Study on a series of water-soluble photoinitiators for fabrication of 3D hydrogels by two-photon polymerization

Article abstract of DOI:10.1016/j.dyepig.2017.02.040

A series of water-soluble benzylidene cyclanone dyes T1-T3 were synthesized. Their photo-physical properties were investigated using UV–Vis spectra, fluorescence spectra and two-photon absorption spectra. The maximum two-photon absorption cross-sections (σ₂) of T1-T3 in deionized water were determined as 567?GM, 808?GM and 231?GM. Using T1-T3 as photoinitiators (PIs) directly, 2D and 3D nano patterns based on two-photon crosslinking or polymerization of bovine serum albumin (BSA), water-soluble acrylic ester monomer (SR610) and hyaluronic acid derivative (HAGM) were successfully fabricated, respectively. Much lower threshold energies and wider fabrication windows were obtained in the formulations containing these PIs compared with commercial ones. Furthermore, the cytotoxicity study showed a favorable biocompatibility of these PIs, which indicated T1-T3 would have application prospects in 3D fabrication of biomaterials.

Full text of DOI:10.1016/j.dyepig.2017.02.040

Accepted Manuscript
Study on a series of water-soluble photoinitiators for fabrication of 3D hydrogels by
two-photon polymerization
Xing Huang, Xiaopu Wang, Yuxia Zhao
PII:
S0143-7208(17)30028-1
10.1016/j.dyepig.2017.02.040
DYPI 5819
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 5 January 2017
Revised Date: 21 February 2017
Accepted Date: 22 February 2017
Please cite this article as: Huang X, Wang X, Zhao Y, Study on a series of water-soluble photoinitiators
for fabrication of 3D hydrogels by two-photon polymerization, Dyes and Pigments (2017), doi: 10.1016/
j.dyepig.2017.02.040.
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ACCEPTED MANUSCRIPT
Study on a series of water-soluble photoinitiators for fabrication of 3D
hydrogels by two-photon polymerization
Xing Huanga,b, Xiaopu Wanga, Yuxia Zhaoa,*
aTechnical Institute of Physics and Chemistry, CAS, 29 Zhongguancun East Road,
Haidian District, Beijing, 100190, China; bUniversity of Chinese Academy of
Sciences, No.19A Yuquan Road, Beijing, 100049, China; *yuxia.zhao@mail.ipc.ac.cn;
phone +86 010-82543569
Abstract
A series of water-soluble benzylidene cyclanone dyes T1-T3 were
synthesized. Their photo-physical properties were investigated using
UV-Vis spectra, fluorescence spectra and two-photon absorption spectra.
The maximum two-photon absorption cross-sections (σ2) of T1-T3 in
deionized water were determined as 567 GM, 808 GM and 231 GM.
Using T1-T3 as photoinitiators (PIs) directly, 2D and 3D nano patterns
based on two-photon crosslinking or polymerization of bovine serum
albumin (BSA), water-soluble acrylic ester monomer (SR610) and
hyaluronic acid derivative (HAGM) were successfully fabricated,
respectively. Much lower threshold energies and wider fabrication
windows were obtained in the formulations containing these PIs
compared with commercial ones. Furthermore, the cytotoxicity study
showed a favorable biocompatibility of these PIs, which indicated T1-T3
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would have application prospects in 3D fabrication of biomaterials.
Keywords：Water-Soluble Photoinitiator; Two-Photon Polymerization;
Biomaterials; Tissue Engineering.
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Introduction
Tissue engineering is a fresh discipline to study the growth of a fully
functional tissue or organ from seed cells and a collagenous scaffold for a
medical purpose. It is a combination of cell biology and material science.
In human tissues, the natural extracellular matrix (ECM) serves as a 3D
substructure, which can be constantly remodeled. Through its intimate
interaction with cells, ECM regulates cell differentiation, proliferation,
exchanging nutrients and metabolites. [1-2] In order to mimick the natural
ECM, study on the development of 3D scaffolds with good
biocompatibility and proper mechanical strength is of very importance.
