Tuning the pKa of Carboxyfluorescein with Arginine-rich Cell

Article Cite This: Anal. Chem. 2019, 91, 9168−9173

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Tuning the pKa of Carboxyfluorescein with Arginine-Rich CellPenetrating Peptides for Intracellular pH Imaging Meng-Chan Xia, Lesi Cai, Yan Yang, Sichun Zhang,* and Xinrong Zhang Department of Chemistry, Tsinghua University, Beijing 100084, P.R. China

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S Supporting Information *

ABSTRACT: 5-Carboxylfluorescein (FAM) is a conventional pH-responsive fluorophore widely used in fluorescence labeling and imaging. Because of its nonfluorescent structure under acidic conditions, FAM has long been limited to pH determination in a neutral−basic environment. Here, we modified the optical properties of FAM with cationic arginine-rich cell-penetrating peptides (CPPs), tuning the pKa value of FAM to adapt well to pH measurement under diverse pH conditions. With increasing length of polyarginine, the pKa value of FAM was tuned from 6.20 ± 0.06 to 5.17 ± 0.05. The key mechanism for pKa variations was attributed to intramolecular electrostatic attraction and the positive charge of cationic CPPs tend to stabilize the fluorescent dianionic form of FAM. Apart from tunable pKa, arginine-rich CPPs also improved the water solubility, membrane permeability, and organelle-specific localization of FAM. Two conjugated probes FAM-R12 and FAM-(Fxr)3 were selected to monitor intracellular pH fluctuations. Compared to FAM-(Fxr)3, highly positively charged FAM-R12 was more effective in lower pH condition and realized targeted visualization of lysosomal pH changes. The arginine-rich CPP-based strategy offers a promising approach to obtain optimized fluorescent pH probes with adjustable pKa values for organelle-specific pH measurement. efficient fluorescent pH probes with suitable pKa values still remains substantially challenging on account of complex chemical structure modifications, poor water solubility, and membrane permeability of small-molecule fluorescent probes.20 Cell-penetrating peptides (CPPs) have achieved rapid progress in bioimaging and therapeutic applications for their high cellular uptake efficiency.21−24 Besides extensive use of CPPs as carriers, CPPs can also be used to modify the optical properties of small-molecule fluorescent probes. Our group has reported that the pH-insensitive dye rhodamine B could be converted to pH-sensitive rhodamine spirolactam through amidation reaction between N-terminal amino group of R12 and rhodamine B.25,26 The potential application of CPPs needs to be further explored and some intrinsic physical-chemical properties of CPPs have not been fully exploited. For instance, most of the CPPs reported are electropositive, especially artificial CPP polyarginines (Rn),27 which carry a net positive charge under physiological conditions.28 Inspired by prior research about the effect of surface charge on ion detection,29 we envisioned that introducing the charge properties of CPPs to the design of small-molecule fluorescent pH probes could modify their detection capability.

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ntracellular pH is actively involved in various physiological processes to maintain cellular homeostasis1 and pH imbalance could trigger cellular dysfunction and severe diseases.2−5 For instance, new evidence suggests that endosomal pH imbalance in brain cells may have the risk of developing Alzheimer’s disease.4 Despite the significance of intracellular pH in biological research and medical diagnosis,6,7 dynamic monitoring of pH distribution and fluctuations inside distinct cellular compartments is still hampered by the lack of efficient fluorescent pH probes with excellent optical properties, reasonable application range, and organelle-specific localization. Among the diverse pH sensors reported, small-molecule fluorescent pH probes play vital roles in live cell imaging.8−14 Because the application ranges of pH-sensitive fluorophores are restricted by their pKa values (pKa ± 1), it is crucial that pH probes with different pKa values get access to a wider pH range. The pKa values of these pH-sensitive fluorophores could be changed through modifications of the conjugated π-electron system.15−18 Sun et al. found that substituting hydrogen atoms with electron-withdrawing fluorine atoms in fluorescein could produce fluorinated fluorescein derivatives with lower pKa values.15 In contrast, introducing electron-donating alkyl group ortho to phenol group was reported to increase pKa value of fluorescein to 6.68.16 Similar methods are applicable to other fluorophores.18 Steric properties of substituents can induce pKa change as well.17,19 Strong steric hindrance near the spirolactam nitrogen provides rhodamine 6G derivative with a much higher pKa value of 6.5.17 Nevertheless, obtaining © 2019 American Chemical Society

