Letter Cite This: ACS Macro Lett. 2019, 8, 79−83
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Single-Polymer−Single-Cargo Strategy Packages Hydrophobic Fluorophores in Aqueous Solution with Retention of Inherent Brightness Rui Liu and Jonathan S. Lindsey* Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204, United States
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S Supporting Information *
ABSTRACT: A strategy for encapsulating hydrophobic organic entities in aqueous solution has been developed through use of a self-assembling heterotelechelic amphiphilic random copolymer. The polymer (∼40 kDa), prepared by living radical polymerization, contains orthogonally reactive terminal groups and pendant hydrophobic (dodecyl), nonionic hydrophilic (PEG9), and ionic hydrophilic (sulfonateterminated) groups. Covalent conjugation of a hydrophobic entity to the polymer terminus has been demonstrated for 8 classes of organic fluorophores. The resulting “pod-fluorophore” architecture is unimeric (∼15 nm in diameter) in aqueous solution with spectral features and fluorescence brightness resembling those of the benchmark fluorophore in organic solution. This strategy separates the functional design of the packaged molecular entity (“cargo”) from the often vexing challenge of water solubilization and in so doing creates a unitary (one-pod−one-cargo) platform architecture for potential applications in cytometry, biomedical imaging, environmental sensing, and supramolecular chemistry.
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workers,4,5 yet going further to include orthogonally reactive terminal groups. The resulting heterotelechelic polymer is designed to assemble into a unimeric particle in aqueous solution with the hydrophobic cargo secured by covalent attachment inside the hydrophobic interior. While our interests are in the use of such single-fluorophore−single-polymer architectures in diverse photochemical studies, the ability to easily package a single, hydrophobic molecular entity for dissolution in water is expected to have far-reaching applications. To pursue an amphiphilic polymer that self-assembles unimerically in aqueous solution, we explored reversible addition−fragmentation chain-transfer (RAFT) polymerizations using hydrophilic and hydrophobic monomers. The polymerizations were carried out under standard conditions using an initiator (AIBN), a chain-transfer reagent (1), and an 1 H NMR marker (mesitylene) in DMF (Figure 1). Upon use of various initial ratios of the monomers lauryl acrylate (LA, hydrophobic) and a polyethylene glycol acrylate (PEGA, hydrophilic/nonionic), exclusive unimer formation was not obtained. Replacing PEGA with a sulfonate-terminated acrylamide (AMPS, hydrophilic/ionic) also failed to exclusively afford unimers.6 Ultimately, we found that combinations
longstanding problem at the interface of chemistry and biology entails working with hydrophobic or only moderately polar compounds in aqueous solution. The problem spans the design and use of enzyme-like catalysts, receptors in supramolecular chemistry, and monomeric or polymeric chemosensors. A widespread manifestation of the problem occurs with diverse organic fluorophores, where aggregation results in spectral alteration and diminished fluorescence intensity.1 Modern approaches to overcome insufficient aqueous solubility typically entail extensive and often idiosyncratic synthesis to add multiple polar (charged or nonionic) groups often with considerable steric bulk, thereby blocking π−π interactions. Such approaches often lead to elaborate superstructures. One approach to solubilize hydrophobic compounds in aqueous solution entails attachment to a water-soluble polymer. In this regard, Zimmerman and co-workers2,3 have reported either hydrophilic or hydrophobic fluorophores attached to the pendant chains of water-soluble polymers. The design protected the fluorophores from photobleaching, but fluorophore quenching was observed despite ostensible protection from the aqueous environment by the amphiphilic polymer. Moreover, sparse attachment to polymer pendant sites leads to an undesirable (e.g., Poissonian) statistical distribution of products. Here, we report an approach that entails the incorporation of a single fluorophore per polymer. The approach relies on the synthesis of an amphiphilic polymer, following the pioneering work of Sawamoto and co© XXXX American Chemical Society
Received: November 27, 2018 Accepted: December 20, 2018
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DOI: 10.1021/acsmacrolett.8b00907 ACS Macro Lett. 2019, 8, 79−83
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ACS Macro Letters
such results are consistent with an extended conformation in an organic solvent but an assembled or folded conformation in aqueous solution. Examination of F-CHO by 1H NMR spectroscopy (in D2O, based on the formyl proton) gave m, n, and p = 22, 21, and 104, respectively. The data cohere with the ratio of m:n:p = 1.0:1.0:5.0 expected from the initial monomer stoichiometry and the observed uniformity of monomer reaction. The calculated molecular weight of FCHO given by the m, n, and p values derived from the 1H NMR measurement is 39.6 kDa, to be compared with the estimated molecular weight of 41.4 kDa for F-Ph inferred from HPLC analysis (Figure S6). Eight classes of chromophores including neutral and hydrophobic (coumarin, BODIPY, perylene, chlorin, bacteriochlorin, phthalocyanine) or charged (cyanine, rhodamine) classes were conjugated to the polymers (seven are shown in Scheme 1). The polymers can be functionalized in three ways: Scheme 1. Fluorophore Building Blocks and Polymer− Fluorophore Conjugates
Figure 1. (A) Synthesis of polymer F-Ph, F-SH, and F-CHO. (B) DLS data of F-Ph in aq NaCl (1.0 M) solution. (C) 2D 1H NMR NOESY data of F-Ph in DMSO-d6. (D) 2D 1H NMR NOESY data of F-Ph in D2O.