[3-6]
Hydrogels are attractive biomaterials for numerous medical
applications. Due to their similar mechanical and diffusivity properties to
ECM, a number of synthetic and natural hydrogels, such as, PEGDA,
[7-11]
Gelatin, Collagen, HA and Lignin,
their potentials in Tissue engineering.
have been developed to explore
Two-photon polymerization (2PP) is a powerful tool to construct 3D
matrixes with micro- and nanometer precision due to its advantages of
large penetration depth and high spatial selectivity. Moreover, with the
low photon energy of near infrared light normally used in 2PP, 3D
manipulation can be performed in the presence of cells, which guides the
formation of biological tissue in all stages of its development. Thus, using
2PP technique to fabricate 3D hydrogels based on natural or artificial
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materials under an aqueous environment has attracted much attention in
recent years. [12-15]
Water-soluble photoinitiators (PIs) with two-photon absorption (2PA)
capability are the first prerequisite for 2PP based on hydrogels. However,
till now the corresponding reports are very limited. Campagnola et al.
studied 2PP of acrylamide and two-photon crosslinking (2PC) of protein
in aqueous solution using commercially available hydrophilic xanthene
dyes, such as Rose Bengal, Eosin and Erythrosine, with amino as
[16]
coinitiator, and fabricated simple 3D models.
However, because the
2PA cross-sections (σ2) of these dyes were very small (only 10 GM at 800
nm), a high laser intensity (100 mW) and long exposure time (300 400
µs) were required in their experiments. By using a water-soluble PI
(WSPI) with a σ2 of 120 GM, Liska et al. successfully fabricated 3D
scaffolds from PEGDA (PEG diacrylates) with a fast scanning speed of
10 mm/s under laser power of 140 mW.[17] Irgacure 2959, a
commercially available hydrophilic PI, was also used for 2PP of 3D
scaffolds due to its good biocompatibility. However, it is only suitable for
2PP at 515 nm wavelength, which may induce the denaturation of
proteins. [18-19] Herein, the exploration of highly active water-soluble PIs
with large σ2 values in near infrared range is a critical requirement.
In our previous work, it was proved that benzylidene cyclanone dyes had
large σ2 values (400 3300 GM) around 800 nm and could be used as
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high-efficient photosensitisers or even photoinitiators directly in 2PP
based on organo-soluble materials. [20-22] Thereafter, through modifying
benzylidene cyclopentanone dyes with sodium carboxylate groups, a
series water-soluble dyes were synthesized. Among them, BSEA
(modified by four sodium carboxylate groups) showed a high efficiency
on initiating 2PP of water-soluble acrylate ester (SR610). [23,24] Liska et al.
also reported similar benzylidene cyclopentanone and cyclohexanone
dyes (P2CK and G2CK), which were modified by two sodium
carboxylate groups at the terminal alkyl chains, and proved their
[25-26]
availability in 2PP or 2PC of biocompatible materials.
These
aforementioned works preliminarily revealed the potential of
water-soluble benzylidene cyclanone dyes in biomedicine fields.
In our latest study on biocompatibility of some hydrophilic dyes, it was
found that the cytotoxicity of a dye was positively correlated with its
amount of cellular uptake. For a similar molecular structure, introducing
more hydrophilic groups is beneficial to decrease cellular uptake and
[27-28]
enhance biological safety.
In this work, we synthesized a series of
hydrophilic dyes using a high-efficient route different from that of BSEA
through introducing four sodium carboxylate groups into different
benzylidene cyclanone moieties (cyclobutanone, cyclopentanone and
cyclohexanone) to further exploit the application of such dyes in tissue
engineering. The specific structures of designed PIs (T1-T3) are shown in
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Scheme 1.
2 Experimental
2.1 Materials
Cyclobutanone,
cyclopentanone,
cyclohexanone
and
glycidyl
methacrylate were purchased from Arcos Organics; acrylic acid, ethanol,
N,N-dimethylformamide(DMF), aniline, phosphorus pentachloride,
p-benzenediol, methyl acrylate, light petroleum, acetic ether, dimethyl
sulfoxide, Eosin and NaOH were bought from Beijing Chemical Co. Ltd.
Chromatographically pure methanol were obtained from Fisher Scientific
Company. Other chromatographically pure reagents were from Xilong
Chemical Co. Ltd. SR610 was purchased from Sartomer Co. Ltd. Bovine
serum albumin (BSA), phosphate buffered solution (PBS), Dulbecco’s
Modified Eagle’s Medium (DMEM), Fetal Bovine Serum (FBS),
Penicillin/Streptomycin and MTT reagent were obtained from Beijing
Solarbio Science & Technology Co. Ltd. HepG2 was donated by Peking
Union Medical College Hospital. Sodium hyaluronate and Poly (ethylene
glycol) diacrylate (PEGDA) were from Aladdin, Hyaluronic acid
derivative (HAGM) with a degree of substitution 61% was prepared
[29]
according to the literature.