Received: April 17, 2019 Accepted: June 21, 2019 Published: June 21, 2019 9168

DOI: 10.1021/acs.analchem.9b01864 Anal. Chem. 2019, 91, 9168−9173

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Analytical Chemistry In the present work, we first established an arginine-rich CPP-based strategy to rationally tune pKa values of 5-carboxyl fluorescein (FAM). The intramolecular interaction between FAM and cationic polyarginines was investigated. As a conventional small-molecule pH-sensitive dye, FAM can show only fluorescent ring-opening form in a neutral−basic environment. Here, conjugating FAM to CPPs extends the application range of FAM to a lower pH value. By simply changing the charge number of CPPs, conjugates with different pKa values could be obtained, adapting well to pH detection under diverse conditions. Additionally, after conjugation to cationic arginine-rich CPPs, not only the water solubility and cell membrane permeability of FAM was greatly enhanced, but also organelle-specific pH detection could be available.

biomineralization method previously reported in the literature.30 The stock solution of Au nanosensors was prepared by adding 20 μL of FAM-R12 (1 mM) to 200 μL of Au nanocluster solution (2.5 mg/mL) and then shaking it well with a half-hour vortex. The quantum dots and the DNA-based sensors were prepared with similar experimental procedure. Cell Culture. For fluorescence imaging, 1 mL of HeLa cells (2 × 104 cells/mL) were cultured on 15 mm glass-bottom culture dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 100 U/mL 1% penicillin and streptomycin (v/v) at 37 °C in a 5% CO2 incubator overnight. The culture medium was removed and the cells were washed twice with DPBS and once with DMEM before use. Intracellular pH Calibration. HeLa cells were stained with 5 μM FAM-R12 for 30 min or 20 μM FAM-(Fxr)3 for 1 h in DMEM without phenol red. Then the original medium was removed and the cells were washed with DPBS buffer three times. The pH values of HeLa cells were clamped by high K+ buffer solutions with 10 μM nigericin at different pH values for another 30 min. Fluorescence imaging experiments were constructed on a confocal laser scanning microscope. FAMR12 was excited by laser at 488 nm and the fluorescence signal was collected from 500 to 545 nm. Localization Analysis of FAM-R12. HeLa cells were treated with 5 μM FAM-R12 for 30 min, followed by 100 nM LysoTracker Red DND-99 for another 30 min. The cells were washed twice with DPBS. For confocal imaging, FAM-R12 was excited by laser at 488 nm and the fluorescence signal was collected from 500 to 545 nm. LysoTracker Red DND-99 was excited by laser at 559 nm and the fluorescence signal was collected from 570 to 650 nm. Drug Stimulation. HeLa cells were treated with 5 μM FAM-R12 for 30 min, followed by 100 nM LysoTracker Red DND-99 for another 30 min. The cells were washed twice with DPBS and then incubated with 100 μM chloroquine for 10 min at room temperature. Fluorescence imaging experiments were performed on confocal laser scanning microscope. The fluorescence signal was collected and analyzed with Olympus software (FV10-ASW). CCK-8 Assay. The cytotoxicity of FAM-R12 and FAM(Fxr)3 was evaluated by cell counting-kit 8 (CCK-8) assay. Briefly, HeLa cells were seeded onto 96-well plates at a density of 5000 cells/well and cultured overnight at 37 °C in a 5% CO2 incubator. The medium was removed and Hela cells were washed twice with PBS solution. Then the cells were treated with DMEM in the presence of 5 μM FAM-R12 (or 20 μM FAM-(Fxr)3). The control cells were cultured in DMEM without probes. After 0.5, 2, 5, and 8 h of incubation, the medium was replaced with 100 μL of DMEM added to 10 μL of CCK-8 solution. The cells were cultured for another 2 h and the absorbance at 450 nm was recorded by a microplate reader M3.

