Subscriber access provided by ECU Libraries
Purification of Semiconducting Polymer Dots by Size Exclusion Chromatography Prior to Cytotoxicity Assay and Stem Cell Labeling Dandan Chen, Ye Yuan, Jiangbo Yu, Daniel T. Chiu, and Changfeng Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00095 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 24, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Purification of Semiconducting Polymer Dots by Size Exclusion Chromatography Prior to Cytotoxicity Assay and Stem Cell Labeling Dandan Chen,† Ye Yuan,‡ Jiangbo Yu,§ Daniel T. Chiu,§ Changfeng Wu
*†
†
Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong 510855, China
‡
State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun, Jilin 130012, China §
Department of Chemistry, University of Washington, Seattle, Washington 98195, United States
*E-mail:
[email protected] ABSTRACT: Semiconducting polymer dots (Pdots) as fluorescent probes have shown promising applications because of their excellent optical properties. However, apparent differences were observed in cytotoxicity assays, which might originate from impurities introduced in polymer synthesis or nanoparticle preparation. In this paper, a simple gel filtration-based purification method was used to address this issue. Purified Pdots displayed obviously decreased cytotoxicity as compared with the same batch of unpurified Pdots. The purified Pdots were further examined in cytotoxicity study on mesenchymal stem cells (MSCs), which are very sensitive to exogenous probes. The results indicated that purified Pdots did not affect the proliferation ability of MSCs, while unpurified Pdots could have obvious cytotoxicity. In addition, the purified Pdots did not show cytotoxicity even after 6-month storage. Our results demonstrated that gel filtration is an effective method for obtaining Pdots with minimal cytotoxicity, which are more suitable for biological applications.
Semiconducting polymer dots (Pdots) are emerging as a class of organic nanoparticles with excellent properties, such as high brightness, fast emission rate and good photostability.1-2 In the past decade, Pdots have shown great potential for a wide variety of applications in biomedical fields,3-4 including specific cell labeling,5-6 targeted tumor imaging,7-8 lymph nodes mapping,9-10 photoacoustic imaging,11-13 cancer phototherapy,14-15 drug delivery,16-18 and biosensing.19-22 The biocompatibility of an exogenous probe is a critical property for live cells and in vivo applications. Cytotoxic assays are a routine measurement in the field of nanomedicine. Although Pdots have been generally claimed to be biocompatible, we found that their concentrations used in cellular assays had large variations, sometimes by an order of magnitude in different studies.9, 13, 2328 Even for the same Pdot species at the same concentration, the cell viability results can vary significantly in different studies. The apparent inconsistency in the cytotoxicity of Pdots is perplexing; however, it is critical to ad-
dress this issue for their widespread applications in biomedicine. Normally, semiconducting polymers were synthesized by sequential organic reactions involving many chemicals. In a typical polymer synthesis by Suzuki coupling,29-30 several monomers were polymerized to obtain semiconducting polymer by using palladium complex catalyst. The resulting polymers were then washed with several solvents to remove monomers, small oligomers, and inorganic salts. In a reprecipitation method to prepare Pdots,31 the polymer in an organic solvent such as tetrahydrofuran (THF) was mixed with water. THF could be removed by partial evaporation under vacuum, yielding aqueous suspension of Pdots. Usually, the resulting Pdots were directly used for cellular assays without careful purification. Therefore, we speculate that some impurities in the precursors and solvents might be retained in the Pdots. There are several potential byproducts or left-over precursors that can be detrimental to cells. For example,
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
