level of intracellular LDs varies greatly []. Therefore, we co-incubated NBMA- co -PEGMA 400 with HeLa and HepG2 cells. The ability of the polymer probe to monitor the level of intracellular LDs was investigated by incubating the cells in different physiological states. As shown in Fig. 7, the levels of LDs in starved HeLa cells were significantly lower than in normal cells, resulting in a faint green fluorescence. Continued addition of oleic acid stimulated the intracellular production of more LDs, and the green channel released bright fluorescence. The above results indicated that NBMA- co -PEGMA 400 had the ability to monitor the levels of LDs in different cells in real time and in situ, which will help to understand the involvement of LDs in early disease monitoring and medical research. 4. Conclusions In conclusion, a novel amphiphilic polymer probe NBMA- co -PEGMA 400 was synthesized by RAFT polymerization method by combining the advantages of benzothiadiazole fluorescent monomer and hydrophilic chain segment PEGMA, and applied to monitor the level of LDs in cells. The photophysical properties of NBMA- co -PEGMA 400 were investigated by spectroscopic tests, and NBMA- co -PEGMA 400 exhibited good water solubility, photostability, and interference resistance. In addition, NBMA- co -PEGMA 400 was extremely sensitive to solvent polarity and showed some solvent discoloration properties. Biological tests showed that NBMA- co -PEGMA 400 has excellent biocompatibility. Lipid droplet co-localization experiments verified that NBMA- co -PEGMA 400 has the ability to illuminate LDs in various living cells. Meanwhile, the dynamic detection of LDs in cells under different physiological conditions using this polymer probe revealed the ability of NBMA- co -PEGMA 400 to monitor LDs in different cells. This work not only provides a preliminary strategy for studying the physiological and pathological processes associated with LDs, but also provides ideas for the rational design of novel polymer probes for the visualization of different intracellular polarities. Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution "Jin-Hua Jiang, Wei-Long Cui, and Yun-Hao Yang wrote the main manuscript text and Jin-Hua Jiang prepared figures 1-3. All authors reviewed the manuscript." 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Correa, Benzothiadiazole Derivatives as Fluorescence Imaging Probes: Beyond Classical Scaffolds, Acc. Chem. Res. 48 (2015) 1560-9, https://doi.org/10.1021/ar500468p. Z.H. Zhang, C.C. Li, W.L. Cui, J. Qu, H. Zhang, K. Liu, X.Z. Zhu, J.Y. Wang, A novel and modified fluorescent amphiphilic block copolymer simultaneously targeting to lysosomes and lipid droplets for cell imaging with large Stokes shift, Eur. Polym. J. 166 (2022) 111030, https://doi.org/10.1016/j.eurpolymj.2022.111030. W. Wan, L. Zeng, W. Jin, X. Chen, D. Shen, Y. Huang, M. Wang, Y. Bai, H. Lyu, X. Dong, Z. Gao, L. Wang, X. Liu, Y. Liu, A Solvatochromic Fluorescent Probe Reveals Polarity Heterogeneity upon Protein Aggregation in Cells, Angew. Chem. Int. Ed. 60 (2021) 25865-25871, https://doi.org/10.1002/anie.202107943. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files NBMAcoPEGMA400SI.docx Scheme1.docx Cite Share Download PDF Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Photochemical & Photobiological Sciences → Version 1 posted Editorial decision: Revision requested 17 Jul, 2025 Reviews received at journal 17 Jul, 2025 Reviews received at journal 07 Jul, 2025 Reviewers agreed at journal 30 Jun, 2025 Reviewers agreed at journal 27 Jun, 2025 Reviewers agreed at journal 27 Jun, 2025 Reviewers agreed at journal 26 Jun, 2025 Reviewers agreed at journal 26 Jun, 2025 Reviewers agreed at journal 25 Jun, 2025 Reviewers invited by journal 25 Jun, 2025 Editor assigned by journal 10 Jun, 2025 Submission checks completed at journal 02 Jun, 2025 First submitted to journal 02 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6800301","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":477755758,"identity":"e8fe49a9-eb42-477c-8b29-0ec46076a930","order_by":0,"name":"Jin-Hua Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYBACxmYQySPBYABkP2BgYwOLShCrhdkAqEWCoBY4AGoBKmdjIKyFuZ352cMvMhZ55hLJzyp/lPHVGRxgPnibh8EuD7fD2MyNZXgkii1npJnd5jnHJmFwgC3ZmochuRiPX8ykJXgkEjfcSDC7zdgG0sJjJs3DcCCxAacW9m9QLenfCn+CtfB/I6CFx0zyA1hLjhkDL8QWNkJayqQZQFrOvCmWBvpFcuZhNmPLOQbJOLUY9h/fJvmzpy5xw/H0jR9/lB3j5zve/PDGmwo73FqAEsy8PXD+MSAXRBvgUA8E8iDH/fgB59fgVjoKRsEoGAUjFgAALZdPpLLVVIcAAAAASUVORK5CYII=","orcid":"","institution":"Shandong Jiaotong University","correspondingAuthor":true,"prefix":"","firstName":"Jin-Hua","middleName":"","lastName":"Jiang","suffix":""},{"id":477755759,"identity":"f0a0b536-8c20-427c-8009-d65c66de4859","order_by":1,"name":"Wei-Long Cui","email":"","orcid":"","institution":"Qilu University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Wei-Long","middleName":"","lastName":"Cui","suffix":""},{"id":477755760,"identity":"5d6eb8e7-414a-4361-b75c-a0c39ad8bace","order_by":2,"name":"Yun-Hao