Water-soluble copolymeric materials: Switchable NIR two-photon fluorescence imaging agents for living cancer cells

Article abstract of DOI:10.1039/c3tb21251c

A simple one-pot multi-component ROMP of hydrophilic, spiropyran (SP) and tert-butyloxycarbonyl-protected (Boc-protected) amino-functionalized norbornenes was developed, and a series of water-soluble copolymeric materials were provided. The photochromic SP moiety endows the copolymers the properties of switchable fluorescence imaging with alternating near-IR (NIR) two-photon and visible single-photon excitations; thereby the copolymers can serve as a promising NIR two-photon fluorescent probe for living cancer cells. The cationic ammonium group enhances the cell transporting capability of the copolymers as well. The present strategy of one-pot ROMP also makes polynorbornene a modular and flexible scaffold in designing and synthesizing specifically biocompatible polymers with applications in imaging, diagnosis and therapy. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c3tb21251c

Journal of
Materials Chemistry B
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PAPER
Water-soluble copolymeric materials: switchable
NIR two-photon fluorescence imaging agents for
living cancer cells†
Cite this: J. Mater. Chem. B, 2014, 2,
502
a
b
b
a
a
Nan Xie, Ke Feng, Bin Chen, Ming Zhao, Shiqi Peng, Li-Ping Zhang,b
*
*
b
b
*
Chen-Ho Tung and Li-Zhu Wu
A simple one-pot multi-component ROMP of hydrophilic, spiropyran (SP) and tert-butyloxycarbonyl-
protected (Boc-protected) amino-functionalized norbornenes was developed, and a series of water-
soluble copolymeric materials were provided. The photochromic SP moiety endows the copolymers the
properties of switchable fluorescence imaging with alternating near-IR (NIR) two-photon and visible
single-photon excitations; thereby the copolymers can serve as a promising NIR two-photon fluorescent
probe for living cancer cells. The cationic ammonium group enhances the cell transporting capability of
the copolymers as well. The present strategy of one-pot ROMP also makes polynorbornene a modular
and flexible scaffold in designing and synthesizing specifically biocompatible polymers with applications
in imaging, diagnosis and therapy.
Received 10th September 2013
Accepted 26th October 2013
DOI: 10.1039/c3tb21251c
www.rsc.org/MaterialsB
can greatly increase the signal-to-noise ratio for higher imaging
quality and target-binding specicity.
Introduction
To date, polymeric imaging materials have been achieved
primarily by two types of strategies, amphiphilic block copoly-
mers19,25,26 and conjugated electrolytes.27–29 Multi-functional
groups can be tethered on polymer backbones through chem-
ical modication step-by-step, however, neither of these strat-
egies avoid tedious and time-consuming multi-step
copolymerization (even coupled with varied polymerization
methods) or post-polymerization treatment. Different from
these strategies, random copolymers can be constructed with
diverse components in a single polymerization step and afford
biocompatible imaging materials,30–32 but a series of problems,
such as functional monomer synthesis, stability of the mono-
mers, tolerance of the initiators and the control of the polymeric
molecular weights and polydispersity indices (PDIs) and yields,
remain to be solved.
Fluorescence imaging is one of the indispensable tools for
highly-sensitive and non-invasive visualization, characteriza-
tion and quantication of normal or pathological processes in
cells, tissues and living organisms.1–5 To support the required
needs for imaging, as well as diagnosis and therapy, there has
been considerable interest in investigating highly specic tar-
geting and labelling materials such as small organic dyes,6–8
inorganic nanoparticles,9–16 liposome assemblies and poly-
mers.17–20 Far from the undesirable leakage of small organic
dyes or the unexpected aggregation of inorganic nanoparticles,
polymeric uorescent materials have attracted increasing
attention due to their uniform characteristics in component,
structure, stability, assembly in biological environment and the
convenience for multipurpose design and synthesis.17–20 On one
hand, polymers can easily integrate more than one component
with multimodal imaging and functionality; on the other hand,
the multivalent effect21 and enhanced permeation and retention
effect,22–24 attributed to the repeat units on the polymer chain,
Ring-opening metathesis polymerization (ROMP) is
a
powerful toolkit for the preparation of random, block and
alternating functionalized copolymers.33–57 Using ruthenium
N-heterocyclic carbene complexes as initiators, controlled
polymer backbones, such as polynorbornenes, have been
obtained from varied function monomers containing chromo-
phores, hydrophilic tails and hydrogen-bond interfaces, oligo-
peptides, ionic and/or metallic compounds.40–57
aSchool of Chemical Biology and Pharmaceutical Sciences, Capital Medical University,
Beijing 100069, P. R. China. E-mail: sqpeng@bjmu.edu.cn; Fax: +86 10 8391 1528;
Tel: +86 10 8391 1528
bKey Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical
Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R.