metal catalyst was used in polymer synthesis and organic solvent was used in nanoparticle preparation. However, it is troublesome to systematically characterize the possible impurities. This is particularly difficult to accomplish because different research laboratories use solvents and chemicals from different vendors. Here, we present a gel filtration-based method to purify Pdots for consistent cellular assays and other biological applications. The biocompatibility of three types of Pdots in three cell lines was examined before and after purification, which revealed significant difference in cytotoxicity results. Purified Pdots were further used for labeling stem cells, which are particularly sensitive to exogenous probes. The proliferation profiles of stem cells incubated with pure Pdots and dye-doped Pdots were discussed. The effect of long-term preservation on cytotoxicity of Pdots was investigated as well. This study outlines the importance of purification of Pdots for consistent cellular assays, which has merits for their further biomedical applications. EXPERIMENTAL SECTION Materials The semiconducting polymer poly [2-methoxy-5-(2ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)] (CNPPV, average molecular weight: 26,000, polydispersity: 2.7) and poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4benzo-{2,1′-3}-thiadiazole)] (PFBT, MW 115,000, polydispersity: 3.2) were purchased from American Dye Source, Inc. (Quebec, Canada). The functional polymer poly(styrene-co-maleic anhydride) (PSMA, cumene ter-
minated, average MW≈1700, styrene content 68%), silicon 2, 3-naphthalocyanine bis(trihexylsilyloxide) (NIR775, dye content 95%), tetrahydrofuran (THF, anhydrous, ≥ 99.9%) were purchased from Sigma-Aldrich. Bovine Serum Albumin (BSA, 98%) was purchased from J&K Chemical Ltd. (Beijing, China). Cell culture agents were purchased from Gibco Laboratories (Grand Island, NY) unless indicated otherwise. Preparation, Purification and Characterization of Polymer Dots The Pdots were prepared by using the reprecipitation method as described previously.31 5 mL of THF mixture containing 100 μg/mL polymer (CN-PPV or PFBT), 20 μg/mL PSMA and 1.0 μg/mL NIR775 were quickly injected into 10 mL of water under sonication. The solvent THF was removed by nitrogen stripping on a 80 oC hot plate for 4 hours, followed by passing the Pdot solutions through a 0.22 μm cellulose membrane filter. Gel filtration columns (Econo-Pac 10DG; Bio-Rad, Herculaes, CA, USA) were used for Pdot purification. The columns were washed with deionized water for 5 times, thereafter, 3 mL of concentrated Pdots solution was added to the column. Purified Pdots were obtained after Pdots solution passing through the column. Particle size and morphology of Pdots were assessed by using a transmission electron microscope (H-600, Hitachi, Japan). Average particle size and zeta potential of Pdots were measured by using Malvern Zetasizer Nano ZS. Optical absorption spectra of Pdots were recorded by using a Shimadzu UV-2550 spectrometer. Fluorescence spectra and quantum yields of Pdots were measured by
Scheme 1. (a) Chemical structures of conjugated polymers (CN-PPV and PFBT), NIR 775 and PSMA. (b) Schematic illustration for preparation and purification of Pdots. ACS Paragon Plus Environment
Page 2 of 9
Page 3 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry using a fluorescence spectrometer (QE-2100, Qtsuka, Japan). Cell Culture Breast cancer cell line MCF-7 and cervical cancer cell line HeLa were ordered from the American Type Culture Collection (ATCC; Manassas, VA, USA). Human umbilical mesenchymal stem cells (MSCs) were obtained from SCLNOW Biological Engineering Co., Ltd (Beijing, China). MCF-7 and HeLa cells were cultured in Dulbecco’s modified Eagle Medium (DMEM) with 10% (v/v) Fetal Bovine Serum (FBS; Tianhang Biotechnology Co., Ltd, Zhejiang, China), 100 U/mL penicillin, and 100 μg/mL streptomycin. MSCs were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 medium (DMEM/F12), supplemented with 10% (v/v) FBS (Biological Industries, Beith Haemek, Israel), 10 ng/mL human fibroblast growth factor-basic (PeproTech, Rocky Hill, NJ, USA), 1% (v/v) MEM Non-essential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, and 55 μM 2mercaptoethanol. The cells were incubated at 37 °C in humidified environment with 5% CO2. The culture medium was changed every 2 days. Cells were detached with 0.25% trypsin-EDTA when cell reached 80% confluence. MTT Assay The viability of cells incubated with Pdots was evaluated by using MTT assay. The cells were seeded into 96-well plates at a number of 4,000 cells per well, then cultured for 18 h prior to the addition of culture medium contain-
