Peptide-Conjugated Aggregation-Induced Emission Fluorogen: Precise and Firm Cell Membrane Labeling by Multiple Weak Interactions

Abstract

Open AccessCCS ChemistryCOMMUNICATION1 Feb 2022Peptide-Conjugated Aggregation-Induced Emission Fluorogen: Precise and Firm Cell Membrane Labeling by Multiple Weak Interactions Juliang Yang, Jing-Jing Hu, Jiaming Wei, Jun Dai, Rui Liu, Fan Xia and Xiaoding Lou Juliang Yang State Key Laboratory of Biogeology and Environmental Geology, Faculty of Materials Science and Chemistry, China University of Geosciences, Wuhan 430078 , Jing-Jing Hu State Key Laboratory of Biogeology and Environmental Geology, Faculty of Materials Science and Chemistry, China University of Geosciences, Wuhan 430078 , Jiaming Wei State Key Laboratory of Biogeology and Environmental Geology, Faculty of Materials Science and Chemistry, China University of Geosciences, Wuhan 430078 , Jun Dai Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 , Rui Liu State Key Laboratory of Biogeology and Environmental Geology, Faculty of Materials Science and Chemistry, China University of Geosciences, Wuhan 430078 , Fan Xia State Key Laboratory of Biogeology and Environmental Geology, Faculty of Materials Science and Chemistry, China University of Geosciences, Wuhan 430078 and Xiaoding Lou *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Biogeology and Environmental Geology, Faculty of Materials Science and Chemistry, China University of Geosciences, Wuhan 430078 https://doi.org/10.31635/ccschem.021.202101349 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The cell membrane is a vital barrier that protects the cell from external damage and is involved in many biochemical processes. Thus, it is of great significance to label the cell membrane to explore its function. However, due to its complex and dynamic nature, precise and firm cell membrane labeling simultaneously is still a challenge. Herein, we report the fabrication of a peptide-conjugated aggregation-induced emission fluorogen (AIEgen), RTP, consisting of three main components: (1) An integrin-targeting peptide (RGD, R), which could bind specifically to integrin αvβ3 on cell membranes through ligand–receptor interaction. (2) An AIE-active tetraphenylethene derivative (T-MY, T) for fluorescent imaging. (3) Palmitic acid-modified peptide (Pal-RRRR, P), in which Pal is inserted into the lipid on the cell membrane by hydrophobic interaction, and RRRR interacted with the negatively charged cell membrane components (proteins and lipids) through electrostatic forces. RTP could precisely label tumor cells with high integrin αvβ3 expression and firmly trace the cell membrane for up to 4 h; it also has a strong resistance to photobleaching. Moreover, RTP achieved in vivo tumor-specific imaging via cell membrane labeling. Thereby, utilizing multiple weak interactions between the fluorescent probe and the cell membrane provided a new strategy for precise and firm imaging of the cell membrane simultaneously. Download figure Download PowerPoint Introduction The cell membrane plays an essential role in many biochemical processes such as maintaining the stability of the intracellular environment, controlling the transportation of substances between the cell and the surrounding environment, regulating signal transduction, and so on.1–4 Therefore, labeling the cell membrane is of great significance to explore its function. In recent years, many fluorescent probes with the advantages of strong selectivity, ultrasensitivity, and real-time detection have been developed for cell membrane labeling.5–14 The labeling mechanisms of these probes rely mainly on the structural characteristics of the cell membrane.15–18 First, many protein receptors are highly expressed on tumor cell membranes; these include integrins,19–24 aminopeptidase P (XPNPEP2) protein,25,26 and others. Using the specific binding force between a receptor and its corresponding ligand, many probes could achieve specific labeling of cell membranes. For instance, Liu's group27 designed double cyclic arginine–glycine–aspartic acid tripeptide (cRGD)-based fluorescent probes that could bind integrin αvβ3 on the cell membrane to realize cell membrane imaging via the detection of integrin αvβ3. Although these probes could specifically target