Open AccessCCS ChemistryRESEARCH ARTICLE6 Jun 2022

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

    Bioconjugation methods offer very important tools in studying biological systems. Synthetic host–guest pairs provide an alternative and complementary conjugation method to bioorthogonal reactions and biological association pairs. Nevertheless, macrocyclic hosts that can be used for in situ capture are limited and often rely on extremely high binding affinities. Herein, we report an alternative bioorthogonal host–guest pair that relies on highly selective molecular recognition in water. The host, namely amide naphthotube, possesses a biomimetic cavity with inward-directing hydrogen bonding sites and shows selective and strong binding to the guest (2-phenyl pyrimidine) even in biological media. Through anchoring the tetraphenyl ethylene-modified hosts to cell surfaces, the bioorthogonal host–guest pair can be applied in cell surface recognition, cell–cell interactions, and tissue imaging in mice. The bioorthogonality is originated from the high binding selectivity of the biomimetic macrocyclic host, which is different from other known host–guest pairs that have been applied in biological systems. This research provides a new noncovalent bioconjugation tool and a new concept for designing bioorthogonal host–guest pairs for biological applications.


    Bioconjugation methods are important tools widely used in chemical biology, molecular biology, and drug discovery. The chemistries underlying bioconjugation should be selective and nonperturbing to biological systems. Therefore, they have been collectively termed bioorthogonal chemistry.1 Over the past two decades, many bioorthogonal reactions2 have been developed for imaging and interrogating biological systems. These reaction-based bioconjugation methods are robust but often suffer from slow reaction kinetics, which limits their applications in animals or humans.3 Noncovalent molecular recognition4 has relatively fast kinetics (diffusion-controlled) and is thus complementary to reaction-based bioconjugation methods. The most widely used noncovalent association pairs are biotin/(strept)avidin pairs which have fast formation kinetics (kon ≈ 107 M−1 s−1) and high association constants (Ka = 1013–1015 M−1).5 Nevertheless, this natural robust binding pair also suffers from several drawbacks: (1) the molecular weight is high (>53 kDa) and it is difficult to modify (strept)avidin; (2) it suffers from background interference and nonspecific binding, limiting its biological applications; (3) the binding pair is irreversible and requires harsh condition to recover the captured proteins. In general, noncovalent association pairs with biological origin are all vulnerable to enzymatic reactions in the biological environment.

    Synthetic host–guest pairs have been proposed to overcome the problems of biological association pairs in terms of large size and fragility.6 They are usually small in size (∼1 kDa) and robust in biological environment. Noncovalent binding pairs are used in two common ways for biological applications: preassembly for subsequent deployment and in situ capture of target molecules. Many host–guest pairs have been used in the manner of preassembly,716 but it is challenging for molecular hosts to capture guests or guest-conjugated targets in complex biological media.1730 It is even more challenging to achieve in situ capture in living animals, and there are only very limited successful examples via the employment of either monovalent31,32 or multivalent33,34 systems. In situ capture requires the host–guest pairs to be bioorthogonal, that is, the host–guest pairs are not interfered by biomolecules and salt ions. Nevertheless, synthetic hosts with a hydrophobic cavity also bind biological molecules in water.3537 To be bioorthogonal, high-affinity host–guest pairs (Ka > 1010 M−1) are usually searched for to overcome the competition in complex biological environment.3841 Recently, the extremely stable binding pair between cucurbit[7]uril and adamantylammonium/ferrocenemethylammonium derivatives (Ka ≈ 1012 ∼ 1015 M−1, in water) has emerged to be a versatile bioorthogonal system for biological applications.38

    In contrast, biomolecular recognition systems usually have moderate binding constants (103–109 M−1) but a high binding selectivity.42 Therefore, similar biomimetic host–guest systems, which possess a high binding selectivity, may be bioorthogonal. However, this type of bioorthogonal host–guest pair with a biomimetic nature has not been demonstrated in vivo via monovalent binding mode. By mimicking the binding pockets of bioreceptors,43 we have developed a pair of macrocyclic hosts—amide naphthotubes—with hydrogen bonding donors inside their deep hydrophobic cavities.4448 These biomimetic hosts are able to selectively recognize a variety of organic molecules in water by combining the hydrophobic effects with shielded hydrogen bonding. The highest association constants of the anti-configured naphthotube reach 106 M−1,49 which is comparable with those of some biomolecular systems. These biomimetic hosts and their complexes show promise to be bioorthogonal but have not been demonstrated in cells or living animals for biological applications. Herein, we report that the host–guest pair between anti-configured amide naphthotube and 2-phenyl pyrimidine is bioorthogonal and their potential for biological applications were demonstrated in cells and living animals. By anchoring the modified hosts to cell surfaces through tetraphenyl ethylene (TPE) sidechains, the bioorthogonal host–guest pair can be successfully applied for cell surface recognition, cell–cell interactions, and tissue imaging in living mice via noncovalent recognition and binding. The bioorthogonality is originated from the moderate binding affinity and high selectivity of the biomimetic macrocyclic host, which is different from other known host–guest pairs that have been applied in biological systems. This research provides a new noncovalent bioconjugation tool through a biomimetic macrocyclic host with an endofunctionalized cavity.

    Experimental Methods

    General method

    The artificial receptors (R1 and R2) and guests (G1, G2, G3, G4, and G5) were synthesized according to Supporting Information Scheme S1, and their intermediates and final products were characterized by standard analytical methods ( Supporting Information Figures S3–S59). 1H and 13C spectra were recorded on a Bruker Avance-400 or 500 NMR (Bruker, Switzerland) spectrometer. All chemical shifts are reported in ppm with residual solvents or sodium methyl sulfonate as the internal standard. Electrospray-ionization time-of-flight high-resolution mass spectrometry (ESI-TOF-HRMS) experiments were conducted on an applied biosystems Elite ESI-quadrupole time of flight (QqTOF) mass spectrometry (Thermo Scientific, United States) spectrometer system. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed on a Bruker Autoflex Speed MALDI-TOF (Bruker Daltonic, United States) equipped with a 337 nm nitrogen laser. α-Cyano-4-hydroxycinnamic acid (HCCA) was used as the matrix. Fluorescence (FL) and UV spectra were obtained on a Shimadzu RF-5301pc (Shimadzu, Japan) spectrometer and a UV–vis spectrophotometer (UV-2600, Shimadzu, Japan), respectively. Absolute emission quantum yields were recorded on Hamamatsu absolute photoluminescence (PL) quantum yield spectrometer C11347 (Hamamatsu, Japan) equipped with an integrating sphere. Isothermal titration calorimetry (ITC) experiments were carried out in 10 mM phosphate-buffered saline (PBS) buffer pH 7.4 (or fetal bovine serum, FBS) at 25 °C on a Malvern VP-ITC instrument (Malvern, United Kingdom). Further details on the titration experiments can be found in the Supporting Information. The size distribution and polydispersity index (PDI) of liposomes were evaluated by dynamic light scattering (DLS) [HORIBA scientific nano particle analyzer SZ-100 instrument (HORIBA, Ltd., Japan)]. The measurements were conducted in triplicate at 25 °C. Further details of the experimental methods for preparation of liposomes can be found in the Supporting Information. In vitro imaging experiments were performed on an inverted confocal laser scanning microscopy (CLSM; Zeiss LSM710, Zeiss, Germany). In vivo imaging experiments were performed on an IVIS Spectrum (Caliper LifeSciences, PerkinElmer, United States) imaging system.