ing Pdots (0, 5, 10, 20, 40, 80, and 160 μg/mL). 24 hours later, 20 μL MTT (5 mg/mL in PBS) was added into plates. After 4 h incubation at 37 oC, the culture medium was removed carefully, followed by adding 150 μL DMSO into each well. The plates were rocked gently for 10 min to fully dissolve formazan, and then the absorbance of formazan at 570 nm was recorded using a microplate reader (Cytation3, Biotek, USA). Cell viability was calculated as a ratio of absorbance of the cells incubated in medium containing Pdots suspension to that of the cells incubated in culture medium only. Results were expressed as the mean ± SD deviation. The proliferation ability of Pdot-labeled MSCs was measured by MTT assays. The MSCs were seeded into 96well plates at a number of 2,000 cells per well. The next day, the cells were incubated with labeling medium containing Pdots at different concentrations for 24 h, followed by further culturing in fresh medium for 24 h and 48 h, respectively. At each time point, MTT assays were performed to assess the cell viability, while the unlabeled MSCs were used as controls. Labeling of Stem Cells with NIR Pdots MSCs were seeded into 12-well plates at a number of 3×104 cells per well. The next day, the Pdots at a certain concentration (0, 5, 10, 20, 40 μg/mL) in culture medium were added to the wells. After 24 hours incubation, the cells were detached by trypsin and resuspended in PBS for flow cytometry analysis or fixed by 4% paraformaldehyde
Figure 1. (a) Normalized absorption spectra and (b) Emission spectra of PFBT dots, CN-PPV Pdots and NIR775-doped CN-PPV Pdots. (c) Hydrodynamic diameter and zeta potential of PFBT Pdots, CN-PPV Pdots and NIR775-doped CN-PPV Pdots. (d) A representative TEM image of NIR775-doped CN-PPV Pdots. (a) and (b) share the same color bar. ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 9
Table 1. Cytotoxicity assay results of Pdots reported in previous studies Nanoparticles (NPs)
Cell Lines
Concentration Pdots
NIR775-doped MEH-PPV NPs
U87MG
PDPPF-BT NPs
Incubation Time
Cell Viability
Reference
100 μg/mL
24 h
~90 %
9
HeLa
100 µg/mL
24 h
~100 %
13
Poly(acrylate)s-F8BT NPs
HEK293
20 μg/mL
24 h
~80 %
23
PFT NPs
MCF-7
2.5 μg/mL
3h
~80 %
24
NIR775 and TCPO doped PFPV NPs
HeLa
18 µg/mL
24 h
~95 %
25
DPP-based NPs
HeLa
20 µg/mL
24 h
~90 %
26
BIBDF-BT NPs
4T1
25 μg/mL
24 h
~90 %
27
NIR720 doped PFBT Pdots
Raw264.7
90 μg/mL
24 h
~92 %
28
(PFA) for fluorescent imaging. Immunofluorescence Staining The MSCs were first fixed with 4% PFA, and permeabilized with 0.5% TritonX-100 followed by blocking with 5% BSA at 26 oC. Then cells were incubated with primary antibodies, including anti-OCT-4 antibody (Abcam) and anti-SSEA-4 antibody (Abcam) at 4 oC. After overnight incubation, cells were washed and further incubated with
of
Alexa Fluor® 488-labeled Goat anti-Mouse IgG (Abcam) and Alexa Fluor® 488-labeled Goat anti-Rat IgG (Abcam), respectively. The nucleus was stained with Hoechst 33258 for 15 min, then cells were observed by confocal scanning microscope. RESULTS AND DISCUSSION Preparation and Characterizations of Pdots Semiconducting polymer dots were prepared via a re-
Figure 2. The viability of HeLa cells, MCF-7 cells and MSCs incubated with unpurified or purified Pdots at different concentrations. (a) Unpurified CN-PPV dots, (b) Unpurified NIR775doped CN-PPV dots, (c) Purified CN-PPV dots, (d) Purified NIR775-doped CN-PPV dots. ACS Paragon Plus Environment