of all three monomers afforded a high extent of unimers. Indeed, with a 1:1:5 ratio of the reactant monomers PEGA:LA:AMPS, the unimer is the predominant species in aqueous solution (Scheme S1, Figure S1). The ratio (1:1:5) of monomers PEGA:LA:AMPS identified through screening was employed in a similar manner but with a carboxy-containing dithiobenzoate chain-transfer reagent (2), affording polymer F-Ph. 1H NMR spectroscopic monitoring of the reaction showed uniform disappearance of the three monomers, with overall conversion of ∼70% (Figure S2). Treatment of F-Ph in DMF with hydrazine hydrate caused cleavage of the thiobenzoyl group7 to give polymer F-SH, which contains a free thiol at the terminus. The absorption spectrum of F-SH lacks the characteristic dithiobenzoate band near 300 nm compared to that of F-Ph (Figure S3). The thiol group of F-SH was selectively derivatized by reaction with pbromomethylbenzaldehyde in DMF to give the formylsubstituted polymer F-CHO (the 1H NMR spectra of three polymers are compared in Figure S4). Each polymer F-Ph, FSH, and F-CHO contains a carboxylic acid at one terminus. F-Ph was dissolved in water containing 1 M NaCl and examined by dynamic light scattering (DLS) (Figure 1B). The data at all concentrations examined (1−10 mg/mL) of F-Ph show exclusively unimeric behavior, with a size distribution peaked at 12−15 nm and without detectable aggregation (Figure S5). 2D 1H NMR NOESY analysis of F-Ph in DMSOd6 (Figure 1C and 1D) revealed no interactions between pendant units, whereas in D2O interactions were observed between LA methyl/LA methylenes, LA methylenes/PEGA methylenes, and AMPS gem-dimethyl/PEGA methyl groups;
(1) reaction of F-CHO with a fluorophore−hydrazide to form a hydrazone; (2) reaction of F-SH with a fluorophore− maleimide to form the thioether; or (3) treatment of F-Ph with ethanolamine (to form the free thiol of F-SH in situ) and the fluorophore−maleimide to form the thioether. All conjugations to form the polymer−fluorophores were carried out in DMF; subsequent dialysis in DMF enabled removal of any free fluorophore. Hydrazone conjugation proceeded to varying degrees of completion (Pod-Chl, 30%; Pod-Coumarin, 9%; Pod-BDPY, 16%; and Pod-Cyanine, 52%), whereas malei80
DOI: 10.1021/acsmacrolett.8b00907 ACS Macro Lett. 2019, 8, 79−83
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ACS Macro Letters mide−thiol conjugation (Pod-Perylene, Pod-BChl, Pod-ZnPc) appeared to go to completion, indicating the superiority of the latter method of conjugation. Control experiments were carried out to test the requirement for a covalent tether of the fluorophore to the polymer (Figure S7). All attempts to incorporate a hydrophobic perylene−monoimide lacking a bioconjugatable group into F-Pheither by addition of the perylene to the assembled unimers of F-Ph in aqueous solution or by combination with F-Ph prior to addition of waterwere unsuccessful. While inclusion of noncovalently bound species may prove feasible and an added feature in some applications, the covalent attachment to the heterotelechelic polymer ensures the single-cargo−single-polymer architecture. DLS studies of all polymer−fluorophore conjugates (except Pod-Chl because the fluorescence interferes with the DLS measurement) were carried out at various concentrations (1− 10 mg/mL) in aqueous NaCl (1 M) solution (Figure 2). All six conjugates exclusively exhibit unimers (Figure S8 and Table S1). Note that the size of the polymer−conjugate increases upon incorporation of the fluorophore (Figure 2A). A
