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Dec 20, 2016 - Cell-Penetrating Peptide Spirolactam Derivative as a Reversible Fluorescent pH Probe for Live Cell Imaging. Meng-Chan Xia, Lesi Cai, Si...
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Cell-Penetrating Peptide Spirolactam Derivative as a Reversible Fluorescent pH Probe for Live Cell Imaging Meng-Chan Xia, Lesi Cai, Sichun Zhang, and Xinrong Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03813 • Publication Date (Web): 20 Dec 2016 Downloaded from http://pubs.acs.org on December 23, 2016

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Cell-Penetrating Peptide Spirolactam Derivative as a Reversible Fluorescent pH Probe for Live Cell Imaging Meng-Chan Xia, Lesi Cai, Sichun Zhang,* and Xinrong Zhang Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing, 100084, P.R. China. ABSTRACT: A colorless and nonfluorescent spirolactam derivative, RhB-R12K, was synthesized by amide condensation between the carboxyl group of rhodamine B (RhB) and the amino group of cell-penetrating peptide (CPP). The fluorescence intensity of RhB-R12K sharply increased with the pH values decreasing from 8.0 to 4.9, which can respond sensitively and reversibly to intracellular pH distribution. This CPP probe was completely water soluble, low cytotoxic, membrane permeable and suitable for pH measurement in various organelles by choosing organelle-specific CPP sequences. Interestingly, CPPs not only acted as carriers but also indispensable parts of fluorophores here. Benefiting from the remaining active groups of peptides, it would be potentially modified with additional dyes to construct multifunctional and ratiometric probes for cell imaging.

Cell-penetrating peptides (CPPs) are a class of unique peptides which can cross plasma membrane without cellular injury.1 The concept of CPPs was first proposed in 1988, which indicated that trans-activator of Transcription (Tat) protein of the Human Immunodeficiency Virus (HIV) could efficiently enter tissue-cultured cells and promote the viral gene expression.2 CPPs are able to act as vectors to facilitate intracellular delivery of diverse compounds, such as small molecules,3-5 oligonucleotides,6,7 siRNA,8 nanoparticles,9-12 proteins13 and other peptides.14,15 Recent years, CPPs have been investigated in cellular imaging. Novel photo-modulatable organic fluorescent probes based on CPPs successfully realized superresolution imaging of F-actin.16 Especially, activatable CPPs (ACPPs) have been widely applied in construction of molecular imaging probes to visualize tumors,17 proteases10 and intracellular processes.18 As reported, fluorescent labeled ACPPs were used in surgery to reduce residual cancer cells17 and thrombin-sensitive ACPPs have potential application in detection and imaging of atherosclerotic plaques.19 CPPs were also conjugated with antitumor drugs for tumor-targeted drug delivery.20 Tat peptide was conjugated onto the surface of the nanoprobes to improve cell permeability and deliver the nanoprobes to the cytosol.21 Organelle-specific delivery was accomplished by some specific localization sequences including nuclear localization signal (NLS),22-26 mitochondria-penetrating peptides (MPP),27-32 lysosomal sorting sequences33 and endoplasmatic reticulum retention sequence.34 However, CPPs were often just used as carriers to facilitate the intracellular delivery of fluorescent dyes, drugs or nanomaterials.

We found that the amino group of CPPs coupled with the carboxyl group of rhodamine B (RhB) to form a colorless and nonfluorescent spirolactam derivative RhB-R12K with ring closure under basic condition. The ring open reaction was carried out when pH values decreased, leading to a dramatic increase of fluorescence intensity. This fluorescent probe possessed excellent cell-membrane permeability, and would be applied to reversible pH measurements in living cells. Although it has been frequently reported that the spirolactam of RhB derivatives was employed to pH and metal-ion measurements, most of them were based on the reactions between RhB and small organic molecules.35-46 Their biocompatibility, cellpenetration and subcellular-localization still need improvement. Based on the preliminary study described above, herein we applied the spirolactam derivative RhBR12K to pH measurements in living cells. The unique analytical features of RhB-R12K make it very promising as a fluorescent probe in pH measurements at cellular and subcellular levels. In comparison to the small-molecule pH probes based on RhB, our novel CPP probes showed many outstanding properties. They overcame the poor water-solubility of those probes and proved to be well biocompatible. Furthermore, the CPP probes were easy to cross the cell membrane in achieving the intracellular imaging. The subcellular localization was successfully realized if we chose organelle-specific CPPs (Table S1). EXPERIMENTAL SECTION Reagents and apparatus. All CPP probes were synthesized and HPLC purified by China Peptides (Shanghai, China). The stock solutions of CPP probes were prepared