The synthesis and characterization of
HAGM were presented in Supporting Information. Y2 and Y3 were
[24]
synthesized on the basis of protocols reported by our group before.
The structures of Y2 and Y3 are shown in Figure 1. All materials were
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used directly without further purification.
2.2 Synthesis and characterization
The synthetic routes towards compounds T1-T3 are shown in Scheme 1.
1
All final compounds were characterized by H-NMR, 13C-NMR,
HR-MS and HPLC analysis, conforming a purity of over 95%.
2.2.1. Dimethyl 3, 3'-(phenylazanediyl) dipropanoate (M1).
Aniline (9.31 g, 0.10 mol), methyl acrylate (26.40 g, 0.30 mol), ethylic
acid (12.12 g, 0.20 mol) and 0.32 g p-benzenediol were added into a 100
ml round-bottom flask and refluxed for 20 hours at 102°C. After the
reaction, excess of the methyl acrylate and ethylic acid were excluded by
vacuum distillation, the compounds were purified by Column
Chromatograph (silica, light petroleum/CH3COOCH2CH3 =8:1) and 25.20
1
g pure liquid M1 was obtained. (Yield 95.05%). H-NMR (400MHz，
CD3OD): δ (ppm): 2.53 (t, J = 8 Hz, 3H), 3.49 (s, 4H), 3.62 (s, 6H),
6.64(t, J = 8 Hz, 1H), 6.69 6.71 (m, 2H), 7.15(t, J = 8 Hz, 2H). HR-MS
(ESI) theoretical m/z [M+1H]+ =266.1392, found 266.1378.
2.2.2. Dimethyl 3, 3'-((4-formylphenyl)azanediyl) dipropanoate (M2).
PCl5 (20.82 g, 0.10 mol) was added slowly to DMF (50 mL) and the
solution was cooled in an ice bath. After stirring at 0°C for 30 min, the
solution was continuously stirred at room temperature for another 15 min,
and then M1 (12.75 g, 0.05 mol) was slowly added. After stirring at 75°C
for 1 h and then cooled at room temperature, the solution was added to
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ice water and neutralized with saturated sodium bicarbonate solution. The
aqueous layer was extracted three times with CHCl3 (3×50 mL) and the
extraction liquid was washed with a large amount of water. The organic
phase was dried over anhydrous MgSO4 and rotary evaporation, the
compounds were purified by Column Chromatograph (silica, light
petroleum/CH3COOCH2CH3 =5:1) to give 13.81 g yellow liquid M2.
(Yield 97.96%). 1H-NMR (400MHz，CD3OD): δ (ppm): 2.69 (t, J = 8 Hz,
4H), 3.65 (s, 6H), 3.82 (t, J = 8 Hz, 4H), 6.87 6.89(d, J = 8 Hz, 2H),
7.72 7.74 (d, J = 8 Hz, 2H), 9.74(s, 1H). HR-MS (ESI) theoretical m/z
[M+1H]+=294.1341, found 294.1328.
2.2.3.
Sodium
3,3',3'',3'''-((((2-oxocyclobutane-1,3-diylidene)bis
(methanylylidene))bis(4,1-phenylene))bis(azanetriyl))tetrapropanoate
(T1)
M2 (1.05 g, 3.58 mmol) and NaOH (0.29 g, 7.25 mmol) were added to 50
ml H2O and stirred for a night at room temperature. Then most of the
water was excluded by rotary evaporation. NaOH (0.07 g, 1.75 mmol)
and cyclobutanone (0.12 g, 1.71 mmol) were added to the above solution
and stirred at 80℃ for 5 h. After the system cooling down to room
temperature, ethanol (50 ml) was added and stirred for 2 h at room
temperature. The deep red compound T1 (0.81 g) was recrystallized from
ethanol. (Yield 70.93%). 1H-NMR (400MHz, D2O): δ (ppm): 2.43 (t, J=8
Hz, 8H), 3.63 (t, J = 8 Hz, 8H), 3.74 (s, 2H), 6.83 6.85 (d, J=8 Hz, 4H),
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13
7.13 (s, 2H), 7.55 7.57 (d, J=8 Hz, 4H). C-NMR (100 MHz, D2O) : δ
(ppm): 193.30, 181.35, 150.33, 139.92, 133.35, 130.23, 122.88, 112.96,
48.47, 36.26, 35.14. HR-MS (ESI) theoretical m/z [1/2(M-2Na)+1H]+
=304.2628, found 304.2608.