representative DLS result of Pod-BChl at 10 mg/mL is shown in Figure 2B. Photophysical properties of the polymer−fluorophore conjugates were studied in aqueous NaCl (1 M) solution and compared to their hydrophobic fluorophore benchmarks8 in organic solution. The absorption and fluorescence features of Pod-BChl in aqueous solution are almost identical with those of Bchl-maleimide (Chart S1) in toluene (Figure 2C). The fluorescence quantum yield (Φf) of Pod-BChl is 0.14 in aqueous solution compared to the benchmark value of 0.15 in toluene. The absorption spectra of Pod-Chl in aqueous solution and the chlorin benchmark Chl-TM in toluene are shown in Figure 2D. The absorption peak at 293 nm of PodChl arises from the hydrazone as well as unreacted F-CHO. Apart from this feature, the absorption and fluorescence features of Pod-Chl in aqueous solution are essentially identical with those of Chl-TM in toluene, including Φf of 0.19 and 0.22, respectively. The absorption spectra, fluorescence spectra, Φf values, and fluorescence band full-width-at-half-maximum (fwhm) values of Pod-Coumarin, Pod-BDPY, Pod-Cyanine, Pod-Perylene, and Pod-ZnPc are shown in Figures S9−S15 and Table S2. All the polymer−fluorophore conjugates in aqueous solution exhibit spectral features resembling the appropriate benchmark in organic solvent, with the exceptions of (1) absorption peaks around 290 nm due to the hydrazone and unreacted F-CHO for Pod-Coumarin and Pod-BDPY; (2) the Pod-Coumarin absorption and fluorescence are slightly bathochromically shifted; (3) the Pod-BDPY shows long-wavelength tailing in the absorption spectrum (of unknown origin) yet essentially unaffected fluorescence properties; and (4) the Pod-Perylene exhibits a bathochromically shifted fluorescence spectrum. Regardless, the Φf values of the polymer−fluorophore conjugates in aqueous solution resemble those of their hydrophobic benchmarks in organic solutions, with the following percentage of the Φf value for the former versus the latter for each fluorophore: BChl, 93%; Chl, 86%; BDPY, 94%; Coumarin, 97%; Cyanine, 230%; Perylene, 89%; ZnPc, 96%. The increase in fluorescence yield observed with the cyanine is typical for such polyenes upon placement in conformationally restricted environments.9 Rhodamine B hydrazide is a fluorogenic reagent with binding specificity to metal ions such as Cu2+, Hg2+, and Cr3+, whereupon hydrolysis unveils the conjugated and fluorescent Rhodamine B.10,11 This conversion from nonfluorescent to fluorescent is very attractive for applications such as heavy metal detection and cell imaging,12,13 but because such applications inevitably take place in aqueous solutions, mixed aqueous/organic solutions including acetonitrile or methanol are required given the hydrophobic nature of Rhodamine-hydrazide. In this regard, we designed PodRhodamine to study metal ion sensing in water without addition of organic solvents (Scheme 2). A solution of Pod-Rhodamine (20 μM) in water was treated with a solution of metal salt (2 mM) in water with stirring at room temperature for 1 h followed by absorption and fluorescence spectroscopy (Figure 3A and 3B). The cations examined were: Au(III), Al(III), Ce(III), Cd(II), Co(II), Cr(II), Cu(II), Fe(III), Ga(III), Hg(II), In(III), Mg(II), Mn(II), Ni(II), Pb(II), Yb(III), and Zn(II). The salts containing Au(III), Cr(II), Cu(II), Fe(III), Hg(II), and In(III) showed a change in absorption, whereas Au(III), Ga(III), Hg(II), and In(III) gave increased fluorescence
Figure 2. (A) Particle size data of three polymer−fluorophores and FPh at 10 mg/mL in aq NaCl solution by DLS; (B) DLS of Pod-BChl at 10 mg/mL in aq NaCl solution; (C) absorption and fluorescence spectra of Pod-BChl in aq NaCl solution compared with benchmark compound in toluene; and (D) absorption and fluorescence spectra of Pod-Chl in aq NaCl solution compared with the benchmark compound in toluene. 81
DOI: 10.1021/acsmacrolett.8b00907 ACS Macro Lett. 2019, 8, 79−83
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ACS Macro Letters
