Letter pubs.acs.org/macroletters
Fluorescent Cell-Conjugation by a Multifunctional Polymer: A New Application of the Hantzsch Reaction Qiang Sun,†,‡ Guoqiang Liu,†,‡ Haibo Wu,‡,§ Haodong Xue,‡,§ Yuan Zhao,‡ Zilin Wang,‡ Yen Wei,‡ Zhiming Wang,§ and Lei Tao*,‡ ‡
The Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China § College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 311400, People’s Republic of China S Supporting Information *
ABSTRACT: Multicomponent reactions (MCRs) can form unique structures with interesting functions, therefore, multifunctional polymers might be simply prepared using MCRs as coupling tools to simultaneously link and generate different functional groups. To verify this concept, a new fluorescent polymer containing phenylboronic acid has been facilely prepared via a one pot method by combining the Hantzsch reaction with reversible addition−fragmentation chain transfer (RAFT) polymerization. The Hantzsch-RAFT system has been found robust to smoothly achieve predesigned multifunctional polymer, which can be used for cell conjugation through the interaction between phenylboronic acid and glycoprotein on cell membrane. The conjugated cells could be directly observed due to the fluorescent Hantzsch moiety in the polymer chain, demonstrating a new application of the old Hantzsch reaction (>130 years) outside organic chemistry. Meanwhile, the conjugated cells remained excellent dispersity in the presence of coagulation protein (lectin), implying that multifunctional polymer a possible anticoagulant for cell separation. We believe that the current research paves a new way to exploit new applications of MCRs in interdisciplinary fields and might prompt the development of other multifunctional polymers based on different MCRs.
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sometimes protective groups are necessary, limiting the largescale manufacture, further studies, and broad applications of those multifunctional polymers though they might have excellent properties. Recently, Meier and co-workers creatively introduced tricomponent Passerini reaction into polymer chemistry.1−3 Afterward, a series of multicomponent reactions (MCRs), some of which are even older than 100 years, such as the Passerini,4,5 Ugi,6−8 Biginelli (>120 years),9,10 Kabachnik-Fields (K− F),11−13 Hantzsch (>130 years),14−16 mecaptoacetic acid locking imine (MALI),17 Mannich,18,19 epoxy-based,20,21 thiolactone-based,22,23 metal-catalyzed multicomponent reactions,24−27 and so on have been reconsidered from the perspective of polymer chemistry to prepare new polymers.7,28,29 Various new functional polymers that contain specific MCR moieties, including α-acyloxy amide, bis-amide, amino-phosphonate, pyrrole,30 and so on, as backbones or side chains have been successfully developed as new members of the polymer family, expanding the application of MCRs outside organic chemistry.31,32
opolymerization of different monomers might be the most effective and direct strategy to synthesize multifunctional polymers. After synthesis of several functional monomers, a multifunctional polymer can be effectively obtained through suitable polymerization process (Scheme 1a). However, synthesis and purification of different functional monomers are normally laborious and time-consuming, and Scheme 1. Comparison of (a) Traditional and (b) One-Pot MCRs-CRPs Approaches To Prepare Multifunctional Polymers
Received: March 22, 2017 Accepted: April 28, 2017
© XXXX American Chemical Society
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Figure 1. (a) One-pot Hantzsch-RAFT process to prepare the polymer A, [AEMA]/[PEGMA]/[CTA]/[ABVN]/[dimedone]/[4formylphenylboronic]/[ammoniu-macetate]/[glycine] = 100/200/3/0.75/100/100/150/15, [AEMA] = 1.0 M in 2.0 mL of CH3CN, 70 °C; (b) the kinetics study of the Hantzsch-RAFT process; (c) 1H NMR spectrum (10% D2O in DMSO-d6) of the purified polymer A (inset spectrum: 11B NMR spectrum of polymer A; photo: fluorescent polymer A solution (10 mg/mL in H2O)).