2.2.4.Sodium
3,3',3'',3'''-((((1E,1'E)-(2-oxocyclopentane-1,3-diylidene)bis(methanylylid
ene))bis(4,1-phenylene))bis(azanetriyl))tetrapropanoate (T2)
Sodium
3,3',3'',3'''-((((1E,1'E)-(2-oxocyclohexane-1,3-diylidene)bis(methanylylid
ene))bis(4,1-phenylene))bis(azanetriyl))tetrapropanoate (T3)
The deep red compounds T2 and T3 were obtained by a similar
procedure as described above for T1.
T2 (yield 88.67%). 1H-NMR (400MHz, D2O): δ (ppm): 2.43 (t, J=7.4 Hz,
8H), 2.76 (s, 4H), 3.54(t, J=7.4 Hz, 8H), 6.74 (d, J=8.8 Hz, 4H), 7.18 (s,
13
2H), 7.46 (d, J=8.8 Hz, 4H). C-NMR (100 MHz, D2O) : δ (ppm):
198.74, 181.26, 149.36, 135.93, 134.37, 127.29, 124.17, 112.85, 48.46,
36.01, 27.03. HR-MS (ESI) theoretical m/z [M−4Na+5H]+ =579.2347,
found 579.2337.
1
T3 (yield 69.33%). H-NMR (400MHz, D2O): δ (ppm): 1.73 (s, 2H),
2.40 (t, J=6 Hz, 8H), 2.85 (s, 4H), 3.59 (t, J=8 Hz, 8H), 6.80 6.82 (d, J=8
13
Hz, 4H), 7.50 7.52 (d, J=8 Hz, 4H), 7.58 (s, 2H). C-NMR (100 MHz,
D2O) : δ (ppm): 193.77, 181.66, 149.17, 139.28, 134.30, 133.05, 123.92,
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112.64, 48.43, 36.18, 28.87, 23.22. HR-MS (ESI) theoretical m/z
1/2[M−4Na+2H]2-=295.1149, found 295.1133.
2.3 Methods
UV–vis spectra were recorded on a JascoV-530 spectrophotometer.
Steady-state fluorescence was performed at room temperature using
Hitachi F-4500 spectrometer. Fluorescence quantum yields (Φf) in water
were measured using rhodamine B dissolved in methanol as a standard
(Φf =0.7). [30] The singlet oxygen quantum yield (Φꢀ) was determined by a
photochemical bleaching method using 1,3-Diphenylbenzofuran (DPBF)
as 1O2 acceptor and methylene blue (MB) as the reference with a yield of
13
0.75 in CH3OH.[31] 1H-NMR and C-NMR spectra were obtained on a
Bruker DPX 400 spectrometer. The HR-MS analyses were carried out on
a Bruker apex IV FT mass spectrometer. 2PP fabricated microstructures
were characterized by scanning electron microscopy (SEM,
HitachiS-4300FEGd).
2PA cross-section (σ2) values of compounds were determined using
two-photon excited fluorescence (2PEF) method with a femtosecond
laser.
In 2PEF measurement, the light source is a Tsunami mode-locked Ti:
sapphire system (750 840 nm, 80 MHz, <130 fs). Up-converted
fluorescence spectra were recorded by a fiber spectrometer (Ocean Optics
USB2000 CCD). The concentrations of samples were 5.0×10-5 M,
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1.0×10-4 M and 1.0×10-4 M for T1-T3, respectively. Rhodamine B in
methanol solution (10-6 M) was used as reference. The 2PA cross-section
of sample was calculated by equations reported previously. [32]
In 2PP experiments, T1-T3 and Eosin were used as PIs, monomers were
SR610, BSA and HAGM. All formulas are showed in table 1. The mixed
photo-curable resins were poured onto glass substrate. The laser (Tsunami
Ti: sapphire, 820 nm, 82 MHz, 80 fs) was tightly focused via an
oil-immersion objective lens (100×, NA=1.45, Olympus) into the sample
which was fixed on a xyz-step motorized stage controlled by a computer.
The fabrication process was monitored by a CCD on live, after the
fabrication, the unpolymerized resin was washed out by deionized water.
2PP linear structures and 2PC patterns were watched by Nikon TI-U
inverted microscope and recorded by Low Temperature refrigeration high
touch bar camera (PIXIS: 100B). The obtained 3D microstructures were
characterized by SEM (HITACHI S-4300F).