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Scheme 1. a) Synthesis of RhB-R12K. b) Proposed mechanism for the fluorescence changes of RhB-R12K in buffer solutions at different pH values.

by dissolving the powder in ultrapure water to 1 mM. RhB and all metal ions of analytical grade were purchased from Sigma Aldrich and used without further purification. H2O2 was obtained from Beijing Chemical Works (Beijing, China). Nigericin, NaOD, DCl were purchased from J&K Chemical Technology (Beijing, China). Dulbecco’s modified eagle media (DMEM), fetal bovine serum (FBS), penicillin and streptomycin (100 U/mL), Trypsin EDTA and phosphate buffered saline (PBS) solution were purchased from GIBICO (Invitrogen, USA). All organelle specific dyes were purchased from Molecular Probes (Invitrogen, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide (MTT) and Albumin Bovine V (BSA) were purchased from Biodee Biotechnology (Beijing, China). Ultrapure water (over 18 kΩ) from Milli-Q water purification system (Millipore) was used throughout the experiment. HeLa cells were obtained from Peking Union Medical College Hospital (Beijing, China). 1H-NMR and 13 C-NMR spectra were recorded on a JNM-ECA600 spectrometer (JEOL, Japan) in D2O. Mass spectra were obtained with an AXIMA-Performance mass spectrometer (Shimadzu, Japan). The absorption spectra were recorded with a U-3900 spectrophotometer (Hitachi, Japan). The excitation and emission spectrum of RhB-R12K and the interference of different species to the fluorescence intensity of RhB-R12K were recorded with a F-7000 fluorescence spectrometer (Hitachi, Japan). Fluorescence spectra of all CPP probes at varied pH values and the absorbance of MTT assay were measured by using a microplate reader M3 (Molecular Devices). Fluorescence imaging experiments were performed on FV-1000 confocal laser scanning microscope (Olympus, Japan) with a 60× objective lens. HeLa cells were incubated in a MCO-5AC CO2 incubator (Panasonic, Japan). B-R buffer solutions at different pH values were achieved by adding NaOH or HCl to the mixture of 40 mM acetic acid, phosphoric acid and boric acid. High K+ buffer solutions were made up of 120 mM KCl, 30 mM NaCl, 20 mM HEPES, 5 mM glucose, 1 mM CaCl2, 1 mM NaH2PO4 and 0.5 mM MgSO4.