Besides introducing “new” organic reactions into polymer area, new polymerization strategies have also been developed to rapidly prepare delicate polymers. For example, controllable radical polymerizations (CRPs) have excellent reputation for their robustness,33−35 therefore, some highly efficient coupling reactions such as Cu(I) catalyzed alkyne−azide cycloaddition (CuAAC), transesterification, alcohol/amine-isocyanate reactions and so on36−39 have been combined with CRPs to construct graceful one-pot systems, where those organic reactions occurred during CRPs to simultaneously generate new monomers and modify polymer chain-ends or growing polymer chains to finally achieve elegant new polymers in one step. Inspired by all the fancy research, our group established a link between the MCRs and CRPs to combine the MCR with CRP in one reactor. As a result, a number of carefully screened MCRs, including the tricomponent Biginelli,9 KF reactions,12 and the tetra-component Hantzsch,14 Ugi reactions40 and so on, have been successfully applied to construct several one-pot MCR-CRP systems. During the study on MCRs, we realized that MCRs might be suitable tools to synthesize multifunctional polymers since they can effectively introduce functional groups like other efficient two-component coupling reactions through the generation of unique multicomponent linkages which have special physicochemical and biological/pharmaceutical properties. To verify that concept, we report herein a straightforward
strategy to synthesize multifunctional polymers by using MCR as a generator of new function and a coupling tool (Scheme 1b). The four-component Hantzsch reaction which creates innately fluorescent 1,4-dihydropyridine (1,4-DHP) was employed as a model MCR and a well-defined fluorescent polymer for cell conjugation was thereof explored. Phenylboronic acid contained (co)polymers have been widely studied as promising biomaterials for drug delivery,41−46 hydrogel,47,48 sensor,49,50 and cell manipulation and separation.51−53 In current work, a well-defined multifunctional polymer can be simply obtained by introducing the phenylboronic acid into the polymer structure via fluorescent 1,4DHP linkages through the one-pot Hantzsch-RAFT process (Figure 1a). This multifunctional polymer can work like other phenylboronic acid polymers while the fluorescent 1,4-DHP group can provide visible information for direct observation. The target polymer was prepared following our previous report.14 Briefly, a commercially available monomer 2(acetoacetoxy)ethyl methacrylate (AEMA), poly(ethylene glycol methyl ether) methacrylate (PEGMA) (comonomer), dimedone, ammonium acetate (1.5 equiv to AEMA for complete Hantzsch reaction), 4-formylphenylboronic, and glycine (catalyst, 15% to aldehyde) were mixed in acetonitrile; then, 2,2′-azobis(2,4-dimethylvaleronitrile) (ABVN, initiator) and 4-cyano-4-(ethylthiocarbonothioylthio) pentanoic acid (chain transfer agent (CTA)) were added. After three 551
DOI: 10.1021/acsmacrolett.7b00220 ACS Macro Lett. 2017, 6, 550−555
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Figure 2. (a) Binding studies of a ternary system consisting of alizarin red s (ARS, I), saccharides, and THBA; (b) binding constants (Ks) of the THBA−saccharides complex (calcd by dividing the value of the KARS by the slope of the [S]/P vs Q plot, according to the Benesi−Hildebrand method).
Figure 3. In vitro cell bonding experiment. (a, a’) L929 cells incubated with polymer A (a: bright field image, a’: fluorescent image); (b, b’) L929 cells incubated with polymer B (b: bright field image, b’: fluorescent image).