Yang","email":"","orcid":"","institution":"Qilu University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yun-Hao","middleName":"","lastName":"Yang","suffix":""},{"id":477755761,"identity":"9ac94d49-bb28-41f4-ab28-acdb96af763b","order_by":3,"name":"Jian-Yong Wang","email":"","orcid":"","institution":"Qilu University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jian-Yong","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-06-02 08:38:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6800301/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6800301/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43630-025-00785-w","type":"published","date":"2025-10-07T15:57:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85649835,"identity":"07a3675f-6808-4244-8618-be65cb4cf54c","added_by":"auto","created_at":"2025-06-30 09:05:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":77089,"visible":true,"origin":"","legend":"\u003cp\u003e(a) \u003csup\u003e1\u003c/sup\u003eH NMR spectra of the polymer probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e and NBMA, PEGMA\u003csub\u003e400\u003c/sub\u003e, CTA in CDCl\u003csub\u003e3\u003c/sub\u003e solvent. (b) UV absorption spectra of polymer probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e (30 μg/mL) and benzothiadiazole fluorescent monomer (NBMA) (10 μM) in DMSO solvent.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6800301/v1/4086d048cdbf6d4ed4b3a0f1.png"},{"id":85649463,"identity":"b506b88a-587c-41fe-8946-de37ff689777","added_by":"auto","created_at":"2025-06-30 08:57:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":120532,"visible":true,"origin":"","legend":"\u003cp\u003e(a)Absorption spectrum of \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e(30 μg/mL) in different solvents.(b) The normalized fluorescent spectra of \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e (30 μg/mL) in solvents with different polarities. The inset shows the above images of different polar solvents under 365 UV lamps.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6800301/v1/f0e1c638617a6aba361c7fa9.png"},{"id":85649466,"identity":"68ab4399-6836-4728-b276-6c663c7a86fa","added_by":"auto","created_at":"2025-06-30 08:57:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68391,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Absorption spectra of the polymer \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e in PBS buffer at different concentrations. (b) Linear fitting curve of absorbance value at 454 nm versus the concentration of fluorescent polymer probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6800301/v1/144953972a5646aa71e975ab.png"},{"id":85649469,"identity":"d310eeaa-c4b6-415e-b9d9-ab56403d16e6","added_by":"auto","created_at":"2025-06-30 08:57:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62873,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The fluorescence spectra of the probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e (30 μg/mL) in different ratios of THF/PBS buffer. (b) Histogram of the relationship between the fluorescence emission intensity of the probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e (30 μg/mL) in different ratios of THF/PBS buffer.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6800301/v1/144243d1553b9e632705d60a.png"},{"id":85649476,"identity":"64e74a07-732d-4e6a-aa4c-cbefd41692db","added_by":"auto","created_at":"2025-06-30 08:57:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":71819,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fluorescence emission intensity of probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e (30 µg/mL) in different pH buffers/THF. (b) Detection of kinetic stability fluorescence spectra of probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e (30 µg/mL) in Toluene and PBS buffers. (c) The fluorescent intensity of \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e in 520 nm with different ions (1, blank; 2, HS\u003csup\u003e-\u003c/sup\u003e；3, NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e；4, CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e；5, TBHP；6, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e；7, CI\u003csup\u003e-\u003c/sup\u003e；8, SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e；9, Ca\u003csup\u003e2+\u003c/sup\u003e；10, HSO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e；11, Cys；12, HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e；13, Mg\u003csup\u003e2+\u003c/sup\u003e；14, Na\u003csup\u003e+\u003c/sup\u003e) (10 mM).