    Cell culture

    All cell lines involved in this research were obtained from American Type Culture Collection (ATCC). All cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% FBS at 37 °C in a humidified atmosphere containing 5% CO2. Cells were subcultured regularly using trypsin/ethylenediaminetetraacetic acid (EDTA). Further details of the experimental methods for biocompatibility analysis of the receptors in vitro, cell membrane anchoring, and molecular recognition in vitro can be found in the Supporting Information.

    In vivo experiment

    All animal experiment procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Southern University of Science and Technology, and approved by the Animal Ethics Committee, Southern University of Science and Technology (Shenzhen, China). Male BALB/c mice at 8–10 weeks and male and female nude BALB/c nude mice at 6–8 weeks were purchased from Vital River Laboratory Animal Technology (Zhejiang, China). Animals were housed in a barrier facility on a 12 h light/dark cycle with food and water provided ab libitum. For building B16 tumor models, male BALB/c nude mice were subcutaneously injected B16 cells on the right flank. For building 4T1 tumor models, female BALB/c nude mice were subcutaneously injected 4T1 cells on the right flank. Further details of the experimental methods for the biocompatibility test of the receptors in vivo and R2 and R1 mediated tumor targeting investigations in vivo can be found in the Supporting Information.

    Statistical analysis

    All of the data are expressed as the mean ± standard error of the mean (s.e.m.) or mean ± standard deviation (SD) as indicated. Statistical analysis was conducted by one-way analysis of variance (ANOVA) for comparison of multiple groups using OriginPro 2015 software. 0.01 < *P ≤ 0.05 was considered significant, **P ≤ 0.01 was considered highly significant, ***P ≤ 0.001 was considered extremely significant.

    Results and Discussion

    Design and synthesis of the receptors and guests

    It has been reported that 2-phenyl pyrimidine shows a relatively high binding affinity to the anti-configured naphthotube in water (Ka = 7.0 × 105 M−1).49 This host–guest pair is very robust and thus selected for biological studies. To be used in a biological environment, the host and guest should be modified. Naphthotube H1 with an alkyne sidechain and G1 with a tetraethylene glycol unit were used to test the binding affinity of the host–guest pair in complex environment after modification. The binding constants between H1 and G1 were determined by ITC in PBS buffer and in FBS to be (1.5 ± 0.2)  × 106 M−1 and ∼105 M−1, respectively ( Supporting Information Figure S2 and Table S1). The 1H NMR experiments ( Supporting Information Figure S1) indicate that the naphthotube binds to the phenyl pyrimidine unit (Figure 1a) rather than the tetraethylene glycol unit, although both of them can work as guests.50 These results suggest that the host–guest pair between the naphthotube and phenyl pyrimidine may be bioorthogonal and used in a complex biological environment.

    Figure 1

    Figure 1 | The structures of artificial receptors, guests, and liposomes, as well as the mechanism of artificial receptors anchored onto cell membrane. (a) Cartoon representations of the binding models of H1 recognition of G1. (b) Cartoon representation of membrane insertion of the artificial receptors. (c) Chemical structures and cartoons of the artificial receptors (R1 and R2). (d) Chemical structures and cartoons of guest molecules. (e) Cartoon representations of the composing and structure of liposomes.

    For biological applications, we thought to anchor the host to cell surfaces. There are several reports that artificial receptors can enter into cell membranes and then undergo cellular internalization.21,5155 Therefore, it is not easy to design an artificial receptor that can be firmly anchored onto cell membranes and retain molecular recognition ability to guest molecules in solution. However, it is known that hydrophobic molecules can be inserted into the cell membrane. Molecules with multiple negatively-charged groups cannot easily cross cell membranes due to the negative intramembranous resting potential of mammalian cells (−10 to −90 mV).56 These two features may be combined to construct artificial receptors that can stably anchor onto cell membranes (Figure 1b).

    Water-soluble naphthotubes carry multiple negatively-charged carboxylates and may not easily cross cell membranes, which may be the reason why their cytotoxicity is low.49 In addition, a hydrophobic moiety is required for membrane insertion. TPE5760—a typical AIE luminogen (AIEgen)—was selected as the membrane anchor for three reasons: (1) it is relatively hydrophobic and would perfectly fit for membrane insertion; (2) it would show enhanced FL when inserted into the cell membrane due to the restricted motion (aggregation-induced emission (AIE) effect)61,62; (3) other AIEgens have shown firm membrane anchoring with a retention time (ca. 6 h) longer than that of commercial cell membrane dye DiD (ca. 1 h).63,64 Consequently, artificial receptors R1 and R2 (Figure 1c) were designed and synthesized by attaching a TPE moiety to the anti-configured naphthotube65 through Cu(I)-catalyzed click reaction ( Supporting Information Scheme S1 and Figures S3–S27). The optical properties of R1 and R2 demonstrate that both of them have good AIE features (R1, λem = 480 nm; R2, λem = 590 nm, Supporting Information Figures S61 and S62). These receptors may be anchored onto cell membranes via hydrophobic TPE moiety. The negatively-charged naphthotube with three carboxylate groups would avoid cellular internalization and is left outside cell surface for molecular recognition (Figure 1b). In addition, guest molecules G2–G5 (Figure 1d) were synthesized for cell and animal experiments ( Supporting Information Figures S46–S59). Guests (G2 and G4) exhibited green FL corresponding to 4-nitro-2,1,3-benzoxadiazole (NBD) in water (λex = 470 nm, λem = 540 nm, Supporting Information Figure S65). G5 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000 (DSPE-PEG2000) were used to prepare guest-modified liposomes (Figure 1e).

    Biocompatibility of the receptors in vitro and in vivo

    The cytotoxicity and acute toxicity of the receptors were first evaluated before cell and animal experiments. The cytotoxicity of R1 and R2 were evaluated with AML-12 (normal mice hepatocytes) and B16 (mouse melanoma cell) cell lines by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.66 Both R1 and R2 displayed minimal cytotoxicity against these two cell lines even at relatively high concentrations, except for R2 against AML-12. The cellular viabilities of AML-12 and B16 treated with R1 (up to 100 μM) were all over 85%, while the survival rates of AML-12 and B16 treated with R2 (100 μM) were over 75% and 95%, respectively ( Supporting Information Figure S66), suggesting that both R1 and R2 have generally good biocompatibility.