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Fluorescence experiments in vitro. RhB-R12K (10 μM) in B-R buffer solutions at varied pH values were used for pH calibration curve. Briefly, 2 μL RhB-R12K (1 mM) and 198 μL B-R buffer solutions at specific pH value were mixed and the fluorescence spectra were recorded by a microplate reader M3. The excitation wavelength was 559 nm. The pH calibration of Other CPP probes and the study of the effect of BSA and ion strength (0.1 mM NaCl) on pKa value of RhB-R12K followed the same procedure. The pH value of RhB-R12K (10 μ M) solution between 5.0 and 8.0 was adjusted back and forth by 5 M NaOH or HCl. The interference of redox specie (0.1 mM H2O2) and metal ions(1 mM Na+, 1 mM K+, 0.1 mM Ca2+, 0.1 mM Mg2+, 50 μM other metal ions (Mn2+, Cu2+, Fe3+, Fe2+, Ag+, Al3+, Zn2+, Co2+, Ni2+, Cr3+, Cd2+, Pb2+, Ba2+)) to the fluorescence intensity of RhB-R12K (10 μM) at pH 5.4 and pH 7.4 was investigated, respectively. Briefly, 2 μL RhB-R12K (1 mM) and 198 μL buffer solutions containing different metal ions were mixed. The fluorescence spectra were recorded by using a fluorescence spectrometer. The excitation wavelength was 559 nm. Cell culture and fluorescence imaging. HeLa cells were cultured in DMEM medium supplemented with 10 % FBS, 100 U/mL 1% penicillin and streptomycin (v/v) at 37 ℃ in a 5% CO2 incubator. Cells were seeded in 15 mm confocal laser culture dishes and cultured with the same medium for 24 h. The medium was removed and cells were washed with PBS (pH = 7.4) before use. For confocal fluorescence imaging, HeLa cells were incubated with RhB-R12K (4 μM), LysoTracker Green DND26 (50 nM) and MitoTracker Deep Red FM (100 nM) in DMEM without FBS at 37 ℃ for 20 min. Then, the medium was removed and the cells were washed with PBS three times. LysoTracker Green DND-26 and MitoTracker Deep Red FM were used to target lysosomes and mitochondria, respectively. Fluorescence imaging experiments were performed on FV1000 confocal laser scanning microscope. The excitation wavelength of LysoTracker Green DND-26 was 488 nm and the fluorescence signal was collected from 500 nm to 545 nm. The excitation wavelength of RhB-R12K was 559 nm and the fluorescence signal was collected from 570 nm to 625 nm. The excitation wavelength of MitoTracker Deep Red FM was 635 nm and the fluorescence signal was collected from 655 nm to 755 nm. The average fluorescence intensity of RhB-R12K at different pH values was calculated with Olympus software by choosing 12 different ROIs and all data expressed as mean ± standard (Table S2). Intracellular pH calibration. The cells were incubated with high K+ buffer solutions in the presence of nigericin (10 μM) at varied pH values in the incubator for 15 min. Then, the fluorescence images were collected and the pH calibration curve was constructed according to the average fluorescence intensity of RhB-R12K in selected ROIs. The pH values of lysosomes, H2O2 tread lysosomes and mitochondria were determined by average fluorescence intensity of RhB-R12K in 12 selected ROIs and the pH calibration curve (Table S3).

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Organelle-specific localization. HeLa cells were incubated with RhB-(Fxr)3-NH2 (4 μM), LysoTracker Green DND-26 (50 nM) and MitoTracker Deep Red FM (100 nM) in DMEM without FBS at 37 ℃ for 20 min. The medium was removed and the cells were washed with PBS three times before use. Fluorescence imaging experiments were performed on FV1000 confocal laser scanning microscope.

The CPPs have more complicated three-dimensional structures and intramolecular interactions than that of small organic molecules. To confirm that the formation of

HeLa cells were incubated with RhB-RPKKKRKV-NH2 (20 μM) in DMEM at 37 ℃ for 4 h. Hochest 33342 (10 μg/mL) were added in the medium for another 15 min. Then, the medium was removed and the cells were washed with PBS three times. Fluorescence imaging experiments were performed on FV1000 confocal laser scanning microscope. MTT assay. The cytotoxicity of RhB-R12K was evaluated by the standard MTT assay. HeLa cells were cultured in 96-well microtiter plates at a density of 8000 cells/well and cultured at 37 ℃ in a 5% CO2 incubator for 24 h. The medium was removed and replaced with DMEM added the CPP probe RhB-R12K (4 μM). The cells were incubated with RhB-R12K for 6 and 12 h, respectively. Then, 100 μL of the MTT solution (0.5 mg/mL) was added to each well. After 4 h, the MTT solution was abandoned and 100 μL of DMSO was added to each well to dissolve the formed formazan. The plates were shaken for 10 min and the absorbance at 490 nm was measured by a microplate reader M3. RESULTS AND DISSCUSION The synthetic route of the RhB-R12K was outlined in Scheme 1a. R12K was a common sequence of CPP formed by polymerization of arginine (R) and lysine (K). The carboxyl group of RhB and the amino group of R reacted in the presence of O-benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate (HBTU) and Nethyldiisopropylamine (DIEA) at room temperature. The ring-closure product of RhB-R12K was colorless and nonfluorescent at alkaline condition. The molecular structure of RhB-R12K was characterized by 1H-NMR, 13C-NMR and MALDI-TOF-MS (Figure S1-S4). The peak of spiro-carbon appeared at δ 77.4 ppm in basic solution (Figure S2). The molecular weight of RhB-R12K in acidic solution exceeded that in basic solution by 1 Da (Figure S3-S4). The spiro ring opened with the increase of the H+ concentration (Scheme 1b). Spectroscopic properties of this probe were studied in Britton-Robinson (B-R) buffer solutions at varied pH values. RhB-R12K had its maximum absorption band at 561 nm and strongest fluorescence emission at 585 nm in aqueous solution (Figure S5). Figure 1a-b showed the changes of fluorescence intensity at different pH values. The fluorescence intensity increased 107 folds with the decrease of pH values from 8.1 to 3.3. The pKa value of this probe was calculated to be 5.00 ± 0.01 by the formula log [(Imax − I)/(I − Imin)] = pH − pKa. The fluorescence quantum yield (Φf) measured in B-R buffer solution at pH 3.3 was 0.56.