polymer was calculated by the integral ratio between the methyl group in the PEGMA block (∼3.10 ppm) and the Hantzsch ring (∼4.78 ppm) as 1:2.2, consistent with the feeding ration (1:2, Table S1). Similarly, different polymers with designed 1,4DHP/PEG ratios could be obtained by simply tuning the feeding ratios (Table S1), confirming the Hantzsch-RAFT system a trustworthy method to precisely prepare multifunctional polymers. To test the combining capacity of the phenylboronic acid moieties in polymer A with saccharides, a small 1,4-DHP molecule (similar structure like the polymer side group) (4-(3(ethoxycarbonyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinolon-4-yl)phenyl) boronic acid (THBA) was synthesized and used as the model to perform a competitive binding experiment (Figure 2a). According to reported assay,54 the binding constant KARS between THBA and Alizarin Red S (ARS), a catechol dye whose fluorescent intensity increases upon binded boronic acid, was detected as KARS = 1611 M−1 (Figure S3), similar to the constant between phenylboronic acid and ARS (∼1250 M−1; reported value: ∼ 1300 M−1).54 Then, the KARS was used to calculate the binding constant (K) between THBA and saccharide. Binding experiments between THBA and different saccharides (fructose, mannitol, and sorbose) were conducted (pH ∼ 7.4), and the binding constants of THBA-fructose, THBA-mannitol, and THBAsorbose were calculated as 96.5, 95.9, and 63.0 M−1, respectively (Figure 2b). The same protocol was also
freeze−pump−thaw cycles to remove oxygen, the mixture was kept in a 70 °C oil bath, samples were taken periodically under N2 atmosphere for 1H NMR and gel-permeation chromatography (GPC) analyses. After 2 h, the polymerization was stopped and the final multifunctional polymer containing both phenylboronic acid group and fluorescent 1,4-DHP structure was obtained after simple precipitation in diethyl ether and named as polymer A. Conversions versus time and the corresponding kinetic curve were presented to evaluate reaction features of the Hantzsch-RAFT system (Figure 1b1). The Hantzsch reaction proceeded smoothly in this one-pot system and finally reached up to ∼97% in 2 h, and the RAFT process finally reached ∼70% conversion in 2 h following a linear pseudo first-order kinetic curve, confirming the compatibility between the Hantzsch reaction and RAFT polymerization. The polymer samples have controlled molecular weights with narrow PDIs (≤1.20; Figure 1b2), indicating the controllable feature of this Hantzsch-RAFT system. From the 1H NMR spectrum of the purified polymer A (Figure 1c), the distinctive Hantzsch ring (4.78 ppm) could be clearly identified, the polymer A also showed an obvious signal in 11B NMR (inserted spectrum, Figure 1c), while its aqueous solution (10 mg/mL) was fluorescent (inset photo, Figure 1c, Figure S1), indicating the phenylboronic acid has been included in the polymer structure via the 1,4-DHP linkage. The prepared polymer A has controlled molecular weight and narrow PDI (∼1.20), and the molar ratio of the 1,4-DHP group in the 552
DOI: 10.1021/acsmacrolett.7b00220 ACS Macro Lett. 2017, 6, 550−555
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ACS Macro Letters conducted to measure the binding constants between phenylboronic acid and saccharides (not presented data), and the bonding constants of phenylboronic acid with saccharides are similar to those of THBA, suggesting the multicomponent compound THBA contained both 1,4-DHP and phenylboronic acid groups also has efficient saccharide-recognition capability like phenylboronic acid. It is well-known the cell membrane is covered with many glycoproteins, and polymers containing phenylboronic acid have been used to recognize and regulate cell behavior through the interaction between glycoprotein and phenylboronic acid.53,55,56 However, complex and expensive analytical methods are normally appealed to detect the conjugated polymers on cell membrane. Therefore, the fluorescent polymer A containing phenylboronic acid might provide a simple and direct way to detect the conjugated polymer on cell surface. To test that hypothesis, an in vitro cell bonding experiment was carried out. A cell line of fibroblast derived from mice (L929) was used as the model since L929 cell has been widely used as an excellent cell model (easy culture and well-known growth law) in the field of biomedical research. The cells were incubated in a PBS solution of polymer A (100 mg/mL, pH ∼ 7.4) for 10 min, then washed with PBS solution (pH ∼ 7.4) twice prior to observation. The blue fluorescent cells can be clearly observed under microscope due to the conjugated polymer A (Figure 3a’). A control polymer was also synthesized using benzaldehyde instead of 4-formylphenylboronic via the same Hantzsch-RAFT process, and named as polymer B which is also fluorescent (Figure S2). When polymer B was used to treat cells through the same process, no fluorescent signal could be observed (Figure 3b’), suggesting the phenylboronic acid moiety in polymer A plays a crucial role to anchor the polymer on cell membrane. Similarly, Hela cells were also used through the same process and the blue fluorescent cell-conjugate can be clearly observed under microscope (Figure S4), which suggested that the polymer A is a universal compound for visible cell-conjugation. Through the reaction between glycoprotein on cell membrane and