    To verify whether the artificial receptors (R1 and R2) could be administered safely in vivo, the acute toxicity and biocompatibility of R1 and R2 were studied on male BALB/c mice. Mice were randomly divided into three groups with five mice in each group, followed by the intravenous (i.v.) tail vein injection of PBS buffer (10 mM, pH 7.4), R1 (100 mg/kg), and R2 (25 mg/kg), respectively, according to the dose-escalation studies in our preexperiment. Then, these mice were observed for 14 days. During the 14-day follow-up, all the mice showed normal increments in body weight without significant differences between the three groups ( Supporting Information Figure S67a). In addition, the levels of the hepatic and renal function biomarkers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Crea), and urea ( Supporting Information Figures S67b and S67c), were similar across all three groups, suggesting that R1 and R2 did not induce toxicity to the kidneys and livers. Furthermore, the major organs’ histopathological analysis of the mice from all three groups also reveals no tissue injuries ( Supporting Information Figure S68). Based on these results, we conclude that the i.v. administrations of R1 (100 mg/kg) and R2 (25 mg/kg) in mice were at a tolerable dose level.

    Cell membrane anchoring and molecular recognition in vitro

    B16 cell line was taken as an example to demonstrate the anchoring abilities of artificial receptors (R1 and R2) onto cell membrane surfaces. B16 cells were incubated with R1 or R2 (40 μM) for 24 h and then were washed with PBS buffer for CLSM analysis. As shown in Figure 2a and Supporting Information Figure S69, the membrane surface of the cells was stained with blue or red FL, suggesting that R1 or R2 can be firmly and specifically anchored onto cell membrane surface. This conclusion was further confirmed by co-localization experiments with a specific cell membrane staining dye Dil. As shown in Figure 2b and Supporting Information Figure S70, the merged image showed co-localization of R1 and Dil on the membrane surface of B16 cells, and the Pearson correlation coefficient (PCC) between them is 0.86. Further experiments show that increasing the incubation time, concentration, and temperature gives rise to stronger fluorescent signals ( Supporting Information Figures S71–S73), suggesting that the number of artificial receptors on cell membrane could be fine-tuned by varying these parameters. R1- or R2-incubated B16 cells were monitored for 21 days to analyze the stability and retention time of the receptors on cell membranes. Surprisingly, the receptors remain on the cell membranes for over 12 days even when cells were dead (Figure 2c and Supporting Information Figures S74 and S75). However, the fluorescent intensity varies during the lengthy incubation because the cells undergo division, proliferation, and death. This astonishing long-term retention of the artificial receptors on cell membrane surface endowed them with a significant potential to anchor onto any selected tissues for targeted delivery in vivo.

    Figure 2

    Figure 2 | Cell membrane anchoring in vitro. (a) CLSM images of B16 cells after treatment with R1 (left, 40 μM) or R2 (right, 40 μM) at 37 °C for 24 h. (b) Fluorescent co-localization of B16 cells stained with R1 (40 μM) and Dil (10 μM). (c) CLSM images of B16 cells stained with R1 (left, 40 μM) and R2 (right, 40 μM) for various durations.

    Moreover, these receptors are also applicable for membrane anchoring of AML-12, Raw 264.7 (mouse leukemia cells of monocyte macrophage), 4T1 (mouse breast cancer cells), LO2 (Human normal hepatocytes), HUVEC (human umbilical vein endothelial cells), 293T (human renal epithelial cells), A549 (human nonsmall cell lung cancer cells), and HepG2 (human hepatocellular liver carcinoma cells) cell lines ( Supporting Information Figures S76–S83). In addition, control experiments by treating 4T1 and HepG2 cells with R1 or R1-encapsulated liposomes ( Supporting Information Figure S84) clearly show that R1 alone stain on the cell membrane surfaces and does not obviously enter cells within 24 h. However, control compounds (C1 and C2) with one carboxylate group can enter cells by showing fluorescent signals intracellularly ( Supporting Information Figure S85). These results support that these receptors are successful cell membrane anchors for a wide scope of cell lines and these three carboxylate groups on the naphthotubes are important to avoid cell internalization.

    Molecular recognition on membrane surface was studied by using B16 cells anchored with R1 and guests G2 or G3. B16 cells were treated with R1 (40 μM) for 24 h, and were then incubated with G2 (40 μM) in DMEM medium for another 30 min before CLMS imaging. As shown in Figure 3a and Supporting Information Figure S86, the FL of R1 and G2 (the PCC of 0.83) is co-localized on the membrane surface of B16 cells, indicating that the naphthotube should be left on the cell surface and is still available for guest binding. The recognition properties of the naphthotubes were almost unaffected by the cell culture medium containing sugars, amino acids and other biological entities, suggesting the bioorthogonal nature of the recognition systems. In contrast, free G2 slowly diffused into cells that were not pretreated with R1 ( Supporting Information Figure S87). Additionally, ditopic guest G3 can conjugate two different cells (B16 and AML-12 cells) together (Figure 3b and Supporting Information Figure S88) to enable cell–cell interaction, further supporting that the recognition property of the naphthotubes is retained after the artificial receptors are inserted onto cell membrane.

    Figure 3

    Figure 3 | Molecular recognition on the membrane surface in vitro. (a) Fluorescent co-localization of B16 cells stained with R1 (40 μM) and G2 (40 μM). (b) CLSM images of B16 (DiO) and AML-12 (Dil) cells conjugations via host–guest interaction (host: R2; guest: G3), the detailed experiments show in Supporting Information Figure S88. CLSM images of the recruitment and uptake of liposomes by B16 cells affected via R1: B16 cells pretreatment with PBS buffer or R1 (80 μM) for 12 h were further incubated with liposomes for 5 min, then analyzed at different time points (5 min, 1 and 2 h). (c) Cells without pretreatment with R1, incubated with 50 μL G5-modified liposome (G5, ∼0.56 mM; DSPE-PEG2000, ∼0.56 mM). (d) Cells with pretreatment with R1, incubated with 50 μL PEG-modified liposome (DSPE-PEG2000, ∼1.12 mM). (e) Cells with pretreatment with R1, incubated with 50 μL G5-modified liposome (G5, ∼0.56 mM; DSPE-PEG2000, ∼0.56 mM). The MFI of Cy 7.5 in these three groups after incubation with liposomes for 5 min was measured by Image J software.