Figure 1. a), c) and e) Fluorescence spectra of RhB-R12K (10 μM), RhB-PKKKRKV-NH2 (10 μM) and RhB-RPKKKRKV-NH2 (10 μM) at different pH values (from 3.3 to 8.1), respectively. b), d) and f) Plots of F585 versus pH value of a), c) and e), respectively. λex = 559 nm and F585 indicates the fluorescence intensity of corresponding probes at 585 nm. The number of parallel experiments was 3.

spiro ring truly caused the unique spectroscopic properties, we chose the CPP PKKKRKV-NH2 for further examination. The fluorescence intensity of RhB-PKKKRKV-NH2 did not change in buffers at different pH values (Figure 1c-d). Nevertheless, when we added an amino acid R to the N-terminal of the CPP, experimental phenomena similar to RhB-R12K appeared again (Figure 1e-f). The Nterminal of PKKKRKV-NH2 was a proline (P), which is the only one imino acid in 20 basic amino acids. The imino group of P could not give out H+ to form spiro ring after it reacted with carboxyl of RhB. When R was added, the amino group of R resulted in the re-formation

Figure 2. a) Reversible fluorescence spectra and b) reversible fluorescence intensity (F585) changes of RhB-R12K (10 μM) between pH = 5.0 and pH = 8.0 in B-R buffer. λex = 559 nm and F585 is the fluorescence intensity of RhB-R12K at 585 nm.

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of spiro ring (Figure S6). We also synthesized another three spirolactam derivatives by coupling RhB to three CPPs with different sequences and their spectroscopic properties were similar to that of RhB-R12K (Figure S7). All experimental results above indicated that the complex structures, properties and interactions of the moiety of CPPs might not affect the unique spectroscopic properties of these CPP probes. Amino acid directly conjugated with RhB would largely determine the formation of spiro ring and the formed spiro ring changed the original pielectron system of RhB, which finally resulted in the typical spectroscopic properties. The CPP probe RhB-R12K can respond to H+ immediately. When it was added to buffers at different pH values, the fluorescence intensity reached saturation in 2.5 minutes (Figure S8). It implied that RhB-R12K was potentially applied to monitor intracellular H+ fluctuation in real time. Notably, the fluorescence emission of this pH probe displayed great reversibility between pH 5.0 and pH 8.0 (Figure 2). The interference of some metal ions and H2O2 on pH measurement was investigated via fluorescence spectroscopy, respectively. From Figure S9, we can see that all these species exhibited negligible interference on fluorescence intensity of this probe, which implied that this probe was potentially used to measure pH values with good selectivity in complex system. We also used buffer solutions with 0.1 mM NaCl to study the effect of ion strength on pKa value of RhB-R12K and we found that pKa was nearly invariable (Figure S10). RhB-R12K showed excellent membrane-permeable as normal CPPs and it dispersed throughout the cellular structure, including the nucleus, which indicated that the probe RhB-R12K had considerable flow ability (Figure 3). The fluorescent probe RhB-R12K was used to determine the pH values in HeLa cells (Figure 3). The pH values of cells and surrounding medium were homogenized by using H+/K+ ionophore nigericin and the intracellular pH of HeLa cells was clamped at desired pH. From Figure 3a, c, e and g, we can see that the fluorescence intensity of RhBR12K gradually decreased with the increase of pH values. Interestingly, the fluorescence intensity of RhB-R12K showed nearly liner relationship (Figure 4j) with pH values over the range of pH 4.9-8.0 (R2 = 0.99413), which was different from that in vitro. This phenomenon might be caused by the influence intracellular bio-molecules, which can change the pKa value. BSA was used as a model protein to explore the effect of intracellular protein on pKa value of RhB-R12K. Figure S11 indicated that the presence of BSA indeed increased the pKa value of RhB-R12K. The cytotoxicity of RhB-R12K in HeLa cells was examined by the MTT assay. The ratio of the absorbance of MTT at 490 nm determined the cell viability and Figure S12 showed that this probe was relatively low toxic to HeLa cells. Figure 4b showed the intracellular pH map in HeLa cells drawn by the fluorescence distribution with RhBR12K probe. For identifying intercellular location purpose, the HeLa cells were also co-stained with commercially available lysosome-specific fluorescent dyes LysoTracker