cells, they are easily internalized into living cells, resulting in an unsatisfying membrane tracing. Second, the membrane phospholipid bilayer constitutes the basic cytoskeleton of the cell membrane. Taking advantage of the lipid molecules such as stearic acid, cholesterol, and palmitic acid, functionalized probes were inserted into the cell membrane through hydrophobic interaction to enable labeling.28–33 Klymchenko et al.34 synthesized a series of fluorescent probes through a click reaction between alkyl chains and various cyanine fluorophores for fast and uniform imaging of cell membranes. Moreover, lipids and proteins of the cell membrane also bestow the cell membrane with surplus negative charges. Therefore, positively charged fluorescent probes could label cell membranes efficiently by electrostatic interaction.35–38 Wang and his collaborators39 devised a cationic probe with rich imidazole groups to accomplish dual-color imaging of cell membranes. Despite the above strategies, due to the complexity of the cell membrane, its components are dynamic,40–42 making it is difficult to achieve precise and firm labeling simultaneously. Herein, taking advantage of multiple weak interactions, we report a peptide-conjugated aggregation-induced emission fluorogen (AIEgen), termed RTP, as a fluorescent probe for the precise and firm cell membrane labeling. As shown in Scheme 1, RTP consists of three components: (1) An integrin-targeting peptide (RGD, R)for specific binding to overexpressed integrin αvβ3 on tumor cell membrane through ligand–receptor interaction. (2) An AIE-active tetraphenylethene derivative (T-MY, T), containing maleimide for thiol-Michael addition reaction. (3) Palmitic acid-modified peptide (Pal-RRRR, P), in which Pal was inserted into the lipid on the cell membrane by hydrophobic interaction, and RRRR bonded with the negatively charged cell membrane through electrostatic interaction. Through these multiple weak interactions with cell membrane components, RTP could successfully distinguish tumor cell membranes from normal cells and image firmly for up to 4 h. Furthermore, RTP could label tumor cell membranes in vivo. Taking advantage of the structural characteristics of the cell membrane and integrating multiple weak interactions, this peptide-conjugated AIEgen is promising for precise and firm imaging of cell membranes simultaneously. Scheme 1 | Chemical structure of RTP and the interactions of RTP and tumor cell membrane. RTP consists of three components: (1) RGD, (2) T-MY, and (3) Pal-RRRR. Through ligand–receptor interaction, hydrophobic interaction, and electrostatic interaction, RTP could precisely and firmly label the tumor cell membrane. Download figure Download PowerPoint Experimental Methods Cell culture Prostatic tumor (PC3) cells were cultured in 1640 medium. Human lung fibroblast (HLF) cells were cultured in Dulbecco's modified Eagle medium/Ham's F-12 (1:1) (DMEM/F-12). Also, cells were co-cultured in a mixture medium containing 50% DMEM/F-12 and 50% 1640 with the same densities. The final culture media for all cells were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and the cells were grown in Petri dishes in an incubator set at 37 °C in a humidified atmosphere of 5% CO2. Incubating living cells with probes For confocal laser scanning microscopy (CLSM) imaging, cells were seeded into glass-bottom cell culture dishes at a density of 1 × 105 in the growth medium. After overnight incubation, the cells were washed with phosphate-buffered saline (PBS). A probe solution dissolved in a medium (20 μM) was then added, and the cells were incubated in a 5% CO2 atmosphere at 37 °C for further usage. The supernatant was then discarded, and the cells were washed twice gently with PBS and immersed in the growth medium prior to optical imaging. Confocal laser scanning microscopy The signals were detected using a Zeiss LSM 880 confocal microscope (ZEISS, Oberkochen, Germany) with a 63× oil-immersion objective. A 405 nm laser was chosen for the excitation of T-MY, RTP, TP (T-MY + Pal-RRRR, i.e., two moieties of RTP without RGD peptide, served as a control), and RT (RGD + T-MY, i.e., two moieties of RTP without the palmitic peptide, served as a control), and the emission was collected at 450–550 nm. A 633 nm laser was chosen for