    To further reveal the applicability of these receptors in complex nanosystems, guest-modified liposomes were studied with R1-anchored B16 cells. 4',6-diamidino-2-phenylindole (DAPI) and Cy 7.5 were employed to stain the nucleus of the cells and phospholipid bilayer of liposomes, respectively. Three groups of experiments were performed: (1) B16 cells pretreated with PBS buffer was incubated with G5-modified liposome (the control group); (2) B16 cells pretreated with R1 were incubated with PEG-modified liposome (the PEG group); (3) B16 cells pretreated with R1 were incubated with G5-modified liposome (the G5 group). After incubation with liposomes, cells were washed by PBS buffer three times and incubated with a fresh DMEM medium, before analysis by CLSM at different time points (5 min, 1 and 2 h). As shown in Figures 3c3e, at 5 min, B16 cells from the G5 group exhibited stronger red FL when compared with the control group according to their mean FL intensity (MFI) of Cy 7.5. The fluorescent response of the control group should be due to nonspecific adsorption of liposomes on cells. The enhanced FL in the G5 group suggests that the artificial receptor can assist the recruitment of liposomes. Surprisingly, the PEG group also displays stronger FL than that of the control group. The low binding affinity between the naphthotube and PEG2000 (ca. 1.6 × 104 M−1, PEG2000:naphthotube = 1:1)50 is usually considered to be insufficient in biological systems. Thus, the observed, improved recognition is likely attributed to the multivalent binding14,15,17 between naphthotubes and PEG2000 ligands of liposome. Thereby, the current experimental results further support the bioorthogonal recognition property of the naphthotubes. In addition, the recruitment efficiency depends on the guest binding affinity: the G5 group shows stronger FL than that of the PEG group because phenylpyrimidine (7.0 × 105 M−1) is a better guest to the naphthotube than PEG2000. Under extended incubation, liposomes were gradually internalized by B16 cells presumably via artificial receptor mediated endocytosis. Therefore, these results further demonstrate that the artificial receptors can also endow cells with the ability to recruit and uptake guest-modified liposome via bioorthogonal molecular recognition.

    Tissue targeting and imaging in living animals

    The success achieved on cell experiments encouraged us to further explore the application of these host–guest pairs for tissue targeting and imaging in living animals. For animal experiments, tumor tissues (B16 or 4T1 tumors) and muscle tissues were selected to demonstrate the anchoring and imaging properties of the receptors. The artificial receptors were first injected into the selected tissues, and the mice were bred for 24 h to allow the receptors to anchor onto the corresponding cell membranes of these tissues. Then, the guests or guest-modified liposomes were administered i.v. (Scheme 1). For guests alone, the mice were sacrificed after 24 h and the B16 tumors were collected and analyzed by IVIS Spectrum imaging system. For guest-modified liposomes, the corresponding tissues (4T1 tumors and left thigh muscle) of the mice were monitored at different time intervals by IVIS Spectrum imaging system. After 96 h, the mice were sacrificed and the corresponding tissues were collected and analyzed.

    Scheme 1

    Scheme 1 | Scheme showing cell membrane anchoring and bioorthogonal molecular recognition of the receptors to promote tumor-targeted of G3 or G5 liposomes via host–guest recognitions.

    To anchor onto the cell membranes of a certain tissue, it would require the receptors to diffuse and uniformly distribute among the cells in the tissue after injecting the receptors in situ. Therefore, multicellular tumor spheroids were selected to simulate the early stage of avascular tumor62 and to reveal the distribution of the artificial receptors. B16 and HepG2 were selected as two cell models. The two cell lines were mixed (the ratio of B16 to HepG2 cells was 5∶1) and then incubated for three days, the three-dimensional (3D) multicellular tumor spheroids were formed without using any matrigel (methylcellulose). After 24 h incubation with R2, the spheroids were washed with PBS buffer. As shown in Figure 4a, the entire 3D multicellular tumor spheroids display red FL of R2, indicating that the receptor can diffuse inside the 3D multicellular tumor spheroids. In addition, all three cross-sections (xy, yz, and xz) show bright FL (Figure 4b), suggesting the uniform distribution of R2 in the complex multicellular tumor spheroids. Thus, these artificial receptors should be able to anchor onto the cell surfaces of the selected tissues in vivo via intratumoral injection (i.t.).

    Figure 4

    Figure 4 | Artificial receptors mediated tissue targeting in vivo. The distribution of R2 in 3D multicellular spheroids: (a) 3D reconstruction of multicellular tumor spheroids after incubating with R2 (80 μM) after 24 h. (b) Cross-sectional images (x–y, y–z, and x–z) of 3D multicellular tumor spheroids. Anchoring ability of R2 to the B16 tumor in vivo. (c) Quantitative analysis of guests accumulation in B16 tumor based on the NBD fluorescent intensity from the ex vivo images in Supporting Information Figure S89. (d) Fluorescent images of B16 tumor sections to prove the ability of R2 to anchor on tumor and the tumor to recruit guests. Tumor targeting ability of R1 to the 4T1 tumor in vivo: (e) In vivo fluorescent images of 4T1-tumor-bearing mice after treatment at different time intervals. (f) The Cy 7.5 fluorescent intensity in the tumor site from the ROI (blue circle showed in (e)). (g) Quantitative analysis of liposome accumulation in 4T1 tumor and left thigh based on the Cy 7.5 fluorescent intensity from the ex vivo images in Supporting Information Figures S93 and S95, respectively. All data are presented as mean ± s.e.m (n = 3) and analyzed by one-way ANOVA (Tukey; 0.01 < *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

    For the in vivo experiments using guests (G2 and G4) alone, the mice were divided into three groups: (1) the control group: the mice were i.v. injected with 150 μL G2 (0.08 mM in PBS buffer) via the tail vein at 24 h after i.t. administration of 60 μL PBS buffer; (2) the G2 group: the mice were injected with 150 μL G2 (0.08 mM) via the tail vein at 24 h after i.t. administration of 60 μL R2 (0.2 mM); (3) the G4 group: the mice were i.v. injected with 150 μL G4 (0.08 mM) via the tail vein at 24 h after i.t. administration of 60 μL R2. After 24 h of guest injection, the mice were sacrificed and the tumor tissues were collected and analyzed by monitoring the FL of NBD of the guests ( Supporting Information Figure S89). As shown in Figure 4c, the fluorescent intensities of NBD for the G4 group and the G2 group are ca. 1.38- and 1.94-fold of that of the control group. For the control group, the FL should originate from nonspecific adsorption of the guest on cell membranes. Obviously, the existence of the receptors in the tissues can significantly improve the recruitment of the guests through bioorthogonal molecular recognition. Again, the brighter FL of the G2 group than the G4 group demonstrated that the accumulation level was mediated by the binding affinity: stronger binding leads to higher recruitment efficiency. Moreover, the red FL with similar intensity over the whole images of the G4 and G2 groups (Figure 4d) suggests that the receptor was uniformly distributed in the tumor tissues. The FL of R2 and NBD of the guests in the tumor sections of the G4 and G2 groups are significantly overlapped, and the fluorescent intensity of NBD of the guests is also related to the binding affinity. Collectively, these results show that the artificial receptors can in situ capture guests with moderate binding constants (1.6 × 104 M−1 for PEG2000 modified guests and 1.6 × 106 M−1 for 2-phenyl pyrimidine modified guests) in a bioorthogonal manner in vivo via monovalent host–guest recognition.