Figure 3. Confocal microscopy images of RhB-R12K (4 μM) in HeLa cells clamped at pH = 4.9 (a-b), pH = 6.0 (c-d), pH = + 6.8 (e-f) and pH = 8.0 (g-h) in high K buffer in the presence of nigericin (10 μM). λex = 559 nm. Scale bars: 20 μm.

Figure 4. Confocal microscopy images of intact and H2O2 treated HeLa cells co-stained with a) and e) LysoTracker Green DND-26 (50 nM), b) and f) RhB-R12K (4 μM) and c) and g) MitoTracker Deep Red FM (100 nM). d) Merged image of (a-c). h) Merged image of (e-g). The excitation wavelength is 488 nm, 559 nm and 635 nm, respectively. These images were collected at 500-545 nm (a and e), 570-625 nm (b and f) and 655-755 nm (c and g). i) Intensity profile of regions of interest (ROI 1) across HeLa cells co-stained with LysoTracker Green (50 nM), RhB-R12K (4 μM) and MitoTracker Deep Red FM (100 nM). j) Intracellular pH calibration curve of RhB-R12K in HeLa cells. k) Average fluorescence intensity of RhB-R12K in lysosomes of intact and H2O2 treated cells. Scale bars: 20 μm.

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Green DND-26 (50 nM) and mitochondrion-specific fluorescent dye MitoTracker Deep Red FM (100 nM). We chose a Region of Interest (ROI 1) across lysosomes and mitochondria in one cell (Figure 4a-d and 4i). As illustrated in Figure 4i, the signal of RhB-R12K in the region colocalized with LysoTracker Green DND-26 was remarkably stronger than that colocalized with MitoTracker Deep Red FM. This phenomenon resulted from the pH difference between lysosomes and mitochondria. Then, the average pH values of lysosomes (5.2 ± 0.4) and mitochondria (7.5 ± 0.3) in the intact HeLa cells (Figure 4b) were respectively determined based on calibration curve in Figure 4j. To investigate the relationship between redox substance and intracellular pH fluctuations, we treated HeLa cells with H2O2. Figure 4k showed that improving the intracellular concentration of H2O2 would lead to the decrease of fluorescence intensity of RhB-R12K in lysosomes. The average lysosomal pH value of H2O2 treated cells was calculated to be 6.2 ± 0.4, which was more basic than that of intact cells. Our experimental results were in agreement with that reported in literature.43,47-51 Oxidative stress can make V-APTase inactive and cause an increase of lysosomal pH values.