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6800301/v1/8c2958f97c44183209ac226b.png"},{"id":85649485,"identity":"2adc1ab8-43f6-4c54-9fbe-6732c74ed039","added_by":"auto","created_at":"2025-06-30 08:57:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":210862,"visible":true,"origin":"","legend":"\u003cp\u003eThe co-localization cell images of the probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e (30 µg/mL) in living HeLa cells (a–e) and HepG2 cells (f–j). (a) Brightfield images of HeLa cells, (b) \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e (30 µg/mL) stain, and (c) Nile Red (5.0 mM) stain. (d) Merged image of (b and c). (e) Fluorescence co-localization curves of (b and c). (f) Brightfield images of HepG2 cells, (g) \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e (30 µg/mL) stain, and (h) Nile Red (5.0 mM) stain. (i) Merged image of (g and h). (j) Fluorescence co-localization curves of (g and h). Green channel for \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e: \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eex\u003c/sub\u003e = 405 nm, collected at 510–570 nm. Scale bar = 25 mm. Red channel for Nile Red: \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eex\u003c/sub\u003e = 550 nm, collected at 570–630 nm. Scale bar = 25 mm.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6800301/v1/6a7c2a4c66f4f51e28084bce.png"},{"id":85649836,"identity":"25d85411-8f63-444a-9e56-ef939dcd364f","added_by":"auto","created_at":"2025-06-30 09:05:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":57783,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence imaging of HeLa cells after different treatments with probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e (10 µg/mL), including starvation treatment, normal culture and oleic acid stimulation. b) Histogram of the mean fluorescence intensity of the cells imaged under different conditions of probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e (10 µg/mL). λex = 405 nm, collected 510–570 nm. Scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"06.png","url":"https://assets-eu.researchsquare.com/files/rs-6800301/v1/ae2def055bdacdbf37ab8e25.png"},{"id":93419757,"identity":"4dd3bdef-f973-4d25-9d1e-c30adeea9984","added_by":"auto","created_at":"2025-10-13 16:07:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1640032,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6800301/v1/11670711-62af-43d2-9294-cba0aa97137e.pdf"},{"id":85649467,"identity":"d8b4d6a2-b538-4168-aaf4-529d7856d13f","added_by":"auto","created_at":"2025-06-30 08:57:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":614496,"visible":true,"origin":"","legend":"","description":"","filename":"NBMAcoPEGMA400SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-6800301/v1/71932442e999542d3a5821da.docx"},{"id":85649464,"identity":"247b518c-85fd-44a1-af06-3568362230f0","added_by":"auto","created_at":"2025-06-30 08:57:33","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":37666,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6800301/v1/5406d39d18efdba7cb5944b3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hydrophilic polymeric fluorescent probes based on benzothiadiazole constructed for real- time monitoring of lipid droplet levels in cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe lipid droplets, also described as a liposome, is a dynamic subcellular organelle within the cell[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is involved in the regulation of neutral lipid storage and metabolism, cell membrane architecture, and intermembrane regulation and transport, and thus this signaling molecule plays an important role in various physiological activities[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Studies have shown that the lipid droplet is a dynamic and complex multifunctional organelle. It interacts with other organelles and plays an important role in processes such as lipid protein degradation and regulation of gene expression[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition, the number and polarity of lipid droplets vary from cell to cell, and even from one physiological state to another within the same cell[\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. By studying and monitoring these differential changes, it helps to understand the determinants of LDs formation and the involvement of LDs in the pathogenesis of metabolic diseases, even cancer[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It has potential applications in areas such as bio-clinical monitoring and early prevention[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently years, fluorescence detection technology has been widely used in the fields of protein labeling[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], analytical monitoring[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and bioimaging[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Compared with traditional detection methods, fluorescent probes have been preferred by experts because of their high sensitivity, high specificity and good signal stability[\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Previously, a large number of fluorescent probes had been widely reported for the specific detection of lipid droplets, including some for the diagnosis and monitoring of lipid droplet pathogenesis[\u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, most