China. E-mail: kefeng@mail.ipc.ac.cn; lzwu@mail.ipc.ac.cn; Fax: +86 10 8254 3580;
Tel: +86 10 8254 3580
Previously, we reported a simple one-pot ROMP using func-
tionalized norbornenes to prepare random copolymers in
water.58 This strategy allows hydrophobic uorescence chro-
mophores through multi-component copolymerization to be
conveniently introduced into an aqueous medium. On this
basis, we synthesized water-soluble imaging random
† Electronic supplementary information (ESI) available: GPC curves, NMR, mass
spectra, decay proles and uorescent images for the intermediates, monomers
and polymers. See DOI: 10.1039/c3tb21251c
502 | J. Mater. Chem. B, 2014, 2, 502–510
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Scheme 1 The preparation of PNB–SPx-co-(NH2$HCl)y-co-P3z water-soluble random copolymers.
copolymers PNB–SPx-co-(NH2$HCl)y-co-P3z in the present work 10 mm linear MZ-Gel SDplus column. The ow rate used for all
(Scheme 1). We selected spiropyran as the uorescence chro- of the measurements was 0.5 mL minꢀ1. No calibration stan-
mophore for the photochromic possession of a nonuorescent dards were used, and dn/dc values were obtained for each
spiropyran (SP) form and a highly uorescent merocyanine injection by assuming 100% mass elution from the columns.
(MC) form, as well as the reported two-photon uorescence,59–62 Mw, Mn and PDIs were calculated using an Astra 5 soware
which has the potential to cause less photo-induced damage package. Commercially available LEDs were employed as UV
and phototoxicity, a low autouorescence background, better (365 nm, input 350 mA, 3.7 V, output ꢁ125 mW cmꢀ2) and
spatial localization and deeper tissue penetration.63–68 As we visible (530 nm, input 350 mA, 3.4 V, output ꢁ120 mW cmꢀ2
)
expected, the polymers exhibited switchable uorescence light sources, and the intensities were determined by a Newport
imaging characteristics with alternating NIR two-photon and (CA, USA) Optical Power/Energy Meter Model 842-PE instru-
visible single-photon excitations in living cancer cells, such as ment. Zeta potential measurements were performed in 10 mM
Bel-7402, HepG2, MCF-7 and A549. Furthermore, a cationic PBS buffer with a pH value of 7.4 at 298 K, using a Nano-2S90
ammonium group was introduced to enhance the cell trans- Zetasizer (Malvern, UK). The zeta potential (z) was calculated
porting capability69 of the copolymers through a standard from the electrophoretic mobility using the Smoluchowski
treatment of the Boc-protected amino group. Importantly, such equation as the average values of three runs.
a one-pot ROMP strategy will make polynorbornene a modular
and exible scaffold to design and synthesize specic biocom-
patible polymers for multipurpose applications in imaging,
diagnosis and therapy.
Materials and synthesis
Exo-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid was synthesized
according to published procedures.70–72 Dulbecco's modied
Eagle's medium (DMEM), RPMI 1640 medium and fetal bovine
serum (FBS) were purchased from Gibco. 3-(4,5-Dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), penicillin,
streptomycin and 4% paraformaldehyde solution in PBS were
Experimental section
General
All of the experiments with air- and moisture-sensitive inter- purchased from Beijing Solarbio Science & Technology Co. Ltd.
mediates and compounds were carried out under an inert The other reagents and solvents were obtained from commer-
atmosphere using standard Schlenk techniques. NMR spectra cial supplies and used as received without further purication.
were recorded on a Bruker Advance DPX 400 MHz spectrometer
N-tert-Butoxycarbonyl-1,6-diaminohexyl-exo-bicyclo[2.2.1]-
and referenced using the residual proton signal of the solvent. hept-5-ene-2-carboxamide (NB–Boc–NH monomer). N-(tert-
Mass spectra were obtained on either a Bruker APEX II or a Butoxycarbonyl)hexyl diamine was synthesized analogously to
Bruker BIFLEX III spectrometer. Elemental analyses were per- the literature.73 At 0 ꢂC, to a solution of 1,6-diaminohexane
formed on a Carlo Erba 1106 elemental analyzer. FT-IR spectra (11.70 g, 101 mmol) in methylene chloride (100 mL), a solution
were taken on an Excalibur 3100 system (varian, USA). UV-Vis of Boc anhydride (4.40 g, 20.2 mmol, 30 mL in CH2Cl2) was
spectra were obtained using a Shimadzu 1601PC spectropho- added dropwise. Then the mixture was stirred at 0 ꢂC for 30
tometer. Fluorescence measurements were run on an F-900 min, and at room temperature overnight. The unreacted
Edinburgh Analytical Instruments spectrometer. Gel-perme- 1,6-diaminohexane was removed under vacuum conditions at
ation chromatography (GPC) analyses were carried out on a 85 ꢂC, and the residue was washed with saturated Na2CO3
multi-angle, digital signal processing light scattering system solution and dried over NaSO4 to afford N-(tert-butoxycarbonyl)-
(Wyatt, USA) using a Shimadzu LC-10A pump coupled to a Dawn hexyl diamine (4.20 g, 19.4 mmol, yield 96%) as a transparent
Heleos II light scattering detector (equipped with a 100 mW oil, and used for the next reaction without further purication.