and both of them improved nuclear import efficiency (Figure S14).26 CONCLUSIONS In summary, we have designed a novel CPP probe RhBR12K to map the intracellular pH distribution. The probe was completely water-soluble and well biocompatible because CPP was the main body of the probe. The Organelle-specific localization was easily realized by changing the sequences of CPPs (Table S1). In this research, CPPs were tactfully used as not only carriers but also indispensable parts of fluorophores. Benefiting from easy modification of CPPs, probes of this kind are promising scaffolds to construct ratiometric52 and multifunctional53 probes which are superior to small-molecule probes. In addition, the CPPs might be replaced by proteins to realize the protein-based fluorescent probes for real-time detection of pH-induced structural variation of proteins and characterization of protein-protein interactions.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website (http://pubs.acs.org.). 1 13 Features of the CPP probe, H-NMR, C-NMR and MALDITOF-MS spectra, additional UV/vis and fluorescence spectra, interference study, cytotoxicity assay, additional fluorescence images and statistics, ESI-MS spectra of all CPP probes and References.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]

Notes The authors declare no competing financial interests. Figure 5. Confocal microscopy images of HeLa cells costained with a) LysoTracker Green DND-26 (50 nM), b) RhB(Fxr)3-NH2 (4 μM), c) MitoTracker Deep Red FM (100 nM). d) Contrast image. e) Merged image of (a-b). f) Merged image of (b-c). g) Merged image of (a-c). The excitation wave-lengths of (a-c) were 488 nm, 559 nm and 635 nm, respectively. These images were collected at a) 500-545 nm, b) 570-625 nm and c) 655-755 nm, respectively. Scale bars: 20 μm.

One of the most attracting properties of CPPs is organelle-specific localization in living cells. The CPPs with specific sequences can successfully deliver various drugs or dyes to nuclear,15 mitochondria,16 lysosomal17 or endoplasmatic reticulum.18 The formation of spiro ring would not affect the fantastic properties of CPPs. In this study, we coupled RhB to a MPP (Fxr)3-NH2 and a NLS RPKKKRKV-NH2, respectively. In Figure 5, HeLa cells were co-incubated with RhB-(Fxr)3-NH2, LysoTracker Green DND-26 and MitoTracker Deep Red FM. The signal of RhB-(Fxr)3-NH2 92% overlapped that of Mito Tracker Deep Red FM and the co-localization coefficient was above 0.85 (Figure S13). The results indicated that the CPP probe RhB-(Fxr)3-NH2 also successfully targeted mitochondria. Another CPP probe RhB-RPKKKRKV-NH2 exhibited similar characteristics as PKKKRKV-NH2 reported

ACKNOWLEDGMENT We thank to the financial support provided by the National Natural Science Foundation of China (21390410 and 21621003), the 973 program (2013CB933804).

REFERENCES (1) Derossi, D.; Joliot, A. H.; Chassaing, G.; Prochiantz, A. J. Biol. Chem. 1994, 269, 10444-10450. (2) Frankel, A. D.; Pabo, C. O. Cell 1988, 55, 1189-1193. (3) Pereira, M. P.; Kelley, S. O. J. Am. Chem. Soc. 2011, 133, 32603263. (4) Ma, Y.; Gong, C.; Ma, Y. L.; Fan, F. K.; Luo, M. J.; Yang, F.; Zhang, Y. H. J. Controlled Release 2012, 162, 286-294. (5) Rousselle, C.; Clair, P.; Lefauconnier, J. M.; Kaczorek, M.; Scherrmann, J. M.; Temsamani, J. Mol. Pharmacol. 2000, 57, 679686. (6) Hassane, F. S.; Saleh, A. F.; Abes, R.; Gait, M. J.; Lebleu, B. Cell Mol. Life Sci. 2010, 67, 715-726. (7) Nakase, I.; Akita, H.; Kogure, K.; Graslund, A.; Langel, U.; Harashima, H.; Futaki, S. Accounts. Chem. Res. 2012, 45, 11321139. (8) Gooding, M.; Browne, L. P.; Quinteiro, F. M.; Selwood, D. L. Chem. Biol. Drug Des. 2012, 80, 787-809. (9) Gupta, B.; Levchenko, T. S.; Torchilin, V. P. Adv. Drug Delivery Rev. 2005, 57, 637-651.

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