of these fluorescent probes for lipid droplets detection were small molecule probes, which inevitably had the limitations of poor water solubility and biocompatibility. In contrast, polymeric probes have been modified to improve overall water solubility and biocompatibility. Polymer probes for the detection of metal ions [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], pH [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], biothiols [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and drug delivery [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] have been widely reported. Among the many polymerization strategies, reversible addition-fragmentation chain transfer polymerization (RAFT) has attracted increasing attention from researchers due to its broad monomer selectivity, easy molecular modification and narrow molecular weight distribution[\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Therefore, it is necessary to combine the advantages of both small-molecule fluorescent probes and polymeric macromolecules to design and construct a novel fluorescent polymer for real-time monitoring of lipid droplet status in various cells.\u003c/p\u003e \u003cp\u003eHerein, we designed and developed a novel lipid droplet-targeted polymer fluorescent molecule (NBMA-co-PEGMA400) by combining the advantages of high quantum yield, good photostability and large Stokes shift of benzothiadiazole[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. As anticipated, the polymer molecule exhibited good pH stability, water solubility and biocompatibility. Simultaneously, this polymer responded to solvent polarity changes and exhibited certain solvent discoloration effects. Furthermore, \u003cb\u003eNBMA-\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003e-PEGMA\u003c/b\u003e\u003csub\u003e\u003cb\u003e400\u003c/b\u003e\u003c/sub\u003e was successfully applied to the imaging detection of lipid droplets in various cancer cells. It was shown that \u003cb\u003eNBMA-\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003e-PEGMA\u003c/b\u003e\u003csub\u003e\u003cb\u003e400\u003c/b\u003e\u003c/sub\u003e could differentiate the levels of different intracellular lipid droplets.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials and Instruments\u003c/h2\u003e \u003cp\u003eUnless otherwise noted, all reagents and drugs are commercially available. They can be used directly without secondary purification. PEGMA\u003csub\u003e400\u003c/sub\u003e was purchased from Shanghai McLean Biochemical Technology Co. and has an average molecular weight of 400. Ultrapure water was used in the experiments. A detailed description of the test equipment and experimental conditions can be found in the ESI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Synthesis of polymeric fluorescent probe \u003cb\u003eNBMA-\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003e-PEGMA\u003c/b\u003e\u003csub\u003e\u003cb\u003e400\u003c/b\u003e\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eThe synthetic scheme and detailed experimental steps for the synthesis of benzothiadiazole fluorescent monomers are described in ESI\u0026dagger; (Scheme S1, ESI\u0026dagger;). The synthesis and characterization of chain transfer agents (CTAs) have been described in detail in previous work [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], and the specific synthesis is shown in scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The starting monomer compound NBMA ( 2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]ethyl-2-methyl-2-propenoate ) ( 0.05 mmol, 15.4 mg ) was combined with the chemical PEGMA\u003csub\u003e400\u003c/sub\u003e ( poly(ethylene glycol) methacrylate with a molecular weight of 400 ) ( 1 mmol, 400 mg ), AIBN ( azodyisobutyronitrile ) ( 0. 005 mmol, 0.8 mg), CDP (2-[( dodecylthiocarbothio ) thio]propionic acid) (0.025 mmol, 10.1 mg) in THF (tetrahydrofuran) (3 mL) and added to a 25 mL Schlenk tube. The reaction system was continuously bubbled with N\u003csub\u003e2\u003c/sub\u003e to exclude O\u003csub\u003e2\u003c/sub\u003e, and the reaction system was frozen with liquid nitrogen, then thawed with a vacuum pump where it was charged with nitrogen, and the freeze-thaw cycle was repeated three times to achieve the effect of oxygen removal. Then the reaction was carried out under N\u003csub\u003e2\u003c/sub\u003e at 75℃ for 24 h to obtain the polymer probe \u003cb\u003eNBMA-\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003e-PEGMA\u003c/b\u003e\u003csub\u003e\u003cb\u003e400\u003c/b\u003e\u003c/sub\u003e, which was purified by dialysis with a 1000 g mol-1 molecular weight cut-off membrane for 72 h.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Design of the polymeric fluorescent probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e.