GaAs laser) and a Optilab rEX interferometeric refractometer 1H NMR (400 MHz, CDCl3) d: 4.70 (br, 1H), 3.02 (m, 2H), 2.62
with THF or DMF (with 10 mM LiBr at 313 K) as eluents on a (m, 2H), 1.37 (s, 15H), 1.26 (m, 4H).
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A mixture of N-(tert-butoxycarbonyl)hexyl diamine (1.10 g, the spiropyran monomer NB–SP, NB–P3 monomer and NB–
5.09 mmol), exo-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid Boc–NH monomer in varied ratios. The reaction mixture was
(0.70 g, 5.07 mmol), PyBOP (benzotriazole-1-yl-oxytripyrrolidino- stirred for 15 min at room temperature. Aer complete
phosphonium hexauorophosphate, 3.20 g, 6.15 mmol) and conversion of the monomer (>99%, monitored by 1H NMR), the
TEA (triethylamine, 1 mL) was stirred in anhydrous DMF (20 mL) polymerization was quenched by the addition of ethyl vinyl
under an inert atmosphere for 1 h. Aer removal of the solvent, ether and stirred for an additional 15 min. The polymer was
the crude product was puried by silica gel column chroma- precipitated with cold ether, and dried at room temperature
tography to give the NB–Boc–NH monomer as a colorless powder under high vacuum.
1
(1.55 g, 4.61 mmol, yield 91%). TLC: Rf ¼ 0.30 (methylene
Polymer PNB–Boc–NH. H NMR (400 MHz, CDCl3, ppm) d:
chloride : ethyl acetate ¼ 4 : 1). ESI-MS: m/z ¼ 359 [M + Na]+, 375 5.78 (br, 1H), 5.27 (m, 2H), 4.90 (br, 1H), 3.15 (m, 2H), 3.05 (m,
[M + K]+. 1H NMR (400 MHz, CDCl3) d: 6.06 (m, 2H), 5.85 (br, 1H), 4H), 2.64 (m, 1H), 2.28 (m, 1H), 2.14 (m, 1H), 1.90 (m, 1H), 1.56
4.63 (br, 1H), 3.20 (m, 2H), 3.06 (m, 2H), 2.87 (m, 2H), 1.96 (m, (m, 1H), 1.41 (m, 12H), 1.28 (br, 4H), 1.10 (br, 1H). FT-IR (KBr): n
1H), 1.86 (m, 1H), 1.69 (m, 1H), 1.45 (m, 4H), 1.40 (s, 9H), 1.30 ¼ 3462, 3332, 3060, 2977, 2933, 2864, 1689, 1646, 1538, 1479,
(m, 6H). 13C{1H} NMR (100 MHz, CDCl3) d: 175.55, 156.01, 1454, 1441, 1387, 1365, 1333, 1275, 1252, 1176, 1048, 1020, 869,
138.07, 135.98, 78.92, 47.15, 46.24, 44.53, 41.47, 40.11, 39.15, 782, 762, 731 cmꢀ1
30.40, 29.92, 29.51, 28.34, 26.18, 26.04. Anal. calcd for
Polymer PNB–SP. 1H NMR (400 MHz, CDCl3, ppm) d: 7.96 (br,
.
C19H32N2O3: C, 67.82; H, 9.59; N, 8.33. Found: C, 67.91; H, 9.39; 2H), 7.14 (br, 1H), 7.05 (br, 1H), 6.86 (br, 2H), 6.66 (br, 2H), 5.83
N, 8.56%. FT-IR (KBr): n ¼ 3460, 3333, 3060, 2976, 2934, 2864, (br, 1H), 5.27 (br, 2H), 4.16 (br, 2H), 3.37 (br, 2H), 2.92 (br, 1H),
1689, 1649, 1539, 1478, 1453, 1442, 1388, 1365, 1333, 1276, 1253, 2.57 (br, 1H), 1.92 (br, 2H), 1.60 (br, 1H), 1.26 (br, 5H), 1.12 (br,
1178, 1050, 1021, 870, 781, 760, 732 cmꢀ1
.
3H). FT-IR (KBr): n ¼ 3421, 3281, 3062, 2968, 2932, 2868, 2855,
2-(30,30-Dimethyl-6-nitro-30h-spiro[chromene-2,20-indo]-10-yl)- 1725, 1654, 1611, 1512, 1483, 1458, 1446, 1382, 1362, 1337, 1297,
ethyl-exo-bicyclo[2.2.1]-hept-5-ene-2-carboxylate (NB–SP mono- 1275, 1170, 1091, 1049, 1028, 955, 920, 810, 750, 719, 683 cmꢀ1
mer). The NB–SP monomer was synthesized according to our
Polymer PNB–P3. 1H NMR (400 MHz, CDCl3, ppm) d: 6.53
.