Page 5 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry precipitation method as previously reported.31 Scheme 1a shows the chemical structures and acronyms of conjugated polymers (CN-PPV and PFBT), near-infrared dye (NIR775) and functional polymer (PSMA) used for preparing Pdots. PFBT Pdots, CN-PPV Pdots, and NIR775-doped CN-PPV Pdots (NIR Pdots) were prepared by quick mixing of a THF solution of hydrophobic polymer with water under sonication. In this process, polymer backbones were folded and collapsed into nanoparticles driven by hydrophobic interactions (Scheme 1b). The PSMA functionalization generates abundant surface carboxyl groups, endowing Pdots with good water-solubility and functional groups for bioconjugation. The Pdots were purified via size exclusion chromatography by passing Pdots solution into a column filled with polyacrylamide gel (Scheme 1b). The properties of Pdots before and after purification were characterized. The absorption spectra and emission spectra of the purified Pdots are very similar to those of the unpurified Pdots (Figure 1a and 1b). From the figure, we can see that the three types of Pdots exhibit broad absorption bands in visible region. The quantum yields of the Pdots were measured to be about 60% for CN-PPV Pdots, 22% for NIR Pdots and 30% for PFBT Pdots, respectively. The hydrodynamic diameter and zeta potential of the purified Pdots have no significant difference as compared to those of unpurified Pdots (Figure 1c). The hydrodynamic diameter of PFBT Pdots, CN-PPV Pdots and NIR Pdots was determined to be ~18 nm, ~16 nm and ~16 nm, respectively.
The zeta potentials of three types of Pdots were all near to -30 mV. A representative TEM image of the Pdots was show in Figure 1d, indicating their spherical morphology with relatively homogenous particle size. Overall, the purification do not affect the optical and physical properties of Pdots. Owing to the small particle size and bright fluorescence, these of Pdots have been demonstrated as promising probes for fluorescent imaging. Cytotoxicity Assay of Pdots We assessed the cytotoxicity of Pdots by a standard MTT assay. In our study, we found the cell viability values varied from batch to batch of the Pdots even for the same polymer species. A literature study also indicates that the nanoparticle concentrations used in cellular toxicity assays are significantly different, sometimes by an order of magnitude in different studies (Table 1). Because Pdots are typically prepared through the solvent mixing method without careful purification, we speculate that the inconsistencies are likely due to some impurities in the precursors and solvents used in the nanoparticle preparation. Therefore, we attempt to test the hypothesis by applying a purification step before cellular assays. First, the density of the Pdots is very close to that of water, thus it is difficult to achieve purification through the common centrifugation method. Density-gradient centrifugation is an effective method of separating solutes with nearequivalent densities. However, gradient medium must be removed from Pdots after centrifugation for subsequent cytotoxicity assays. Dialysis is time-consuming and it is
Figure 3. The proliferation profile of MSCs after incubation with Pdots. (a) Unpurified CNPPV Pdots, (b) Unpurified NIR775-doped CN-PPV Pdots, (c) Purified CN-PPV Pdots, (d) Purified NIR775-doped CN-PPV Pdots. ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
also hard to choose the molecular weight cutoff for unknown species. It is reported that using size-exclusion column chromatography to purify nanomaterials to decrease the cytotoxicity of materials.32-33 It is a fractionation method which utilizes the “reversed” sieving properties of a bed of porous gel granules, by which solutes in solution are separated by their size or molecular weight.34-35 Based on the size difference of Pdots from molecular impurities, gel filtration is a feasible method for removing cytotoxic agents from Pdot solution. In our previous studies, we have used Sephacryl gels to remove excess in Pdot bioconjugation.36-37 In this study, we use Econo-Pac 10DG because it is a pre-packed column, which is convenient and cost-effective for removing small molecules (