\u003c/h2\u003e\n \u003cp\u003eThe benzothiadiazole with excellent photophysical properties and a highly water-soluble poly(ethylene glycol) chain were selected for copolymerization. Secondary ammonia and nitro on the NBMA monomer formed a typical D-\u0026pi;-A structural model through the benzene ring, which greatly enhanced the ICT process of the system. The structure of the probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e was characterized by \u003csup\u003e1\u003c/sup\u003eH NMR and UV absorption spectroscopy. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Figure S6 (ESI\u0026dagger;), the characteristic peaks of the benzene ring and amino group on the benzothiadiazole monomer were present in the NMR spectrum of the polymer probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e. In addition, the disappearance of the characteristic peak of olefin on NBMA was another evidence of the successful polymerization of NBMA with other monomers (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). Meanwhile, the characteristic peaks of other monomers PEGMA and CTA could be found in the H NMR spectrum of the polymer \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e. In addition to the 1H NMR studies, the UV absorption and fluorescence emission behaviors of \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e were investigated, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb, S2. The polymer and the monomer NBMA had an identical absorption peak around 460 nm, and the fluorescence spectra also showed some agreement. This indicated that \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e retained the luminescent properties of benzothiadiazole, which proved the successful preparation of the fluorescent polymer. In addition, the polymerization of this fluorescent polymer was tested by GPC, and the test analysis revealed a single peak, compound normal distribution for the \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e polymer probe, indicating a homogeneous polymer with a single structure. PDI\u0026thinsp;=\u0026thinsp;1.53, \u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e=7800, \u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003e=5100 (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e, Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Photophysical properties of polymer probes \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e.\u003c/h2\u003e\n \u003cp\u003eFirst, we investigated the absorption and emission behavior of \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e in different solvent environments (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, S2). The maximal absorption peaks of the polymer probes in different solvents were around 460 nm and red-shifted with increased solvent polarity (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). Meanwhile, the relative fluorescence intensity of \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e was weak in anionic solvents such as PBS buffer, methanol and DMSO, while the relative fluorescence intensity was significantly higher in lipid-soluble solvents such as THF, 1,4-dioxane and ethyl acetate (Figure S2). During the test, the polymer probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e was found to be solvent-chromatic because its maximum fluorescence emission wavelength was polar (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb), which shifted to red with increasing solvent polarity. Such observation could be explained by the intramolecular charge transfer (ICT) mechanism upon irradiation due to the dipole moment caused by the electron donating secondary amino group and the electron withdrawing chromonitro group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb) [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]. The above experimental results showed that \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e is sensitive to the solvent polar environment and can analyze and detect the polarity of the microenvironment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Study of the water solubility of probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e.\u003c/h2\u003e\n \u003cp\u003eWater is an indispensable component of living organisms. It is an important factor in maintaining normal physiological functions of cells. To compensate for the poor water solubility of the fluorescent monomer, the introduction of the hydrophilic chain segment PEGMA improved the overall water solubility of the polymer. The water solubility of \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e was then characterized for different concentrations. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, the absorbance of the polymer probe increased with the concentration of the probe in water, and the absorbance was linear with the probe concentration within 300 \u0026micro;g/mL, and the R\u003csup\u003e2\u003c/sup\u003e of the fitted curve was 0.996 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). This indicated that the polymer probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e had better water solubility in the in vitro environment, which laid the foundation for the probe to cope with the complex intracellular environment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Study of the water solubility of probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e.