phenylboronic acid in polymer A, the polymer chains can surround cells like a blanket to shield the recognition sites on cell surface, leading to the cell separation in the presence of coagulation proteins (Figure 4a). Therefore, a cell agglutination experiment was conducted to evaluate the screening effect. L929 cells were treated with polymer A as above-mentioned (Figure 4b), then lectin (a model coagulation protein) was added to the cell suspension. The polymer A conjugated cells remained separation with existing lectin, which can be easily observed by visible fluorescence (Figure 4b’), suggesting the polymer masked the cell surface well as an anticoagulant to prevent the agglutination. Subsequently, high concentrated fructose solution (10 mg/mL) was added to the mixture to partly peel off the polymer A from cells’ surface, ∼5% polymer A was removed according to the fluorescence analysis (Figure S5) and cells were observed to aggregate immediately (Figure 4b”), verifying that lectin is a really efficient protein for cell coagulation, even only 5% of the polymer A was removed from cells’ surface, the amount of exposed glycoprotein on the cell surface is enough to be recognized by lectin, leading to quick cell aggregation. As a control, the polymer B was also tested through the same process. Cell aggregation occurred after addition of lectin (Figure 4c’), and the aggregated cells could
Figure 4. (a) Schematic diagram of the anticoagulation of conjugated polymer on cell surface; (b, b’, b”) images of cells by different treatments, b: cell + polymer A, b’: cell + polymer A + lectin, b”: cell + polymer A + lectin + fructose; (c, c’, c”) images of cells by different treatments, c: cell + polymer B, c’: cell + polymer B + lectin, c”: cell + polymer B + lectin + fructose.
not be separated by concentrated fructose solution (Figure 4c”). Similar phenomena were also observed after adding lectin to native cells (Figure S6), further confirming the anticoagulation effect of conjugated polymer A on cell surface. Additionally, a double staining experiment was performed to evaluate the viability of cells after the conjugation of polymer A. After treatment in polymer A solution, as above-mentioned, L929 cells were further double stained using fluorescein diacetate (FDA, 10 μg/mL) and propidium iodide (PI, 10 μg/mL) mixed PBS solution (pH ∼ 7.4). It is clear to see almost all the treated cells kept high viability (green; Figure 5a), preliminary suggesting the polymer A on cell surface is safe for
Figure 5. (a) Double staining experiment and (a’, a”) flow cytometry of L929 cells incubated with the polymer A; (b) double staining experiment and (b’, b”) flow cytometry of native L929 cells. 553
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cell survival. Furthermore, flow cytometry was used to quantitatively calculate the cell viability, and more than 95% of the cells were found survive safely after the treatment of polymer A (Figure 5a’,a”), similar to the result of native L929 cells (∼97%, Figure 5b,b’,b”), suggesting that the conjugation of polymer A on cell membrane is harmless to cells. Similarly, Hela cells were also used through the same viability process and ∼97% Hela cells were found to survive after conjugation by polymer A (Figure S7), confirming the safety of polymer A to cells. In conclusion, MCRs can be looked at as effective tools to simultaneously link and generate different functional groups, providing an alternative method for synthesis of multifunctional macromolecules. In this research, the Hantzsch reaction has been employed as a specific example to prepare well-defined multifunctional polymers on the platform of RAFT polymerization, leading to the facile preparation of a fluorescent phenylboronic acid polymer via a one-pot manner. This multifunctional polymer could be used for visible cell conjugation due to the unique 1,4-DHP linkage by the Hantzsch reaction. Compared to other phenylboronic acid polymers which were used to block cell adhesion, the polymer A and related cell-polymer A conjugate could be easily detected without resorting to complicate analytical methods, demonstrating a new application of the old Hantzsch reaction in different research fields. Besides the Hantzsch reaction, other MCRs such as the Biginelli, Passerini, Mannich, K−F reactions, and so on have also been reassessed by polymer chemists to exploit new multicomponent polymers with unique structures and interesting properties. The current report of multifunctional polymer for visible cell conjugation is only a primary example of the potential of multicomponent polymers, which might prompt deeper understanding of MCRs to develop different multifunctional polymers.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00220.
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Detailed polymerization procedures, small molecule synthesis, competitive binding and cell experiments, and so on (PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Lei Tao: 0000-0002-1735-6586 Author Contributions †
These authors contributed equally to the manuscript.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the National Science Foundation of China (21574073, 21534006, 21372033). 554
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ACS Macro Letters
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DOI: 10.1021/acsmacrolett.7b00220 ACS Macro Lett. 2017, 6, 550−555