    The artificial receptors mediated tissue targeting and imaging of guest-modified liposomes were further studied on tumor tissues in vivo to reveal the applicability the receptors in complex systems. Muscle tissues were also studied at the same mice to confirm the reliability. Dye Cy 7.5 was encapsulated in the guest-modified liposomes (Figure 1e) for fluorescent imaging. To demonstrate the broad applicability in different tissues, 4T1 tumor and muscle tissues were selected instead of B16 tumor cells for the liposome-injection experiments. Other experimental procedures were similar to those for the guest injection experiments. The mice were also divided into three groups: the control group, the PEG group, and the G5 group. After i.v. administration of liposomes, the average Cy 7.5 fluorescent signals of the tumors were analyzed with an IVIS Spectrum in vivo imaging system at different time intervals (Figure 4e and Supporting Information Figures S90–S92). The quantitative fluorescent data of the tumor sites from the region of interest (ROI) are shown in Figure 4f. After 96 h, the tumor tissues and muscle tissues were collected and analyzed, and the data are shown in Figure 4g.

    As shown in Figure 4f, the areas under the curve (AUC) of the PEG group and the G5 group are ca. 1.30- and 1.58-fold of that of the control groups. The PEG group and the G5 group show better targeting efficiency than the control group whose FL should originate from Enhanced Permeability and Retention (EPR) effect of the tumor tissues. This again suggests that the artificial receptor can efficiently improve the tumor targeting efficiency of guest-modified liposomes due to bioorthogonal molecular recognition. The accumulation of liposomes in the tumor tissues (Figure 4f) can be divided into three stages: growth stage (1–24 h), plateau stage (the G5 group, 24–48 h; the PEG group, 24–36 h; the control group, 24–60 h), and clearance stage. During the growth stage, the fluorescent intensities of tumors preanchored with the artificial receptors increased linearly and rapidly (G5 group, y = 0.057+ 1.61, R2 = 0.93; PEG group, y = 0.036x + 1.52, R2 = 0.96), and reached the maximum at 24 h (G5 group, 1.63-fold increase from 1 to 24 h; PEG group, 1.44-fold increase from 1 to 24 h). This is in contrast to the very low intensity and poor linear regression exhibited by the control group (y = 0.008+ 1.37, R2 = 0.62), as well as the very modest increase from 1 to 24 h (1.17-fold increase). The fluorescent intensity in the tumor tissues of the PEG group and the G5 group are ca. 1.35- and 1.67-fold of that of control group, thus demonstrating the advantage of having artificial receptors as the targeting sites for improving tumor targeting efficiency of liposomes via multivalent binding effects. Conversely, the passive targeting via EPR effects of liposomes to the tumor was rather poor. During the plateau stage, the fluorescent intensity became steady, likely attributed to the saturation of nonspecific adsorption and specific adsorption based on multivalent host–guest recognition. Over time, the fluorescent intensity decreased during the clearance stage, due to the tumor metabolism and natural clearance of nanoparticles. Even at 96 h, the fluorescent intensities of the tumors preanchored with the artificial receptors are still stronger than that at 1 h (G5 group, 1.46-fold of that at 1 h; PEG group, 1.29-fold of that at 1 h). This is in contrast to the low fluorescent intensity of the tumor from the control group (1.06-fold of that at 1 h). Moreover, the fluorescent intensity of the PEG group and the G5 group are still ca. 1.34- and 1.66-fold of that of the control group, after 96 h.

    To accurately quantify the improvement in the targeting efficiency mediated by the artificial receptor, the tumor tissues were collected after 96 h and their fluorescent signals of Cy 7.5 were analyzed by an IVIS Spectrum imaging system ( Supporting Information Figure S93). The average fluorescent intensity of the tumors (Figure 4g) was consistent with previously discussed for the in vivo data. The G5 group showed significant enhancement when compared with the control group, and the fluorescent intensity of the former is 1.65-fold of that of the latter. The PEG group is also better than the control group with 1.45-fold enhancement. In addition, the experiments with the tumor sections further support this conclusion ( Supporting Information Figure S94). The experimental results from the muscle tissues confirmed the results and conclusion from the tumor tissues. The average fluorescent intensities of the left thighs (Figure 4g and Supporting Information Figure S95) in the PEG group and the G5 group are ca. 1.58- and 1.91-fold of that of the control group. The experiments with the muscle sections show a similar trend ( Supporting Information Figure S96). Therefore, the artificial receptors can also efficiently improve the muscle targeting efficiency of guest-modified liposomes via multivalent host–guest recognition. However, the FL intensities in the muscle tissues are almost half of that of the tumor tissues of the corresponding groups. In the tumor tissues, EPR effect can enhance the adsorption of liposomes67; while in the muscle tissues, the background interaction is only from nonspecific adsorption. Overall, the two different tissues show consistent trends of improvement in targeting efficiency. In all experiments, the targeting efficiency was mediated by the binding affinity: stronger binding leads to higher targeting efficiency. All these experimental results lead to a reliable conclusion: these artificial receptors can improve the tissue targeting of guests and guest-modified liposomes through bioorthogonal molecular recognition.


    We reported a bioorthogonal host–guest pair between anti-configured amide naphthotube and 2- phenyl pyrimidine. This biomimetic binding pair is held together through shielded hydrogen bonding and hydrophobic effects and is thus very selective to each other even in complex biological media. By anchoring the modified host through a TPE moiety, the charged amide naphthotube is left on cell surfaces for molecular recognition. These receptors showed excellent capabilities to be stably anchored onto cell membranes (lasting for over 12 days). Cell experiments demonstrate that the bioorthogonal host–guest pair is able to recognize each other on cell surfaces and enable cell–cell interactions. Moreover, the artificial receptors can work as targeting sites to significantly improve the targeting efficiency of guest species and guest-modified liposomes via monovalent and multivalent host–guest recognition in living animals, respectively, when compared with the mere EPR effects of passive targeting. This can be applied for cell targeting and tissue imaging. Surprisingly, improved targeting efficiency was even observed for the guest (polyethylene glycol) with a low binding affinity (∼104 M−1). Such low binding affinity is usually considered to be insufficient for biological application, but this result further supports the bioorthogonal nature of the naphthotube-based host–guest pairs. This research provides a new noncovalent bioconjugation tool and a new concept for designing bioorthogonal host–guest pairs for biological applications.

    Supporting Information

    Supporting Information is available and includes experimental details, general information about materials and methods, synthesis and characterizations, NMR and mass spectra of the compounds, ITC titration data, and X-ray single-crystal data for (E)-Bis(OH)-TPE and (Z)-Bis(OH)-TPE, cytotoxicity of artificial receptors, CLSM data for anchoring ability of artificial receptors to the membrane surface of cells, biocompatibility evaluation of artificial receptors in a mouse model, Ex vivo fluorescent images of major organs, tumors and left thigh, and in vivo fluorescent images of the 4T1-tumor-bearing mice at different time intervals.

    Conflicts of Interest

    The authors declare no competing financial interest.


    This research was financially supported by National Natural Science Foundation of China (nos. 21772083 and 21822104), the Shenzhen Special Funds (no. JCYJ20180504165810828), the Guangdong Provincial Key Laboratory of Catalysis (no. 2020B121201002), the University of Macau (no. MYRG2019-00059-ICMS), the Shenzhen “Pengcheng Scholar” Program, and Guangdong High-Level Personnel of Special Support Program (no. 2019TX05C157). The authors thank SUSTech-CRF for the technical support.