previous reports.58,74 EI-MS: m/z ¼ 472 [M]+. H NMR (400 MHz, (br, 2H), 5.35 (m, 2H), 4.88 (br, 2H), 4.10 (br, 6H), 3.81 (m, 4H),
CDCl3, ppm) d: 8.01 (m, 2H), 7.21 (t, 1H, J ¼ 10.6 Hz), 7.09 (d, 1H, 3.76 (m, 2H), 3.69 (m, 6H), 3.63 (m, 12H), 3.52 (m, 6H), 3.34 (br,
J ¼ 7.2 Hz), 6.91 (m, 2H), 6.72 (m, 2H), 6.13 (m, 1H), 6.07 (m, 1H), 9H), 3.10 (m, 1H), 2.75 (m, 1H), 2.53 (br, 1H), 2.03 (br, 2H), 1.66
5.89 (d, 1H, J ¼ 10.4 Hz), 4.29 (m, 1H), 4.21 (m, 1H), 3.53 (m, 1H), (br, 1H), 1.13 (br, 1H). FT-IR (KBr): n ¼ 3059, 2974, 2928, 2875,
3.43 (m, 1H), 2.92 (m, 1H), 2.90 (s, 1H), 2.14 (m, 1H), 1.86 (m, 1H), 2817, 1728, 1591, 1505, 1455, 1439, 1366, 1349, 1333, 1248,
1
1.46 (m, 1H), 1.34 (m, 2H), 1.29 (m, 3H), 1.18 (m, 3H). 13C{1H} 1234, 1199, 1112, 1027, 942, 853, 724, 702 cmꢀ1
NMR (100 MHz, CDCl3, ppm) d: 176.06, 159.36, 146.64, 141.01,
Copolymer PNB–SP20-co-(Boc–NH)20-co-P360. 1H NMR
.
138.06, 138.01, 135.59, 135.54, 128.24, 127.77, 125.89, 122.70, (400 MHz, CDCl3, ppm) d: 7.96 (br, 0.4H), 7.13 (br, 0.2H), 7.04
121.75, 119.84, 118.39, 115.49, 106.74, 106.45, 62.36, 52.79, 46.49 (br, 0.2H), 6.85 (br, 0.4H), 6.72 (br, 0.4H), 6.66 (br, 1.2H), 5.89
& 46.41 (isomers), 46.28, 42.99 & 42.96 (isomers), 42.40, 41.53, (br, 0.2H), 5.20 (m, 2H), 4.94 (m, 1.4H), 4.10 (m, 4H), 3.81
30.38 & 30.34 (isomers), 25.80, 19.80. Anal. calcd for C28H28N2O5: (m, 2.4H), 3.76 (m, 1.2H), 3.69 (m, 3.6H), 3.63 (m, 7.2H), 3.52
C, 71.17; H, 5.97; N, 5.93. Found: C, 70.82; H, 5.95; N, 5.87%. FT- (m, 3.6H), 3.34 (m, 5.8H), 2.99 (m, 2H), 2.54 (m, 1.6H), 2.05
IR (KBr): n ¼ 3420, 3279, 3062, 2967, 2933, 2868, 2855, 1725, 1653, (m, 1.8H), 1.72 (m, 1H), 1.43 (m, 5.6H). FT-IR (KBr): n ¼ 3470,
1612, 1512, 1483, 1458, 1445, 1383, 1363, 1337, 1298, 1275, 1170, 3369, 3248, 3059, 2932, 2872, 2819, 1729, 1711, 1650, 1632,
1091, 1049, 1029, 955, 921, 810, 750, 720, 683 cmꢀ1
3,4,5-Tris-(1,4,7,10-tetraoxaundecyl)benzyl-exo-bicyclo[2.2. 1]- 1350, 1337, 1298, 1273, 1250, 1199, 1167, 1112, 1092, 1043,
hept-5-ene-2-carboxylate (NB–P3 monomer). The NB–P3 mono- 1028, 954, 851, 810, 749 cmꢀ1
.
1608, 1594, 1544, 1520, 1509, 1483, 1458, 1440, 1384, 1364,
.
mer was synthesized according to our previous report.58 EI-MS:
The copolymers were deprotected in a mixture of 95% TFA,
m/z ¼ 714 [M]+. 1H NMR (400 MHz, CDCl3, ppm) d: 6.57 (s, 2H), 2.5% H2O and 2.5% triisopropylsilane for 20 min, then
6.10 (m, 2H), 4.99 (s, 2H), 4.14 (t, 6H, J ¼ 4.6 Hz), 3.83 (m, 4H), precipitated in cold ether. The crude products were collected by
3.77 (m, 2H), 3.70 (m, 6H), 3.63 (m, 12H), 3.52 (m, 6H), 3.35 (s, centrifugation and puried by dialysis (Spectra/Por
1
9H), 3.04 (m, 1H), 2.91 (m, 1H), 2.25 (m, 1H), 1.91 (m, 1H), 1.50 membrane, MWCO 6000–8000) against water for 5 days at 4 ꢂC.
(m, 1H), 1.36 (m, 2H). 13C{1H} NMR (100 MHz, CDCl3, ppm) d: Aer lyophilisation, the resulting copolymers were obtained as
175.49, 152.30, 137.99, 137.69, 135.31, 131.24, 107.49, 71.91, yellow solids.