the excitation of DID (a lipophilic carbocyanine cell-labeling or delivery fluorescent dye), and the emission was collected at 640–680 nm. RTP was the test agent; T-MY, TP, and RT were used as controls. Cytotoxicity assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays were used to assess PC3 cell viability after incubation with the probes (RTP, TP, or RT at 20 μM). The cells were seeded in 96-well plates and incubated with the probes for 24 h. Then MTT diluted in PBS (10 μL of 5 mg/mL) was added to each well. After incubation for 4 h, the supernatant was discarded, and the precipitate was dissolved in dimethyl sulfoxide (DMSO) (150 μL) with gentle shaking. The absorbance of MTT at 570 nm was monitored using a microplate reader (Infinite M200 Pro, Tecan, Austria). Animals and tumor-bearing mouse model Female BALB/c nude mice (4 weeks old, ca. 20 g body weight) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animal care and handling procedures were in agreement with the guidelines evaluated and approved by the ethics committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). PC3 cells (3 × 106) were injected onto the underarm of mice. On the 15th day, the mice were treated through the caudal vein injection with 100 μL of probes at 200 μM concentration in PBS. After 4 h, the mice were sacrificed. Subsequently, the tumor tissues were harvested and fixed with 4% paraformaldehyde, followed by hematoxylin and eosin (H&E) staining and frozen sections preparation. Finally, the frozen sections were imaged by CLSM. Calculation method We utilized bilayer membranes built by CHARMM-GUI for molecular simulations, solvated in a TIP3P water model with 0.1 M NaCl, in a size of 59.5 × 59.5 × 75.6 Å3 containing about 19,000 atoms. The ratio of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) is 7:3 (56 DOPC and 24 DOPS, while total number of lipids is 80). The RTP was implemented by a g-membed module in GROMACS to embed the proteins in the membrane bilayer, and 4 DOPC and 2 DOPS were removed from the top leaflet of the bilayer. We carried out unbiased molecular dynamics (MD) simulation by GROMACS with an amber99SB-ILDN force field for 100 and 600 ns to equilibrate the system for analysis. Results and Discussion Synthesis and characterization of RTP, TP, and RT The AIE-active molecule, T-MY, was synthesized through five-step reactions, with the synthetic routes shown in Supporting Information Scheme S1–S4. The successful synthesis of the intermediate products in each step was verified by nuclear magnetic resonance spectra (NMR) and high-resolution mass spectra (HRMS) ( Supporting Information Figures S1–S8). The final RTP product was obtained through the thiol-Michael addition reaction between T-MY and RP-C with an 80% yield ( Supporting Information Scheme S5). To better demonstrate the function of RTP, two control probes TP (T-MY + Pal-RRRR) and RT (RGD + T-MY), were designed and synthesized in a similar fashion ( Supporting Information Scheme S6 and S7 and Table S1). RTP, TP, and RT were purified by high-performance liquid chromatography (HPLC) (Figure 1a). HRMS (Figure 1b and Supporting Information Figures S9–S11) proved that RTP, TP, and RT were synthesized successfully. Then their optical properties in solution were tested (Figure 1c). We found that RTP, TP, and RT showed similar absorption spectra (300–350 nm) as that of T-MY. The fluorescence spectrum revealed that the emission of T-MY was negligible, which might be due to the exciton annihilation process in its maleimide unit (Figure 1d); we presumed that after the T-MY covalently bound to peptide, n-π electronic conjugation of the maleimide group was destroyed.43,44 Thus, RTP, TP, and RT could emit fluorescence in the range of 400–700 nm. The fluorescence intensity of RTP was greatly improved when the proportion of water increased to more than 70%, indicating that RTP had obvious AIE characteristics ( Supporting Information Figure S12). The successful synthesis and photophysical properties of these probes are significant for further exploration of their imaging ability on living cell membranes. Figure 1 | Synthesis and characterization of probes. (a) HPLC purification results of RTP, TP, and RT. (b) Mass spectra of RTP, TP, and RT. (C) UV–vis absorption spectra of RTP (10 μM), TP (10 μM), RT (10 μM), and T-MY (10 μM) in DMSO/water mixture (v/v = 1/99). (d) Photoluminescence (PL) spectra of RTP (10 μM), TP (10 μM), RT (10 μM), and T-MY (10 μM) in DMSO/water mixture (v/v = 1/99). λex = 330 nm. Download figure Download PowerPoint The mechanism of Pal-RRRR and RGD in cell membrane labeling and the precise cell membrane labeling of RTP To study the cell membrane imaging properties of RTP in living cells, PC3 cells with overexpressed αvβ3 integrin were selected. After incubation with T-MY, RTP, TP, and RT, the fluorescence signals of PC3 cells were observed by CLSM. As shown in Supporting Information Figures S13 and S14, T-MY ultimately entered the cells within 30 min of incubation, demonstrating that T-MY could not label the cell membrane on the surface. In contrast, when RGD peptide or Pal-RRRR or both were modified in T-MY, the RT, TP, and RTP obtained demonstrated apparent localization on the cell membrane, displaying their capability for cell membrane imaging. Further, we investigated the mechanism of Pal-RRRR in RTP labeling cell membrane by pretreating the PC3 cells with RTP, TP, and RT. After three times washes with PBS, the PC3 cells were further incubated with DID (Figure 2a). DID, as mentioned earlier, is a commercial fluorescent probe for cell membrane imaging. The chemical structure of DID shows a long lipophilic hydrocarbon chain with positive charges to the P-involved probes (RTP and TP) ( Supporting Information Figures S15 and S16). The membrane imaging ability of DID is a benefit both from electrostatic and hydrophobic interactions.45,46 As shown in Figures 2b and 2c and Supporting Information Figure S17, compared with the negligible red fluorescence of DID in RTP or TP pretreated PC3 cells, bright red fluorescence could be observed in RT pretreated cells. Since there is a competitive relationship between DID and Pal-RRRR when labeling cell membrane, DID could not be labeled on the cell membrane of PC3 cells pretreated with RTP and TP. On the contrary, RGD binds to the cell membrane through ligand–receptor interaction, which does not compete with DID. Therefore, DID could still label cell membrane after PC3 pretreatment of RT. In turn, we also conducted a study in PC3 cells pretreated with DID, followed by incubation with RTP, TP, and RT probes (Figure 2d). The CLSM results showed that, compared with the untreated PC3 cells, the fluorescence of RTP and TP in DID pretreated PC3 cells decreased, while the fluorescence of RT remained almost unchanged (Figures 2e and 2f and Supporting Information Figure S18), proving that RTP and TP had similar dual interactions as DID. Figure 2 | The mechanism of Pal-RRRR and RGD in cell membrane labeling and the cell membrane labeling precisely of probes. (a) The scheme shows the procedure for incubating cells with probes and the data analysis in Figure c. The PC3 cells were treated with blank, RTP (20 μM), TP (20 μM), or RT (20 μM) for 30 min, followed by incubated with DID (20 μM) for 30 min, then (b) CLSM images and (c) the corresponding intensity of DID were analyzed. (d) Scheme of the procedure of incubating cells with probes and the data analysis in Figures e and f. The PC3 cells were treated without DID or with DID (20 μM) for 30 min, followed by incubated with RTP (20 μM), TP (20 μM), or RT (20 μM) for 30 min, then (e) CLSM images and (f) the corresponding intensity of RTP, TP, and RT were analyzed. (g) CLSM images and (h) the corresponding intensity of PC3 cells, PC3 cells pretreated with RGD (20 μM) for 30 min, and HLF cells incubated with RTP (20 μM), TP (20 μM), or RT (20 μM) for 30 min. (i) CLSM images and (j) the FPC3/FHLF intensity ratio of co-cultured PC3 cells and HLF cells incubated with RTP (20 μM), TP (20 μM), RT (20 μM), or DID (20 μM) for 30 min. The white circles indicate HLF cells. Scale bars: 20 μm. Data are expressed as mean ± SD; n.s.: no significant difference, *p < 0.05, **p < 0.01. Download figure Download PowerPoint Subsequently, we used PC3 cells, RGD pretreated PC3 cells, and HLF cells (αvβ3 low expression) to study the role of RGD peptide in the probe ( Supporting Information Figures S19 and S20). As displayed in the fluorescence cells, the intensity of RTP and RT decreased in RGD pretreated PC3 cells and HLF cells (Figures 2g and 2h). This