    • 1. Prescher J. A.; Bertozzi C. R.Chemistry in Living Systems.Nat. Chem. Biol.2005, 1, 13–21. Google Scholar
    • 2. Algar W. R.; Dawson P. E.; Medintz I. L.Chemoselective and Bioorthogonal Ligation Reactions: Concepts and Applications; Wiley-VCH: Weinheim, 2017. Google Scholar
    • 3. Rossin R.; Robillard M. S.Pretargeted Imaging Using Bioorthogonal Chemistry in Mice.Curr. Opin. Chem. Biol.2014, 21, 161–169. Google Scholar
    • 4. Chatterji D.Basics of Molecular Recognition; CRC Press: Boca Raton, FL, 2016. Google Scholar
    • 5. McMahon R. J.Avidin-Biotin Interactions: Methods and Applications; Humana Press: Totowa, NJ, 2008. Google Scholar
    • 6. Schreiber C. L.; Smith B. D.Molecular Conjugation Using Non-Covalent Click Chemistry.Nat. Rev. Chem.2019, 3, 393–400. Google Scholar
    • 7. Lee J.-H.; Chen K. -J.; Noh S. -H.; Garcia M. A.; Wang H.; Lin W. -Y.; Jeong H.; Kong B. J.; Stout D. B.; Cheon J.; Tseng H.-R.On-Demand Drug Release System for in Vivo Cancer Treatment through Self-Assembled Magnetic Nanoparticles.Angew. Chem. Int. Ed.2013, 52, 4384–4388. Google Scholar
    • 8. Wang L.; Li L. -L.; Fan Y. -S.; Wang H.Host–Guest Supramolecular Nanosystems for Cancer Diagnostics and Therapeutics.Adv. Mater.2013, 25, 3888–3898. Google Scholar
    • 9. Gao C.; Cheng Q.; Wei J.; Sun C.; Lu S.; Kwong C. H. T.; Lee S. M. Y.; Zhong Z.; Wang R.Bioorthogonal Supramolecular Cell-Conjugation for Targeted Hitchhiking Drug Delivery.Mater. Today2020, 40, 9−17. Google Scholar
    • 10. Zhou J.; Rao L.; Yu G.; Cook T. R.; Chen X.; Huang F.Supramolecular Cancer Nanotheranostics.Chem. Soc. Rev.2021, 50, 2839–2891. Google Scholar
    • 11. Wang H.; Yan Y.-Q.; Yi Y.; Wei Z.-Y.; Chen H.; Xu J.-F.; Wang H.; Zhao Y.; Zhang X.Supramolecular Peptide Therapeutics: Host–Guest Interaction-Assisted Systemic Delivery of Anticancer Peptides.CCS Chem.2020, 2, 739–748. AbstractGoogle Scholar
    • 12. Wang H.; Wu H.; Yi Y.; Xue K.-F.; Xu J.-F.; Wang H.; Zhao Y.; Zhang X.Self-Motivated Supramolecular Combination Chemotherapy for Overcoming Drug Resistance Based on Acid-Activated Competition of Host-Guest Interactions.CCS Chem.2021, 3, 1413–1425. AbstractGoogle Scholar
    • 13. Peck E. M.; Liu W.; Shaw S. K.; Spence G. T.; Davis A. P.; Destecroix H.; Smith B. D.Rapid Macrocycle Threading by a Fluorescent Dye-Polymer Conjugate in Water with Nanomolar Affinity.J. Am. Chem. Soc.2015, 137, 8668–8671. Google Scholar
    • 14. Peck E. M.; Battles P. M.; Rice D. R.; Roland F. M.; Norquest K. A.; Smith B. D.Pre-Assembly of Near-Infrared Fluorescent Multivalent Molecular Probes for Biological Imaging.Bioconjugate Chem.2016, 27, 1400–1410. Google Scholar
    • 15. Roland F. M.; Peck E. M.; Rice D. R.; Smith B. D.Preassembled Fluorescent Multivalent Probes for the Imaging of Anionic Membranes.Bioconjugate Chem.2017, 28, 1093–1101. Google Scholar
    • 16. Yu G.; Chen X.Host-Guest Chemistry in Supramolecular Theranostics.Theranostics2019, 9, 3041–3074. Google Scholar
    • 17. Xu Z.; Jia S.; Wang W.; Yuan Z.; Ravoo B. J.; Guo D.-S.Heteromultivalent Peptide Recognition by Co-Assembly of Cyclodextrin and Calixarene Amphiphiles Enables Inhibition of Amyloid Fibrillation.Nat. Chem.2019, 11, 86–93. Google Scholar
    • 18. Agasti S. S.; Liong M.; Tassa C.; Chung H. J.; Shaw S. Y.; Lee H.; Weissleder R.Supramolecular Host–Guest Interaction for Labeling and Detection of Cellular Biomarkers.Angew. Chem. Int. Ed.2012, 51, 450–454. Google Scholar
    • 19. Murray J.; Sim J.; Oh K.; Sung G.; Lee A.; Shrinidhi A.; Thirunarayanan A.; Shetty D.; Kim K.Enrichment of Specifically Labeled Proteins by an Immobilized Host Molecule.Angew. Chem. Int. Ed.2017, 56, 2395–2398. Google Scholar
    • 20. Kim K. L.; Sung G.; Sim J.; Murray J.; Li M.; Lee A.; Shrinidhi A.; Park K. M.; Kim K.Supramolecular Latching System Based on Ultrastable Synthetic Binding Pairs as Versatile Tools for Protein Imaging.Nat. Commun.2018, 9, 1712. Google Scholar
    • 21. Li M.; Lee A.; Kim K. L.; Murray J.; Shrinidhi A.; Sung G.; Park K. M.; Kim K.Autophagy Caught in the Act: A Supramolecular FRET Pair Based on an Ultrastable Synthetic Host-Guest Complex Visualizes Autophagosome-Lysosome Fusion.Angew. Chem. Int. Ed.2018, 57, 2120–2125. Google Scholar
    • 22. An J.; Kim S.; Shrinidhi A.; Kim J.; Banna H.; Sung G.; Park K. M.; Kim K.Purification of Protein Therapeutics via High-Affinity Supramolecular Host–Guest Interactions.Nat. Biomed. Eng.2020, 4, 1044–1052. Google Scholar
    • 23. Sasmal R.; Saha N. D.; Pahwa M.; Rao S.; Joshi D.; Inamdar M. S.; Sheeba V.; Agasti S. S.Synthetic Host–Guest Assembly in Cells and Tissues: Fast, Stable, and Selective Bioorthogonal Imaging via Molecular Recognition.Anal. Chem.2018, 90, 11305–11314. Google Scholar
    • 24. Sung G.; Lee S.-Y.; Kang M.-G.; Kim K. L.; An J.; Sim J.; Kim S.; Kim S.; Ko J.; Rhee H.-W.; Park K. M.; Kim K.Supra-Blot: An Accurate and Reliable Assay for Detecting Target Proteins with a Synthetic Host Molecule–Enzyme Hybrid.Chem. Commun.2020, 56, 1549–1552. Google Scholar