1
71.55, 70.43, 70.30, 70.14, 69.33, 68.53, 65.83, 58.58, 46.24,
Copolymer PNB–SP20-co-(NH2$HCl)20-co-P360. H NMR (400
45.95, 42.74, 41.25, 30.02. Anal. calcd for C36H58O14: C, 60.49; H, MHz, DMSO-d6/CDCl3 (v/v, 3 : 1), ppm) d: 7.99 (br, 0.4H), 6.98
8.18. Found: C, 60.65; H, 8.13%. FT-IR (KBr): n ¼ 3062, 2975, (m, 0.4H), 6.68 (br, 0.4H), 6.49 (br, 1.6H), 5.83 (br, 0.2H), 5.13
2930, 2875, 2820, 1728, 1592, 1504, 1456, 1439, 1367, 1349, 1334, (m, 2H), 4.87 (m, 1.4H), 4.01 (m, 4H), 3.72 (m, 2.4H), 3.66
1247, 1233, 1200, 1111, 1026, 942, 852, 723, 702 cmꢀ1
.
(m, 1.2H), 3.59 (m, 3.6H), 3.52 (m, 7.2H), 3.41 (m, 3.6H), 3.23
(m, 5.8H), 3.00 (m, 2H), 2.76 (m, 1.2H), 2.32 (m, 0.4H), 1.89
(m, 1.8H), 1.60 (m, 1H), 1.23 (m, 3.8H). FT-IR (KBr): n ¼ 3413,
3252, 3051, 2935, 2871, 2820, 1730, 1649, 1607, 1592, 1546,
General polymerization and deprotection procedure
A solution of Grubbs' third-generation initiator37 (ꢁ0.01 M) was 1518, 1509, 1487, 1457, 1440, 1381, 1363, 1350, 1332, 1298,
added to a methylene chloride solution (ꢁ0.05 M) containing 1249, 1199, 1162, 1111, 1090, 1027, 952, 851, 811, 749 cmꢀ1
.
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Ltd), and incubated overnight in 5% CO2 at 37 ꢂC. Then the
polymeric probes were added from a concentrated stock solu-
tion in PBS at a nal concentration of 0.025 mg mLꢀ1 and
incubated for 1 h. Aer discarding the media and rinsing three
times with PBS, cultured cells were placed in fresh warm serum-
free growth medium. Cell imaging was performed with an
OLYMPUS ZX81 confocal laser scanning microscope equipped
with a 63ꢃ oil-immersion objective lens and a Leica TCS SP5 MP
confocal laser scanning microscope equipped with a 63ꢃ oil-
immersion objective lens, a temperature control module, an
argon ion laser and a Coherent Chameleon Ti:sapphire laser as
the two-photon excitation source, which could be tuned over the
range of 720–950 nm. For the single-photon excitation uores-
cence imaging, cells were excited at 488 nm and emissions were
collected using a 630/30 nm lter set. For the two-photon exci-
tation uorescence imaging, cells were illuminated using the
NIR laser (750 nm) and single-photon laser (488 nm) alternately
to intensify and erase the red uorescence signals, respectively.
Cell culture
Human breast adenocarcinoma (MCF-7) cells, human hepa-
toma (Bel-7402) cells and human lung adenocarcinoma cells
(A549) were cultured in RPMI 1640 medium, and human liver
carcinoma (HepG2) cells were cultured in Dulbecco's Modied
Eagle Medium (DMEM). Both of the media contained 10% fetal
bovine serum (FBS), 100 mg Lꢀ1 streptomycin and 100 U mLꢀ1
penicillin. The cells were maintained in a humidied atmo-
ꢂ
sphere of 5% CO2 at 37 C.
Cytotoxicity assay
The cytotoxicity studies were measured using the 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Cells growing in the log phase were seeded into 96-well cell-
culture plates at a density of 5 ꢃ 104 per well, and incubated
overnight in 5% CO2 at 37 ꢂC. The polymeric probes were added
to the wells of the treatment group at concentrations of 0.00625,
0.0125, 0.025, 0.050, 0.10 and 0.20 mg mLꢀ1. The cells were
incubated for 24 h, and then 5 mg mLꢀ1 MTT (25 mL per well) in
PBS solution was added to each well. Aer incubation for an
additional 4 hours, the formazan crystals generated by dehy-
drogenases in live cells were dissolved in DMSO and the
absorbance was recorded at 492 nm by using a microplate
reader (Multiskan MK3, Thermo Electron Corporation). The
viability of the cells can be calculated as the ratio of the mean
absorbance for the treatment group to the mean absorbance for
the control group. MTT assays of cells incubated in the medium
without polymers were set as the control.
Results and discussion
The norbornenyl monomer NB–Boc–NH was obtained via the
typical condensation reaction between exo-norbornenyl
carboxylic acid70–72 and N-(tert-butoxycarbonyl)hexyl diamine73
in the presence of PyBOP and TEA in DMF, with an excellent
yield of 91%. The spiropyran monomer NB–SP and hydrophilic
monomer NB–P3 were synthesized according to our previous
report.58 For the preparation of our designed copolymers, the
living character of the polymerization for the NB–Boc–NH and
NB–P3 monomers was rstly investigated. Both were subject to
ROMP individually using Grubbs' third-generation initiator at
room temperature in degassed methylene chloride. Fig. 1 shows
the molecular weights (Mn) of these homopolymers versus the
monomer to initiator ratios (Fig. S1 and Table S1, ESI†). Four
different polymerizations were carried out with monomer to
initiator ratios of 25 : 1, 50 : 1, 75 : 1 and 100 : 1. A linear plot
suggests a high degree of control over the polymerization.