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EXPERIMENTAL SECTION Reagents and Apparatus. All CPP-based fluorescent probes with purity over 95% were synthesized by SynPeptide Co., Ltd. (Shanghai, China) and China Peptides Co., Ltd. (Shanghai, China). NaCl and nigericin were supplied by J&K Chemical Technology (Beijing). 5-Carboxyfluorescein (FAM), chloroquine, HAuCl4·3H2O, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich and used without further purification. CdSe/ZnS quantum dots were prepared by Suzhou Xingshuo Nanotech Co., Ltd. (Suzhou, China). DNA oligonucleotides were synthesized and purified by Sangon Biotechnology Co., Ltd. (Shanghai, China); the sequence (5′ to 3′) is CGCTTATCTAGGGGTATAAGCTGGTC. Ultrapure water (over 18 MΩ/cm) used in the experiment was purified by Milli-Q integral water purification system (Millipore). All cell culture products and Dulbecco’s phosphate buffered saline (DPBS) solution were purchased from GIBCO (Invitrogen, USA). Britton-Robinson (B-R) buffer solutions and high K+ buffer solutions of varied pH values were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing). The human cervical cancer cell line HeLa was obtained from Peking Union Medical College Hospital (Beijing) and cultured in a Panasonic MCO5AC CO2 incubator. The excitation and emission spectra of FAM and FAM-Rn were detected by an Hitachi F-7000 fluorescence spectrometer. The fluorescence lifetime and quantum yields of FAM and FAM-Rn were measured by using an Edinburgh Instruments FLS920 steady state and transient state fluorescence spectrometer. Fluorescence spectra of all probes at B-R solutions of varied pH were recorded with a Molecular Devices microplate reader M3. Cell imaging was conducted on an Olympus FV1000 confocal laser scanning microscope with 60× oil-immersion objective lens. Spectroscopic Properties of FAM-CPP. Stock solutions of these conjugated pH sensors were prepared by ultrapure water at a concentration of 1 mM and stored at 4 °C. Working solutions were prepared by diluting stock solutions to a concentration of 10 μM with B-R buffer solutions. The fluorescence spectra of FAM-CPP at varied pH values were recorded by a microplate reader at ambient temperature (25 ± 1 °C). The excitation wavelength was 488 nm. Curves were plotted by fluorescence intensity at 527 nm versus pH value. The effect of ionic strength of solutions on the pKa value of FAM-R12 was explored by a similar experimental procedure and the ionic strength of buffer solutions was modified by different concentrations of NaCl. Preparation of Au Nanosensors. BSA-protected Au nanoclusters (AuNCs) were prepared with a classical

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RESULTS AND DISCUSSION FAM was conjugated to polyarginines with varied polymerization degree (Rn) by 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) method and the synthetic routes were outlined in Scheme 1. The conjugated probes (FAM-Rn) with purity higher than 95% were confirmed by HPLC and ESI-MS (Figures S1−S6, Supporting Information). Some photophysical properties of FAM and FAM-Rn were measured and are presented in Table S1. The polypeptide 9169

DOI: 10.1021/acs.analchem.9b01864 Anal. Chem. 2019, 91, 9168−9173

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Analytical Chemistry Scheme 1. (a) Conjugation of FAM to Rn; (b) Reversible Ring-Open and Ring-Closed Structure of FAM Triggered by pH Variations