    • 25. Cao W.; Qin X.; Wang Y.; Dai Z.; Dai X.; Wang H.; Xuan W.; Zhang Y.; Liu Y.; Liu T.A General Supramolecular Approach to Regulate Protein Functions by Cucurbit[7]uril and Unnatural Amino Acid Recognition.Angew. Chem. Int. Ed.2021, 60, 11196–11200. Google Scholar
    • 26. Som A.; Pahwa M.; Bawari S.; Saha N. D.; Sasmal R.; Bosco M. S.; Mondal J.; Agasti S. S.Multiplexed Optical Barcoding of Cells via Photochemical Programming of Bioorthogonal Host-Guest Recognition.Chem. Sci.2021, 12, 5484–5494. Google Scholar
    • 27. Schrader T.; Bitan G.; Klärner F.-G.Molecular Tweezers for Lysine and Arginine—Powerful Inhibitors of Pathologic Protein Aggregation.Chem. Commun.2016, 52, 11318–11334. Google Scholar
    • 28. Malik R.; Meng H.; Wongkongkathep P.; Corrales C. I.; Sepanj N.; Atlasi R. S.; Klärner F.-G.; Schrader T.; Spencer M. J.; Loo J. A.; Wiedau M.; Bitan G.The Molecular Tweezer CLR01 Inhibits Aberrant Superoxide Dismutase 1 (SOD1) Self-Assembly in Vitro and in the G93A-SOD1 Mouse Model of ALS.J. Biol. Chem.2019, 294, 3501–3513. Google Scholar
    • 29. Herrera-Vaquero M.; Bouquio D.; Kallab M.; Biggs K.; Nair G.; Ochoa J.; Heras-Garvin A.; Heid C.; Hadrovic I.; Poewe W.; Wenning G. K.; Klärner F.-G.; Schrader T.; Bitan G.; Stefanova N.The Molecular Tweezer CLR01 Reduces Aggregated, Pathologic, and Seeding-Competent α-Synuclein in Experimental Multiple System Atrophy.BBA-Mol. Basis. Dis.2019, 1865, 165513. Google Scholar
    • 30. Di J.; Siddique I.; Li Z.; Malki G.; Hornung S.; Dutta S.; Hurst I.; Ishaaya E.; Wang A.; Tu S.; Boghos A.; Ericsson I.; Klärner F.-G.; Schrader T.; Bitan G.The Molecular Tweezer CLR01 Improves Behavioral Deficits and Reduces Tau Pathology in P301S-Tau Transgenic Mice.Alzheimers Res. Ther.2021, 13, 6. Google Scholar
    • 31. Zou L.; Braegelman A. S.; Webber M. J.Spatially Defined Drug Targeting by in Situ Host–Guest Chemistry in a Living Animal.ACS Cent. Sci.2019, 5, 1035–1043. Google Scholar
    • 32. Li M.; Kim S.; Lee A.; Shrinidhi A.; Ko Y. H.; Lim H. G.; Kim H. H.; Bae K. B.; Park K. M.; Kim K.Bio-Orthogonal Supramolecular Latching Inside Live Animals and Its Application for in Vivo Cancer Imaging.ACS Appl. Mater. Interfaces2019, 11, 43920–43927. Google Scholar
    • 33. Spa S. J.; Welling M. M.; van Oosterom M. N.; Rietbergen D. D. D.; Burgmans M. C.; Verboom W.; Huskens J.; Buckle T.; van Leeuwen F. W. B.A Supramolecular Approach for Liver Radioembolization.Theranostics2018, 8, 2377–2386. Google Scholar
    • 34. Welling M. M.; Spa S. J.; van Willigen D. M.; Rietbergen D. D. D.; Roestenberg M.; Buckle T.; van Leeuwen F. W. B.In Vivo Stability of Supramolecular Host-Guest Complexes Monitored by Dual-Isotope Multiplexing in a Pre-Targeting Model of Experimental Liver Radioembolization.J. Control. Release2019, 293, 126–134. Google Scholar
    • 35. Barrow S. J.; Kasera S.; Rowland M. J.; Del Barrio J.; Scherman O. A.Cucurbituril-Based Molecular Recognition.Chem. Rev.2015, 115, 12320–12406. Google Scholar
    • 36. Escobar L.; Ballester P.Molecular Recognition in Water Using Macrocyclic Synthetic Receptors.Chem. Rev.2021, 121, 2445–2514. Google Scholar
    • 37. Dong J.; Davis A. P.Molecular Recognition Mediated by Hydrogen Bonding in Aqueous Media.Angew. Chem. Int. Ed.2021, 60, 8035–8048. Google Scholar
    • 38. Shetty D.; Khedkar J. K.; Park K. M.; Kim K.Can We Beat the Biotin–Avidin Pair?: Cucurbit[7]uril-Based Ultrahigh Affinity Host–Guest Complexes and Their Applications.Chem. Soc. Rev.2015, 44, 8747–8761. Google Scholar
    • 39. Liu W.; Samanta S. K.; Smith B. D.; Isaacs L.Synthetic Mimics of Biotin/(Strept)avidin.Chem. Soc. Rev.2017, 46, 2391–2403. Google Scholar
    • 40. Murray J.; Kim K.; Ogoshi T.; Yao W.; Gibb B.The Aqueous Supramolecular Chemistry of Cucurbit[n]urils, Pillar[n]arenes and Deep-Cavity Cavitands.Chem. Soc. Rev.2017, 46, 2479–2496. Google Scholar
    • 41. Park K. M.; Murray J.; Kim K.Ultrastable Artificial Binding Pairs as a Supramolecular Latching System: A Next Generation Chemical Tool for Proteomics.Acc. Chem. Res.2017, 50, 644–646. Google Scholar
    • 42. Houk K. N.; Leach A. G.; Kim S. P.; Zhang X.Binding Affinities of Host-Guest, Protein-Ligand, and Protein-Transition-State Complexes.Angew. Chem. Int. Ed.2003, 42, 4872–4897. Google Scholar
    • 43. Yang L.-P.; Wang X.; Yao H.; Jiang W.Naphthotubes: Macrocyclic Receptors with a Biomimetic Cavity Feature.Accounts Chem. Res.2020, 53, 198–208. Google Scholar
    • 44. Huang G.-B.; Wang S.-H.; Ke H.; Yang L.-P.; Jiang W.Selective Recognition of Highly Hydrophilic Molecules in Water by Endo-Functionalized Molecular Tubes.J. Am. Chem. Soc.2016, 138, 14550–14553. Google Scholar
    • 45. Wang L.-L.; Chen Z.; Liu W.-E.; Ke H.; Wang S.-H.; Jiang W.Molecular Recognition and Chirality Sensing of Epoxides in Water Using Endo-Functionalized Molecular Tubes.J. Am. Chem. Soc.2017, 139, 8436–8439. Google Scholar