Moreover, the 1H NMR spectra (Fig. S2†) show complete
disappearance of the carbene signal of the initiator at 19.1 ppm,
AbsTreatment group
Cell viability ð%Þ ¼
ꢃ 100:
AbsControl group
The statistical analysis was compared using Student's t-test
over ve nominally identical measurements. The results were
expressed as the mean ꢄ SD% and considered to be statistically
signicant when p < 0.05.
Flow cytometry
Bel-7402 cells were plated in a 6-well plate at a density of 1 ꢃ 106 and at the same time the formation of two new broad carbene
per well and grown overnight in 5% CO2 at 37 ꢂC. Then the
polymeric probes were added from a concentrated stock solu-
tion in PBS at a nal concentration of 0.025 mg mLꢀ1 and
incubated for 1 h. Aer discarding the media and rinsing three
times with PBS, the cells were harvested by trypsinization. Aer
centrifugation, the cell pellets were washed carefully with PBS,
re-suspended in PBS and analyzed immediately using a BD
FACSAria ow cytometer (BD Biosciences) operating at 488 nm
excitation and with a 610/20 nm emission lter set. The cell
suspension was illuminated for 30 s to convert the SP polymers
to MC polymers using a 365 nm UV LED before ow cytometric
analysis.
Cellular imaging
The cells were seeded at a density of 1 ꢃ 105 per well on each
Fig. 1 Plot of Mn vs. the monomer to initiator ratios for the ROMP of
35 mm glass bottom culture dish (NEST Biotechnology Co., NB–Boc–NH and NB–P3 monomers.
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Table 1 GPC and zeta potential data for the PNB–SPx-co-(Boc–NH)y-co-P3z and PNB–SPx-co-(NH2$HCl)y-co-P3z water-soluble random
copolymers
Polymera
Mn/kDa
Mw/kDa
PDI
z potentialb/mV
PNB–SP20-co-P380
77.2
70.5
60.4
60.3
60.2
44.1
43.2
93.8
88.7
76.8
81.5
76.9
56.4
58.1
1.22
1.26
1.27
1.35
1.28
1.28
1.34
ꢀ2.43
—
—
PNB–SP20-co-(Boc–NH)10-co-P370
PNB–SP20-co-(Boc–NH)20-co-P360
PNB–SP20-co-(Boc–NH)30-co-P350
PNB–SP20-co-(NH2$HCl)10-co-P370
PNB–SP20-co-(NH2$HCl)20-co-P360
PNB–SP20-co-(NH2$HCl)30-co-P350
—
+13.2
+13.7
+15.6
a
b
Measured in DMF with 10 mM LiBr at 313 K. Measured in 10 mM PBS buffer (pH 7.4) with the concentration of 1.0 mg mLꢀ1 at 298 K.
signals at 18.7 and 18.4 ppm. These observations clearly
The chemical structure for copolymers and their compo-
demonstrate a living fashion of ROMP for the Boc-protected nents were investigated by FT-IR (Fig. 3). The spiropyran
amino monomer NB–Boc–NH and the hydrophilic monomer homopolymer PNB–SP (Fig. 3c, blue) exhibited an almost
NB–P3.45,46
identical spectrum as its monomer NB–SP,75 presenting typical
Based on our previous study, water-soluble PNB–SPx-co-(Boc– NO2 asymmetric and symmetric stretching vibrations at 1512
NH)y-co-P3z copolymers were prepared empirically from suit- and 1337 cmꢀ1, alkyl-N stretching at 1297 cmꢀ1, phenyl-N
able feeding proportions of the photochromic spiropyran stretching at 1275 cmꢀ1, O–C–N stretching at 955 cmꢀ1, and
monomer NB–SP, amino monomer NB–Boc–NH and hydro- aromatic C–H out-of-plane deformation at 810 and 750 cmꢀ1
.
philic monomer NB–P3. As the degree of polymerization (DP) Besides, the vibration peak at 3062 cmꢀ1 is attributed to alkene
was xed at 100, i.e. the molar ratio of the total feeding amounts C–H stretching on the polynorbornene backbone or aromatic C–
of the monomers to Grubbs' third-generation initiator was H. Other vibration peaks were observed at 1725, 1170 and
100 : 1, the spiropyran monomer proportion x was chosen as 20 1091 cmꢀ1 for C]O, alkyl-O and phenyl-O stretching, respec-
and the NB–Boc–NH monomer proportion y was varied from tively. The hydrophilic homopolymer PNB–P3 (Fig. 3e, purple)
0 to 30. The GPC data is listed in Table 1. The PDIs of the showed strong vibration peaks attributed to alkyl C–H stretch-
resulting PNB–SPx-co-(Boc–NH)y-co-P3z copolymers were rela- ing at 2800–3000 cmꢀ1, C]O stretching at 1728 cmꢀ1, alkyl-O
tively narrow, ranging from 1.22 to 1.35, and the molecular stretching at 1199 cmꢀ1 and phenyl-O stretching at 1112 cmꢀ1
,
weight Mn was in the range of 60–77 kDa, which showed a which should be assigned to the repeated oligo-ethylene glycol
reasonable relationship with the total feeding amounts of the units. Bearing the Boc-protected amino group, the PNB–Boc–
monomers.