chain improved the water solubility of FAM and FAM-Rn was completely soluble in 100% aqueous solutions. The emission spectra of FAM-Rn have a slight red shift compared to that of free FAM, which show a characteristic fluorescence emission peak at 527 nm. pKa Change after Conjugation to Polyarginines. The fluorescence spectra of FAM and FAM-Rn at different pH values were measured in B-R buffer solutions. As indicated in Figure 1, the formed CPP-conjugated probes had similar fluorescence spectra as free FAM but shifted pKa values. Consistent with free FAM, all these conjugated probes are nonfluorescent in their spirocyclic forms and show increased fluorescence intensities with pH values. The response curves were plotted with the maximal emission and fitted by Henderson−Hasselbalch equation log[(Imax − I)/(I − Imin)] = pH − pKa (Figures S7 and S8). The pKa values of these conjugates were determined by the corresponding nonlinear fitting curves. Figure 1e−h shows that the pKa values of FAMRn decreased with the increase of the length of arginine chain. Especially, conjugation to R12 made the pKa value of FAM drop to 5.17 ± 0.05 from 6.20 ± 0.06, remarkably changing the functional application range for pH measurement (pKa ± 1). In comparison to free FAM and other conjugated probes, FAM-R12 has much stronger fluorescence intensities at pH 4.5, indicating its spirocyclic form is gradually opening when the pH value is low at 4.5. The results indicate that FAM-R12 is more sensitive to pH change under low pH condition and easier to ionize to fluorescent form than free FAM at the physiological pH range. Meanwhile, FAM-R3 has a similar pKa value as free FAM and the ring-opening reaction occurs at near neutral condition. The arginine-rich CPPs could tailor the pKa value of FAM and thus a different functional range for pH sensing can be obtained by tuning the sequence length of polyarginines. On the basis of previous research about the sensitivity of fluorescein to its surrounding chemical environment,31−34 we hypothesized that the pKa change after conjugation was strongly associated with the electrostatic environment created by the positive-charged CPPs. As shown in Scheme 1, polyarginines carry net positive charges in buffer solutions because of the cationic guanidine residues of the basic amino acid arginine. Through intramolecular electrostatic attraction, the dense positive charges of polyarginines tend to stabilize the fluorescent dianionic form of FAM in lower pH values, causing the decrease of the pKa value. Even if polyarginines were replaced with other cationic CPPs, similar experimental results could be obtained. FAM were attached to three other cationic

Figure 1. Fluorescence spectra of (a) FAM-R12 (10 μM), (b) FAMR6 (10 μM), (c) FAM-R3 (10 μM), and (d) FAM (10 μM), respectively. (e−h) Plots of emission maxima in (a−d) versus pH value and corresponding fitting curves. pKa was determined by the average value of three parallel experiments. λex = 488 nm.

CPP sequences, namely, (Fxr)3, PKKKRKV, and GPKKKRKV, with varied positive charges. As demonstrated in Figure 2a and Figures S9 and S10, the pKa value of FAM still decreases with the increased charge number of CPPs. Meanwhile, the insertion of neutral glycine (G) to PKKKRKV has no effect

Figure 2. Calibration plots of the conjugated probes and free FAM dissolved in B-R buffer solutions clamped at varied pH values. (a) Replacing polyarginines with other cationic cell-penetrating peptides, namely, PKKKRKV, GPKKKRKV, and (Fxr)3. (b) Neutralizing the positive charge of R12 with negative-charged BSA-AuNCs, CdSe/ZnS, and DNA. 9170

DOI: 10.1021/acs.analchem.9b01864 Anal. Chem. 2019, 91, 9168−9173

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Analytical Chemistry on the pKa value. In combination with Figures 1 and 2a, we concluded that when the charge amount of CPPs is maintained, the pKa value is nearly irrelevant to the specific sequence of peptides. To further verify our hypothesis, we attempted to eliminate the effect of electrostatic attraction and investigated whether the pKa value of the conjugates changed inversely when the positive charges of polyarginines were shield by densely negative-charged substances. Anionic materials, including gold nanoclusters (BSA-AuNCs), quantum dots (CdSe/ZnS), and oligonucleotide were prepared and characterized (Figures S11 and S12). FAM-R12 were adsorbed on these anionic materials to form complexes through electrostatic interaction and their fluorescence responses to changes in pH were measured (Figure S13). Figure 2b shows that the fitting curve of all complexes shifted to higher pH value compared to that of FAM-R12. Especially for oligonucleotide, the pKa values increased to 6.80 ± 0.03. The fluorescent dianionic form of FAM is supposed to be repelled by negative charges, resulting in increasing pKa values. Electrostatic attraction between anionic fluorophores and cationic polyarginines diminishes as the distance between them increases. Therefore, we explored the influence of distance to pKa value. To adjust the distance, polyarginines and FAM were separated by a different number of short spacer 6-aminocaproic acids (Acp). As depicted in Figures S14 and S15, the pKa values have a slow upward trend with the rising number of spacers. However, the pKa values of these conjugates are still much lower than that of free FAM, which may be due to the randomly coiled and twisted structure of polyarginines and spacers in the solution. Spacers with a more rigid and longer structure could be more effective to improve the pKa values. The pKa values of all conjugated pH probes were calculated and are summarized in Table 1.