    • 46. Yao H.; Ke H.; Zhang X.; Pan S.-J.; Li M.-S.; Yang L.-P.; Schreckenbach G.; Jiang W.Molecular Recognition of Hydrophilic Molecules in Water by Combining the Hydrophobic Effect with Hydrogen Bonding.J. Am. Chem. Soc.2018, 140, 13466–13477. Google Scholar
    • 47. Wang L.-L.; Quan M.; Yang T.-L.; Chen Z.; Jiang W.A Green and Wide-Scope Approach for Chiroptical Sensing of Organic Molecules through Biomimetic Recognition in Water.Angew. Chem. Int. Ed.2020, 59, 23817–23824. Google Scholar
    • 48. Chai H.; Chen Z.; Wang S.-H.; Quan M.; Yang L.-P.; Ke H.; Jiang W.Enantioselective Recognition of Neutral Molecules in Water by a Pair of Chiral Biomimetic Macrocyclic Receptors.CCS Chem.2020, 2, 440–452. AbstractGoogle Scholar
    • 49. Ma Y.-L.; Quan M.; Lin X.-L.; Cheng Q.; Yao H.; Yang X.-R.; Li M.-S.; Liu W.-E.; Bai L.-M.; Wang R.; Jiang W.Biomimetic Recognition of Organic Drug Molecules in Water by Amide Naphthotubes.CCS Chem.2020, 2, 1078−1092. Google Scholar
    • 50. Ke H.; Yang L.-P.; Xie M.; Chen Z.; Yao H.; Jiang W.Shear-Induced Assembly of a Transient yet Highly Stretchable Hydrogel Based on Pseudopolyrotaxanes.Nat. Chem.2019, 11, 470–477. Google Scholar
    • 51. Chen J.; Li S.; Wang Z.; Pan Y.; Wei J.; Lu S.; Zhang Q.-W.; Wang L.-H.; Wang R.Synthesis of an AIEgen Functionalized Cucurbit[7]uril for Subcellular Bioimaging and Synergistic Photodynamic Therapy and Supramolecular Chemotherapy.Chem. Sci.2021, 12, 7727–7734. Google Scholar
    • 52. Liang G.; Lam J. W. Y.; Qin W.; Li J.; Xie N.; Tang B. Z.Molecular Luminogens Based on Restriction of Intramolecular Motions through Host–Guest Inclusion for Cell Imaging.Chem. Commun.2014, 50, 1725–1727. Google Scholar
    • 53. Yang K.; Wen J.; Chao S.; Liu J.; Yang K.; Pei Y.; Pei Z.A Supramolecular Photosensitizer System Based on the Host-Guest Complexation between Water-Soluble Pillar[6]arene and Methylene Blue for Durable Photodynamic Therapy.Chem. Commun.2018, 54, 5911–5914. Google Scholar
    • 54. Pan Y.-C.; Barba-Bon A.; Tian H.-W.; Ding F.; Hennig A.; Nau W. M.; Guo D.-S.An Amphiphilic Sulfonatocalix[5]arene as an Activator for Membrane Transport of Lysine-Rich Peptides and Proteins.Angew. Chem. Int. Ed.2021, 60, 1875–1882. Google Scholar
    • 55. Barba-Bon A.; Pan Y.-C.; Biedermann F.; Guo D.-S.; Nau W. M.; Hennig A.Fluorescence Monitoring of Peptide Transport Pathways into Large and Giant Vesicles by Supramolecular Host-Dye Reporter Pairs.J. Am. Chem. Soc.2019, 141, 20137–20145. Google Scholar
    • 56. Yang M.; Brackenbury W. J.Membrane Potential and Cancer Progression.Front. Physiol.2013, 4, 185. Google Scholar
    • 57. Mei J.; Leung N. L. C.; Kwok R. T. K.; Lam J. W. Y.; Tang B. Z.Aggregation-Induced Emission: Together We Shine, United We Soar!Chem. Rev.2015, 115, 11718–11940. Google Scholar
    • 58. Feng G., Kwok R. T. K.; Tang B. Z.; Liu B.Functionality and Versatility of Aggregation-Induced Emission Luminogens.Appl. Phys. Rev.2017, 4, 021307. Google Scholar
    • 59. Qian J.; Tang B. Z.AIE Luminogens for Bioimaging and Theranostics: From Organelles to Animals.Chem2017, 3, 56–91. Google Scholar
    • 60. Xu Sh ; Duan Y.; Liu B.Precise Molecular Design for High-Performance Luminogens with Aggregation-Induced Emission.Adv. Mater.2020, 32, 1903530. Google Scholar
    • 61. Wang D.; Su H.; Kwok R. T. K.; Hu X.; Zou H.; Luo Q.; Lee M. M. S.; Xu W.; Lam J. W. Y.; Tang B. Z.Rational Design of a Water-Soluble NIR AIEgen, and Its Application in Ultrafast Wash-Free Cellular Imaging and Photodynamic Cancer Cell Ablation.Chem. Sci.2018, 9, 3685–3693. Google Scholar
    • 62. Shi L.; Liu Y.-H.; Li K.; Sharma A.; Yu K.-K.; Ji M. S.; Li L.-L.; Zhou Q.; Zhang H.; Kim J. S.; Yu X.-Q.An AIE-Based Probe for Rapid and Ultrasensitive Imaging of Plasma Membranes in Biosystems.Angew. Chem. Int. Ed.2020, 59, 9962–9966. Google Scholar
    • 63. Jia H.-R.; Wang H.-Y.; Yu Z.-W.; Chen Z.; Wu F.-G.Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy.Bioconjugate Chem.2016, 27, 782–789. Google Scholar
    • 64. Jia H.-R.; Zhu Y.-X.; Xu K.-F.; Pan G.-Y.; Liu X.; Qiao Y.; Wu F.-G.Efficient Cell Surface Labelling of Live Zebrafish Embryos: Wash-Free Fluorescence Imaging for Cellular Dynamics Tracking and Nanotoxicity Evaluation.Chem. Sci.2019, 10, 4062–4068. Google Scholar
    • 65. Yao H.; Wang X.; Xie M.; Wang Y.-M.; Quan M.; Yang L.-P.; Jiang W.Mono-Functionalized Derivatives and Revised Configurational Assignment of Amide Naphthotubes.Org. Biomol. Chem.2020, 18, 1900–1909. Google Scholar
    • 66. Mosmann T.Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays.J. Immunol. Methods1983, 65, 55–63. Google Scholar
    • 67. Maeda H.; Wua J.; Sawaa T.; Matsumurab Y.; Horic K.Tumor Vascular Permeability and the EPR Effect in Macromolecular Therapeutics: A Review.J. Control. Release2000, 65, 271–284. Google Scholar