NH homopolymer exhibited strong N–H stretching and bending
For the activation of the Boc-protected amino group, the peaks at 3332 and 1646 cmꢀ1, respectively (Fig. 3d, green), and
PNB–SPx-co-(Boc–NH)y-co-P3z copolymers were deprotected by a the carbonyl stretching vibration of the amide linkage shied to
standard treatment with a mixture of 95% TFA, 2.5% H2O and 1689 cmꢀ1, which has an obvious difference to that of the ester.
2.5% triisopropylsilane. Here, triisopropylsilane served as a The FT-IR spectrum for the PNB–SP20-co-(Boc–NH)20-co-P360
radical scavenger for stabilizing the deprotected amino group.
Aer counterion exchange with NaCl and dialysis in a refriger-
ator, the activated PNB–SPx-co-(NH2$HCl)y-co-P3z copolymers
were obtained as yellow solids. The GPC measurements
provided clear evidence for the decrease of the molecular
weights (Fig. 2), while the corresponding retention volumes
were obviously greater than those before the Boc-deprotection.
Fig. 2 Normalized GPC curves of the PNB–SPx-co-(Boc–NH)y-co-
P3z (solid line) and PNB–SPx-co-(NH2$HCl)y-co-P3z (dashed line) Fig. 3 FT-IR spectra of the deprotected PNB–SP20-co-(NH2$HCl)20
-
copolymers: x ¼ 20, y ¼ 0, z ¼ 80 (black); x ¼ 20, y ¼ 10, z ¼ 70 (red); x co-P360 (a, black), PNB–SP20-co-(Boc–NH)20-co-P360 (b, red), PNB–
¼ 20, y ¼ 20, z ¼ 60 (green); x ¼ 20, y ¼ 30, z ¼ 50 (blue).
SP (c, blue), NB–Boc–NH (d, green) and PNB–P3 (e, purple) polymers.
506 | J. Mater. Chem. B, 2014, 2, 502–510
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copolymer was well represented as a composite of the above
three components (Fig. 3b, red). Aer standard treatment with
TFA, the vibration peaks for N–H stretching at 3369 cmꢀ1, C]O
stretching at 1729 cmꢀ1 and N–H bending at 1650 cmꢀ1 were
greatly decreased in the activated PNB–SP20-co-(NH2$HCl)20-co-
P360 copolymer (Fig. 3a, black), suggesting the removal of the
Boc pretecting group.
The similarity in chemical structure for the activated and
Boc-protected copolymers was also corroborated by 1H NMR,
shown in Fig. 4. The spectrum of the PNB–SP20-co-(Boc–NH)20
-
co-P360 copolymer resembles a stoichiometric superposition of
its PNB–SP, PNB–Boc–NH and PNB–P3 homopolymer compo-
nents (see Fig. S22–24, ESI† for details). A single peak at
1.43 ppm clearly indicates the existence of the tertiary butyl
group in the Boc-protected copolymer. Aer deprotection this
signal peak disappears, suggesting the formation of an
ammonium group in the activated PNB–SP20-co-(NH2$HCl)20-co-
P360 copolymer.
Fig. 5 UV-Vis absorption and normalized fluorescence spectra of the
non-fluorescent SP form (black) and fluorescent MC form (red) of
PNB–SP20-co-(NH2$HCl)20-co-P360 with a concentration of 0.082
mg mLꢀ1; and the normalized fluorescence spectrum of deprotected
PNB–SP20-co-(NH2$HCl)20-co-P360 (blue) with a concentration of
0.087 mg mLꢀ1 in 10 mM PBS buffer.
Consistently, both the activated PNB–SP20-co-(NH2$HCl)20
-
co-P360 and Boc-protected PNB–SP20-co-(Boc–NH)20-co-P360
copolymers exhibit excellent photochromism in aqueous
media. Reversible photoisomerization of the SP units was
clearly evidenced by UV-Vis absorption spectra, shown in Fig. 5
(see also Fig. S3, ESI†). On irradiating the aqueous solution of
PNB–SP20-co-(NH2$HCl)20-co-P360 by 365 nm UV light for 6
seconds, the photostationary states were reached. A strong
absorption peak at 559 nm, characteristic of the MC isomer,
appeared (Fig. S3, ESI,† top), and the colorless solution became
deep blue. On irradiating the blue solution with 530 nm visible
light, the MC unit isomerized back to the SP isomer in 200 s,
and the absorption spectrum almost reverted to the original
spectrum (Fig. S3, ESI,† bottom). No Stokes shis were
observed, which implies no aggregation or environmental
changes to the SP or MC isomers during photoisomerization.