\u003c/h2\u003e\n \u003cp\u003eThe optical properties of the probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e in the THF/PBS binary mixture system were next investigated. In addition to the large amount of cytoplasm within the cells of living organisms, there was also a large amount of lipid media. The ability to label LDs in these complexed environments became an important indicator of polymeric probes. The emission of \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e showed an exponential increase with increasing THF content in the system (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb), accompanied by a slight blue shift (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). The affinity of the polymer \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e for lipid-soluble solvents was further demonstrated, providing theoretical support for experiments using this polymer to label intracellular lipid droplets.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Investigation of the stability and selectivity of polymer \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e.\u003c/h2\u003e\n \u003cp\u003eNext, we explored the emission behavior of the polymer probe under different pH environments in vitro. As showed in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, the fluorescence emission of \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e was almost unaffected by the pH solution environment, which was consistent with the data in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea and showed excellent acid and base resistance. Then, after continuous irradiation of the polymer under Toluene and PBS buffer system for 4500 s under 520 nm excitation, \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e still maintained stable fluorescence emission. In addition, the fluorescence intensity of \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e changed slightly in the configuration of solvents containing competing molecules such as metal ions, reactive oxygen species, and anions. This indicated that the polymer probe was highly resistant to interference. The above results indicated that the polymer probe has good photostability, pH resistance and excellent selectivity, and was suitable for application in intracellular lipid droplet imaging.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6. The co-localization fluorescence imaging of the copolymer \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e.\u003c/h2\u003e\n \u003cp\u003eThe biocompatibility of the polymer \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e was tested using the MTT method prior to cell imaging. As showed in Figure S5, different concentrations of \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e were co-incubated with HeLa cells for 24 hours. The cell survival rate was above 85% in all groups tested, indicating that the polymer had low biotoxicity. This was consistent with our design strategy and allowed \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e to be used for cell imaging.\u003c/p\u003e\n \u003cp\u003eEncouraged by the above test results, we applied \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e and the commercially available targeted lipid droplet dye Nile Red to HeLa and HepG2 cells. \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e showed bright spherical spots in the yellow and green channels after incubation in HeLa and HepG2 cells, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb, S6b). The combined channels showed good overlap after co-incubation of the polymers with Nile Red dye (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed, S6d). The Pearson\u0026apos;s correlation for the two compounds was 0.91 and 0.80 in HeLa and HepG2 cells, respectively. The above results indicate that \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e has the ability to localize LDs in HeLa and HepG2 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7. Imaging of LDs levels by polymer probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e detection.\u003c/h2\u003e\n \u003cp\u003eThe number and polarity of LDs vary in different cells. Even when the same cell is in different physiological states, the level of intracellular LDs varies greatly []. Therefore, we co-incubated \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e with HeLa and HepG2 cells. The ability of the polymer probe to monitor the level of intracellular LDs was investigated by incubating the cells in different physiological states. As shown in Fig. 7, the levels of LDs in starved HeLa cells were significantly lower than in normal cells, resulting in a faint green fluorescence. Continued addition of oleic acid stimulated the intracellular production of more LDs, and the green channel released bright fluorescence. The above results indicated that \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e had the ability to monitor the levels of LDs in different cells in real time and in situ, which will help to understand the involvement of LDs in early disease monitoring and medical research.