was due to the differences in their integrin αvβ3 expression. Meanwhile, the fluorescence of different cells, including PC3 cells, RGD pretreated PC3 cells, and HLF cells incubated with TP, were almost the same. These results confirmed that RGD endowed the probes (RTP and RT) with the ability to achieve the tumor cell membrane imaging through ligand–receptor interaction. To prove the synergistic effect of RGD and Pal-RRRR inducing precise cell membrane imaging, we established a co-culture model of PC3 cells and HLF cells and incubated them with the probes (Figure 2i). By comparing the fluorescence intensity of the PC3 cell membrane (FPC3) with that of the HLF cell membrane (FHLF), we found that the imaging intensity ratio of FPC3 to FHLF was RTP > RT > TP ≈ DID (Figure 2j). This indicated that under the joint action of RGD and Pal-RRRR, RTP had significant advantages in precise imaging of tumor cell membranes. To better demonstrate this, the co-cultured PC3 cells and HLF cells were pretreated with RGD and Pal-RRRR ( Supporting Information Figure S21). In the case of RGD pretreatment, the ratios of FPC3 to FHLF were almost the same in the RTP, TP, and DID group, while PC3 cells and HLF cells had nearly no fluorescence of the RT group. These results suggested that RGD allowed the probe to target tumor cells with high expression of integrin αvβ3. After Pal-RRRR preincubation, there was no fluorescence in TP and DID groups, unlike the ratios of RTP and RT that were the same. It demonstrated that the probes could effectively bind to the cell membrane under the action of Pal-RRRR. Collectively, the above results, shown in Figure 2, indicated that RGD ensured that RTP precisely targeted tumor cells with high expression of integrin αvβ3 at the molecular level, and Pal-RRRR provided the RTP cell membrane imaging ability at the level of subcellular organelles. Firm cell membrane labeling and photobleaching resistance of RTP Next, the ability of the RTP probe for firm imaging was validated. The cells were incubated with probes at varying times and then observed by CLSM (Figure 3a). With the extension of the incubation time, the total fluorescence intensity of RTP, TP, RT, and DID was increased (Figure 3b). We further analyzed the ratio of fluorescence intensity on the cell membrane (FM) to that of the whole cell (FW) (Figure 3c). After incubation for 4 h, the ratio of RTP remained at a high level (75%), while that of TP and DID were decreased to about 55%, implying that RTP remained bound to the cell membrane rather than internalized by cells. Although the FM to FW ratio of RT did not change appreciably, its ratio was significantly than that of RTP, its further in cell membrane imaging. These results indicated that, compared with TP, RT, and DID, RTP bound more firmly to the cell membrane. This could be to the multiple interactions between RTP and cell membrane under the action of RGD and Pal-RRRR. Figure | The cell membrane firm labeling and photobleaching resistance of the probes. (a) CLSM (b) the corresponding total intensity total intensity of the membrane and the internalized and (c) the intensity ratio of PC3 cells. PC3 cells were incubated with RTP (20 μM), TP (20 μM), RT (20 μM), or DID (20 μM) for 1, 2, and 4 h. FM and FW to the intensity of the probes on the cell membrane and the intensity of the total probes. (d) Scheme of the procedure of incubating cells with probes and the method of data analysis in Figures PC3 cells were incubated with RTP (20 μM), TP (20 μM), RT (20 μM), or DID (20 μM) for h, then washed three times with PBS and incubated with medium for 1, 2, and 4 h. (e) CLSM and (f) the corresponding intensity ratio of the probes on the PC3 cells membrane. the fluorescence intensity of the probe on the cell membrane at h. (g) HPLC analysis of the cell culture medium. A was the of the absorption of the was the of the absorption of the probes at 1 h. (h) CLSM images of PC3 cells with RTP (20 μM) and DID (20 μM) for 30 min, then scanning with a laser of nm. (i) The corresponding intensity of RTP and DID with the of scanning laser intensity is shown in Supporting Information Figure Scale bars: 20 μm. Download figure Download PowerPoint Furthermore, the final of the probes after cell incubation was PC3 cells were incubated with