The MC uorescence of PNB–SP20-co-(NH2$HCl)20-co-P360 was
given as the red curve in Fig. 5 with a maximum emission peak
at 638 nm and a lifetime of 2.87 ns (Fig. S4, ESI†). It shows no
obvious difference from that of PNB–SP20-co-(Boc–NH)20–co-
P360, with a maximum emission peak at 636 nm and a lifetime
of 1.73 ns (Fig. S5, ESI†).
Cytotoxicity is essential for the application of poly-
norbornenes in uorescence imaging. Here, the effect of PNB–
SP20-co-(NH2$HCl)20-co-P360 on the proliferation of Bel-7402,
HepG2, MCF-7 and A549 cells was accessed using a modied
MTT assay. When the concentration of the polymer is lower
than 0.10 mg mLꢀ1, the cell viabilities are greater than 96%;
when the concentration of the polymer is as high as
0.20 mg mLꢀ1 the cell viabilities remain at 91%, as shown in
Fig. 6. This observation suggests that the cytotoxicity of the
polynorbornenes is very low and may benet the single- and
two-photon in vitro uorescence imaging.
Cell transporting capability is also essential for the applica-
tion of polynorbornenes in uorescence imaging. The activated
ammonium group can signicantly enhance the cell uptake
efficiency of these multi-component polymers. The uorescence
Fig.
(bottom) in CDCl3 and deprotected PNB–SP20-co-(NH2$HCl)20-co- (purple) and A549 (green) cells after incubation with the PNB–SP20
P360 (top) in DMSO-d6/CDCl3 (v/v, 3 : 1). The residual solvents are co-(NH2$HCl)20-co-P360 copolymer at various concentrations
4
1H NMR spectra for PNB–SP20-co-(Boc–NH)20–co-P360 Fig. 6 Metabolic viability of Bel-7402 (red), HepG2 (blue), MCF-7
-
marked with stars.
for 24 h.
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potential of ꢀ2.43 mV exhibits no cell transporting capability.
Since cell membranes are weakly negatively charged, electro-
static interactions drive the cationic PNB–SPx-co-(NH2$HCl)y-co-
P3z polynorbornenes to easily anchor to cell membranes and
internalize into cells through a charge-mediated endocytosis
process.69 In addition, the control experiment at 4 C (Fig. S7,
ꢂ
ESI†) suggests that the internalization of PNB–SP20-co-
(NH2$HCl)20-co-P360 is dramatically inhibited at low tempera-
ture, which indicates that the uptake of polynorbornenes by Bel-
7402 cells is through an energy-dependent pathway.32
The application of multi-component polynorbornenes for
cellular uorescence imaging was investigated by confocal laser
scanning microscopy. Bearing a photo-responsive SP compo-
nent, the PNB–SP20-co-(NH2$HCl)20-co-P360 copolymer exhibits
switchable uorescence imaging characteristics with alternating
NIR two-photon and visible single-photon excitations in Bel-
7402, HepG2, MCF-7 and A549 cells, as shown in Fig. 8. A cycle of
optical switching begins with excitation of the non-uorescent
SP form within a single scan with a 750 nm NIR laser, eliciting an
excited-state isomerization to the uorescent MC form. Subse-
quent excitation of MC using a 488 nm visible laser induces
either red uorescence or gradual photoisomerization back to
the non-uorescent SP form. Such switching cycles regulated by
NIR and visible laser illumination could be repeated for more
than ten cycles in Bel-7402 cells (Fig. 9), as well as in HepG2,
MCF-7 and A549 cells (Fig. S9–11 in ESI†). The uorescent signal
from the cytoplasm is observed clearly to distinguish the cell
Fig. 7 Flow cytometry histograms of the Bel-7402 cells after 1 h
incubation with 0.025 mg mLꢀ1 PNB–SPx-co-(NH2$HCl)y-co-P3z
copolymers: control experiment (black), x ¼ 20, y ¼ 0, z ¼ 80 (red); x ¼
20, y ¼ 10, z ¼ 70 (green); x ¼ 20, y ¼ 20, z ¼ 60 (blue); x ¼ 20, y ¼ 30, z
¼ 50 (purple), illuminated with a 365 nm UV LED for 30 s.
proles of the PNB–SPx-co-(NH2$HCl)y-co-P3z copolymers was
quantitatively studied by ow cytometry in Bel-7402 cells, and
the histograms are shown as Fig. 7. With more cationic ammo-
nium groups on the polynorbornene backbones, the cell uptake
of the PNB–SPx-co-(NH2$HCl)y-co-P3z copolymers is more effi-
cient, which is in agreement with the increasing of the positive z
potentials listed in Table 1. In the absence of activated ammo-
nium groups, the neutral PNB–SP20-co-P380 copolymer with a z
Fig. 8 NIR two-photon fluorescence imaging and time-dependent single-photon fluorescence decay for Bel-7402 (a), HepG2 (b), MCF-7 (c)
and A549 (d) cells.
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