Figure 3. (a) Calibration plots of the emission maxima of FAM-R12 against pH value in B-R buffer solutions with varied ionic strengths tuned by different concentrations of NaCl. (b) Corresponding pKa values under different ionic strengths.

concentration exceeds 100 mM. The experimental results indicate that ionic strength in physiological range cannot affect the pKa of the FAM-R12 and the conjugate probes have strong application potential for intracellular pH sensing. Intracellular pH Calibration. Cationic CPPs optimized the water solubility and membrane permeability of FAM, allowing the conjugates to be used in intracellular pH measurement. Two conjugated pH probes, FAM-R12 and FAM-(Fxr)3 with different positive charges, were applied in intracellular pH calibration. The cytotoxicity of these two conjugated probes was evaluated and Figure S17 demonstrates that they have weak toxicity to HeLa cells. For intracellular pH calibration, HeLa cells were clamped at specific pH value in high K+ buffer solution mixed with nigericin. The intracellular pH calibration curves of FAM-R12 and FAM-(Fxr)3 were plotted, respectively. As depicted in Figure 4, more cationic CPP still leads to lower pKa value, which agrees well with the experiment results in buffer solutions. Thus, these two conjugated probes have different application range of pH sensing in HeLa cells. FAM-R12 with lower pKa value had a significant positive linear correlation with pH value in the

Table 1. pKa Values of FAM and the CPP-Based Conjugated pH Probes probe FAM FAM-R3 FAM-R6 FAM-R12 FAM-R12/CdSe/ZnS FAM-R12/BSA-AuNCs FAM-R12/DNA FAM-Acp-R12 FAM-(Acp)2-R12 FAM-(Acp)3-R12 FAM-(Fxr)3 FAM-PKKKRKV FAM-GPKKKRKV

pKa 6.20 6.08 5.70 5.17 6.08 6.71 6.80 5.32 5.47 5.51 6.02 5.62 5.64

± ± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.06 0.05 0.05 0.06 0.03 0.03 0.02 0.06 0.02 0.02 0.04 0.06

Figure 4. Fluorescent images of HeLa cells at varied pH values pretreated with the conjugate probe (a) FAM-R12 and (b) FAM(Fxr)3, respectively. HeLa cells were stained with 5 μM FAM-R12 for 30 min in DMEM without phenol red. Then the original medium was removed and the cells were incubated in high K+ buffer solutions added to 10 μM nigericin at different pH values for another 30 min before confocal imaging. (c, d) Intracellular pH calibration curve plotted by fluorescence intensity of FAM-R12 versus pH values. (e, f) Intracellular pH calibration curve plotted by fluorescence intensity of FAM-(Fxr)3 versus pH values. The fluorescence signal was collected from 500 to 545 nm. Scale bar: 10 μm.

For the sake of practical application in intracellular pH measurement, the influence of ionic strength should be taken into consideration. The conjugate FAM-R12 was chosen as a representative to explore the effect of ionic strength on the pKa value of these charged fluorescent pH probes (Figure 3 and Figure S16). Varied concentrations of NaCl were used to modify the ionic strength of buffer solution. Figure S16 shows that the buffers with NaCl concentration lower than 100 mM have little interference with the pKa value of FAM-R12. The pKa value of FAM-R12 has no obvious difference until the NaCl 9171

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Analytical Chemistry

S19). The results suggest that FAM-R12 can monitor the pH fluctuations inside acidic lysosomes with high sensitivity.