RTP, TP, RT, and DID for and washed with PBS, and then a culture medium was added (Figure With the of the incubation time, we that probes on the cell membrane might have three cells, binding on the cell membrane, from the cell membrane, and into the culture medium. Thus, at different incubation the of the probes in the cell was observed by and the of the probes into the medium was analyzed by CLSM imaging revealed that at 4 h, of RTP remained on the cell membrane, while this ratio was with TP, with RT, and with DID (Figures and that RTP was more firmly labeled on the cell membrane than the three probes. After the of the probes in the we found that as the incubation the of RTP was significantly than that of TP, RT, and DID, indicating that of RTP into the medium compared with the control probes ( Supporting Information Figure and Figure The results of CLSM and HPLC confirmed that under the synergistic action of RGD and Pal-RRRR, RTP probe could bound to the cell membrane more firmly by the of probes cells and of probes from the cell membrane. For cell membrane fluorescent and is the the of the is also AIEgen has photobleaching resistance making it for imaging. Therefore, the of RTP was We used a laser to the shown in the white in Figure at different After 20 scanning the fluorescence intensity of the white of RTP remained almost that of DID was decreased and difficult to the photobleaching results of RTP and DID with different laser also showed that RTP had better photobleaching resistance than DID (Figure and Supporting Information Figure the cell viability of the probes was using the MTT As shown in Supporting Information Figure at a concentration of 20 μM membrane RTP almost no its with of RTP in vivo and of interaction between RTP and the cell membrane cell membrane probes are used for in assays in vivo due to the complex by the precise and firm cell membrane labeling ability of RTP in we evaluated the of RTP in vivo. PC3 tumor-bearing mice model was when the were about they were into three groups = and RTP, TP, or RT, at 200 μM concentration was injected via the vein for 4 h, the tumor tissues were (10 were analyzed. the results of CLSM in Figure and the intensity analysis in Figure better tumor were observed with RTP and RT than TP, proving that RTP and RT could target tumor tissues through the RGD In the fluorescence intensity of images showed that RTP was more on the cell membrane compared with RT. This was due to the that RTP was bound to the cell membrane not through ligand–receptor interaction of RGD peptide also through hydrophobic and electrostatic interactions of Pal-RRRR. these results showed that RTP was precisely and firmly labeled in the cell membrane, in an in complex Figure 4 | The of probes in vivo and the of the interaction between RTP and cell membrane. (a) CLSM images and staining of a of PC3 cells. tumor was removed after injection of RTP μM), TP μM), or RT μM) through the vein for 4 h. (b) The corresponding mean intensity of RTP, TP, and RT. Data are expressed as mean ± SD; < (c) of RTP components binding to the cell membrane. is is is white is and is (d) The between RTP and the of the cell membrane. Download figure Download PowerPoint Finally, in addition to the results, simulation was further used to the multiple weak interactions of RTP (Figure A system of RTP probe binding to a bilayer membrane was After the system for 600 the RTP probe was found in the bilayer membrane, that the RGD and moieties of RTP could with the cell membrane. Moreover, after the between RTP and the of the cell membrane, we found stability in the their interactions (Figure which a for the of RTP in cell membrane imaging. We designed a peptide-conjugated AIEgen as a fluorescent probe for precise and firm imaging of cell membrane simultaneously. In of the specific and negatively charged characteristics of the cell membrane, we two (RGD and Pal-RRRR) to and RTP with multiple weak The ligand–receptor interaction of RGD and integrin αvβ3 on the cell membrane the probe to target tumor cells with high integrin αvβ3 expression. The hydrophobic and electrostatic interactions of Pal-RRRR allowed the probe to bind efficiently to the cell membrane. under the synergistic effect of RGD and Pal-RRRR,

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