In summary, we have developed a strategy for solubilizing hydrophobic organic fluorophores by packaging with a selfassembling polymer derived from three monomers. The use of a heterotelechelic polymer ensures a “one-cargo−one-polymer” paradigm, enabling studies of homogeneous unitary constructs rather than statistical mixtures of variously substituted polymers. The use of the polymer to impart aqueous solubility separates the functional design of the packaged entity from the longstanding (and often idiosyncratic and tedious) challenge of designing a suitable polar superstructure. Members of the perylene, tetrapyrrole, and phthalocyanine families are particularly notorious for aggregation even in organic solvents. Solubilization in organic solvents has been achieved for such large π-chromophores by synthetic installation of multiple bulky substituents, but aqueous solubilization has presented substantially greater challenges. Here, facile aqueous solubilization of such hydrophobic structures is achieved by one-step covalent attachment to the amphiphilic polymer. The polymers examined herein (∼40 kDa) are several times the size of myoglobin (∼17 kDa),14 which contains a single “packaged” heme. An open issue concerns whether more compact architectures can be constructed that self-assemble and incorporate a wide variety of molecular cargo. Conversely, the upper size limit on packaged entities for polymers of a given size remains to be determined. Much remains to be explored and understood concerning the single-cargo−singlepolymer architectures. Regardless, the present heterotelechelic self-assembling polymers package diverse compounds, retain inherent fluorophore brightness,15 and also contain a single bioconjugatable functional group, which taken together already suggest a platform technology for aqueous applications encompassing diverse fields across the life and physical sciences.
Scheme 2. Metal-Catalyzed Hydrolysis of Fluorogenic PodRhodamine in Water
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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.8b00907.
Figure 3. Absorption (Panel A) and fluorescence (Panel B) spectra of Pod-Rhodamine in water with various metal cations; comparison of absorbance and fluorescence intensity of Pod-Rhodamine metal complexes versus nonmetal Pod-Rhodamine (Panel C). Fluorescence titration of Hg(II) (Panel D) and the linear relationship between fluorescence intensity and concentration of Hg(II) (Panel E).
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intensity compared to the blank control. Loss of fluorescence with Cu(II) and Fe(III) samples was observed. Figure 3C shows the absorbance and fluorescence intensity changes upon introduction of various metal ions compared to Pod-Rhodamine itself in water. Pictures of various reaction solutions with or without illumination are shown in Figure S16. Fluorescence titration was carried out with Hg(II) (Figure 3D) and Au(III) (Figure S17) with 10 μM Pod-Rhodamine and 0−1.0 μM cations (excitation at 510 nm). A linear relationship between the concentration of Hg(II) or Au(III) and the fluorescence intensity was observed (Figure 3E), which suggests application for quantitative detection of such ions in water. Treatment of Pod-Rhodamine-Hg(II) with excess EDTA showed only a slight decrease in fluorescence intensity (Figure S18). The ability to bind metals indicates the chromophore is not completely sequestered in the inner confines of the polymer but does interact with the environment.
Complete experimental section, HPLC characterization data, and spectral data (1H NMR, absorption, and fluorescence) (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Rui Liu: 0000-0003-2927-3080 Jonathan S. Lindsey: 0000-0002-4872-2040 Notes
The authors declare the following competing financial interest(s): J.S.L. is cofounder of NIRvana Sciences, which develops fluorophores for use in life sciences applications.
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ACKNOWLEDGMENTS This work was supported by the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Award No. DESC0001035. Characterization measurements were carried out 82
DOI: 10.1021/acsmacrolett.8b00907 ACS Macro Lett. 2019, 8, 79−83
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ACS Macro Letters in the Molecular Education, Technology, and Research Innovation Center (METRIC) at NC State University.
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