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn conclusion, a novel amphiphilic polymer probe \u003cb\u003eNBMA-\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003e-PEGMA\u003c/b\u003e\u003csub\u003e\u003cb\u003e400\u003c/b\u003e\u003c/sub\u003e was synthesized by RAFT polymerization method by combining the advantages of benzothiadiazole fluorescent monomer and hydrophilic chain segment PEGMA, and applied to monitor the level of LDs in cells. The photophysical properties of \u003cb\u003eNBMA-\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003e-PEGMA\u003c/b\u003e\u003csub\u003e\u003cb\u003e400\u003c/b\u003e\u003c/sub\u003e were investigated by spectroscopic tests, and \u003cb\u003eNBMA-\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003e-PEGMA\u003c/b\u003e\u003csub\u003e\u003cb\u003e400\u003c/b\u003e\u003c/sub\u003e exhibited good water solubility, photostability, and interference resistance. In addition, \u003cb\u003eNBMA-\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003e-PEGMA\u003c/b\u003e\u003csub\u003e\u003cb\u003e400\u003c/b\u003e\u003c/sub\u003e was extremely sensitive to solvent polarity and showed some solvent discoloration properties. Biological tests showed that \u003cb\u003eNBMA-\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003e-PEGMA\u003c/b\u003e\u003csub\u003e\u003cb\u003e400\u003c/b\u003e\u003c/sub\u003e has excellent biocompatibility. Lipid droplet co-localization experiments verified that \u003cb\u003eNBMA-\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003e-PEGMA\u003c/b\u003e\u003csub\u003e\u003cb\u003e400\u003c/b\u003e\u003c/sub\u003e has the ability to illuminate LDs in various living cells. Meanwhile, the dynamic detection of LDs in cells under different physiological conditions using this polymer probe revealed the ability of \u003cb\u003eNBMA-\u003c/b\u003e\u003cb\u003eco\u003c/b\u003e\u003cb\u003e-PEGMA\u003c/b\u003e\u003csub\u003e\u003cb\u003e400\u003c/b\u003e\u003c/sub\u003e to monitor LDs in different cells. This work not only provides a preliminary strategy for studying the physiological and pathological processes associated with LDs, but also provides ideas for the rational design of novel polymer probes for the visualization of different intracellular polarities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e\"Jin-Hua Jiang, Wei-Long Cui, and Yun-Hao Yang wrote the main manuscript text and Jin-Hua Jiang prepared figures 1-3. All authors reviewed the manuscript.\"\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThis work was financially supported by Natural Science Foundation of China (21801145), Natural Science Foundation of Shandong Province (ZR2017BB012).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eN. Krahmer, , R.V. Farese, Jr., T.C. Walther, Balancing the fat: lipid droplets and human disease, EMBO Mol. Med. 5 (2013) 973-983, https://doi.org/10.1002/emmm.201100671.\u003c/li\u003e\n \u003cli\u003eT.C. Walther, R.V. Farese, Jr., Lipid droplets and cellular lipid metabolism, Annu. Rev. Biochem. 81 (2012) 687-714, https://doi.org/10.1146/annurev-biochem-061009-102430.\u003c/li\u003e\n \u003cli\u003eS.M. Storey, A.L. McIntosh, S. Senthivinayagam, K.C. Moon, B.P. Atshaves, The phospholipid monolayer associated with perilipin-enriched lipid droplets is a highly organized rigid membrane structure, Am. J. Physiol. Endocrinol. 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Ed. 60 (2021) 25865-25871, https://doi.org/10.1002/anie.202107943.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"photochemical-and-photobiological-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ppss","sideBox":"Learn more about [Photochemical \u0026 Photobiological Sciences](https://link.springer.com/journal/43630)","snPcode":"43630","submissionUrl":"https://www.editorialmanager.com/ppss/","title":"Photochemical \u0026 Photobiological Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"RAFT polymerization, Fluorescent copolymers, Lipid droplets ","lastPublishedDoi":"10.21203/rs.3.rs-6800301/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6800301/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA water-soluble polymer probe \u003cstrong\u003eNBMA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-PEGMA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e400\u003c/strong\u003e\u003c/sub\u003e was constructed by selecting benzothiazole as a fluorescent parent and modifying it with hydrophilic PEGMA\u003csub\u003e400\u003c/sub\u003e moiety by reversible addition-breakage chain transfer polymerization (RAFT). 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