range of pH 4.0−6.2 and FAM-(Fxr)3 with higher pKa value showed positive linear correlation with pH value in the range of pH 5.3−7.8. Compared to FAM-(Fxr)3, more positivecharged conjugated probe FAM-R12 is more suitable to detect pH variations in an acid environment. Colocalization Analysis and Drug Stimulation. The intracellular localization of FAM-R12 was analyzed. HeLa cells were costained with FAM-R12 and a commercially available lysosome-targeted sensor LysoTracker Red DND-99 (LTR, Figure 5). Figure 5i shows that FAM-R12 colocalized well with

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CONCLUSIONS In summary, we proposed a rational strategy to obtain pKa tunable florescent pH probes through conjugation of FAM to cationic arginine-rich CPP. Stabilization of fluorescent dianionic form of FAM by the increasing positive charges of cationic CPPs is the main reason for pKa decrease after conjugation. Cationic arginine-rich CPPs expanded the potential application of FAM under acidic conditions. Through adjustment of the sequences lengths of polyarginines, the pKa value of conjugated pH probes vary from 5.17 ± 0.05 to 6.08 ± 0.06. The conjugated pH probes worked well in intracellular pH measurement and FAM-R12 with lower pKa value was successfully employed in measuring lysosomal pH change after drug stimulation. Considering the organelle-specific localization of some special CPPs, many targeted pH probes with suitable pKa value could be accessible. Moreover, the conjugated probes with lower pKa values are more valuable for fluorescent labeling of proteins because of more fluorescein molecules in fluorescent form in the physiological pH range. Further studies about the charge effect of cationic arginineCPPs on property modification of other small-molecule fluorescent probes are worth delving into.

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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/acs.analchem.9b01864. Figure 5. Fluorescence imaging of HeLa cells stimulated by chloroquine. HeLa cells were treated with 5 μM FAM-R12 for 30 min, followed by 100 nM LTR for another 30 min. Then the cells were washed twice with PBS and treated with 100 μM chloroquine for 10 min at room temperature before confocal imaging. (a−d) Control group. (e−h) Cells stimulated by chloroquine. (a, e) Fluorescence signal of FAM-R12 collected from 500 to 545 nm. (b, f) Fluorescence signal of LTR was collected from 570 to 650 nm. (c, g) Merged images of green channel and red channel. (d, h) Corresponding images of bright field. (i) Colocalization analysis between FAM-R12 and LTR. (j) Corresponding fluorescence intensity of FAM-R12 localized in lysosomes of HeLa cells stimulated with or without chloroquine. Scale bar: 10 μm.

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ESI-MS and HPLC spectra of FAM-Rn, photophysical properties of FAM and FAM-Rn, TEM images of CdSe/ ZnSe and BSA-AuNCs, pKa change with the increase of the length of spacer, localization analysis and intracellular pH determination, and additional ESI-MS spectra of other conjugated probes (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (S.Z.). ORCID

Sichun Zhang: 0000-0001-8927-2376 Notes

LTR. The overlap and the Pearson’s coefficient of FAM-R12 and LTR are both greater than 0.85 (Figure S18), which verifies that FAM-R12 is located in the lysosomes. The localization capability of FAM-R12 was analyzed and attributed to the endocytosis-based cellular uptake mechanism of polyarginine at low concentration.35−37 FAM-R12 was then applied to detect pH changes in lysosomes (Figure 5a−h). Chloroquine, an endosomal acidification inhibitor, accumulates inside the lysosomes and inhibit lysosomal degradation pathway, for example, autophagy.38,39 Here, we treated HeLa cells with chloroquine to raise the lysosomal pH. As shown in Figure 5j, the fluorescence intensity of FAM-R12 greatly enhanced after 10 min of stimulation, indicating rapid pH increase in lysosomes. According to the intracellular calibration curve of FAM-R12, the lysosomal pH values of untreated and treated cells were determined to be 4.6 ± 0.2 and 5.7 ± 0.2, respectively (Figure

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21621003). REFERENCES

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DOI: 10.1021/acs.analchem.9b01864 Anal. Chem. 2019, 91, 9168−9173