The resins R2-R4 (listed in Table 1) were used in one-photon
polymerization experiments. The mixed solution was poured into a glass
cell made in our laboratory, the thickness of the sample was 0.5 mm. A
532 nm laser was used as light source. Its intensity of irradiation was 60
mW/cm2. The conversion rate of double bonds during the photocuring of
the formulation was recorded by real-time Fourier transform infrared
spectroscopy (Nicolet Magna 5700 real-time FTIR). The polymerization
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conversion efficiency was calculated by the change of the double bond
absorption peak area at 6082-6230 cm-1 in near-IR region.
The evaluation of biocompatibility of T1-T3 was carried out by MTT
assay using hepatoma cells HepG2, while Y2 and Y3 were used as
contrasts. The cells were incubated in the culture media of DMEM
containing 10% FBS and 1% Penicillin/Streptomycin. HepG2 cells were
seeded onto a 96 well plate at a density of 1×104 cells per well in 100 µl
culture media and incubated for 24 h for cells to attach in an incubator in
a humid atmosphere with 5% carbon dioxide at 37°C. After that, 100 µl
culture media containing different concentrations of PIs (T1-T3, Y2 and
Y3) were added into the 96 well plates to achieve the final PIs’
concentrations of 0.6, 0.9, 1.3, 2.0 and 3.0 mM in wells, respectively. The
culture media and the culture media plus HepG2 were used as blank
group and control group, respectively. After another 24 h incubation, 20
µl MTT reagent (dissolved in PBS, 5 g/L) was added into the plates and
well-distributed, then the 96 well plates were put back into the incubator
for additional 4 h incubation. Afterwards, the culture media was removed
and all wells were washed three times with PBS, 150 µl dimethyl
sulfoxide was added into the wells to dissolve the formazan crystals. The
plates were incubated on a shaker in the dark at room temperature for 20
min, and the absorbance in each well was measured at 570 nm on a
Multi-mode microplate detecting instrument (Perkin Elmer).
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3. Results and discussions
3.1 Photo-physical properties
The normalized absorption spectra and the florescence emission spectra
of T1-T3 in deionized water are shown in Figure 2. Their comprehensive
photo-physical characteristics are listed in Table 2. It is shown that all of
the three compounds present a strong intramolecular charge transfer (ICT)
absorption band within 400 650 nm. This absorption band is attributed to
symmetrical charge transfer from the electron-donating amino groups on
both ends to the central electron-withdrawing carbonyl group. Compared
with T1, the absorption peaks of T2 and T3 exhibit a blue-shift of 9 nm
and 44 nm, respectively, while their emission peaks also have similar
shifts to the short wavelength region, which should be due to the increase
of the ring size. From T1 (cyclobutanone) to T3 (cyclohexanone), the
planarity of the cross-conjugated system decreases, which diminishes the
delocalization of the π electrons.
The Stokes shift of T3 is much larger than that of T1 and T2, which
means that the geometry change between the ground state and the first
excited state of T3 is much greater compared with the other two
compounds. A decrease in fluorescence quantum yield (Φf) can be
observed with an increase of the size of the middle ring from T1 to T3,
which should be due to the better flexibility of the larger ring, thus
promoting non-radiative relaxation of electronic excited states.
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The Φꢀ value of a compound is positively correlated with its
photocytotoxicity after being taken by cells or tissues when it is exposed
to light. All of T1-T3 present a low Φꢀ data within 0.01 0.02, which
ensures a certain security when they are applied in biological fields.
3.2 2PA cross-sections
Using the 2PEF method, the two-photon-excited spectra of compounds
T1-T3 in deionized water within 750 840 nm were measured using
Rhodamine B as a reference. The results are showed in Figure 3. It can
be seen that the 2PA peaks of three compounds within the measured range
all appear in 820 nm. The maximum σ2 values of T1-T3 are 567 GM, 808
GM and 231 GM, respectively, which are much higher than the
corresponding data of commercial hydrophilic initiators. It is known that
the 2PA intensity of a compound is strongly dependent on its
intramolecular charger transfer (ICT), which is critically relative with its
co-planarity. This result further confirms that the cross-conjugated system
is distorted by the flexible six-membered ring of cyclohexanone.
3.3 One-photon polymerization
The monomer’s double bond conversion (DBC) is an efficient indicator
to character the initiation capacity of a PI. A higher conversion of
monomers means better initiation efficiency of PIs. The conversion
efficiency of three resins R2-R4 with SR610 as monomer containing
T1-T3 as PI, respectively, was characterized by plotting the DBC during
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the polymerization against the time (Figure 4). It can be easily found that
in the same formula of resins, T2 exhibits the best initiation efficiency.
3.4 Two-photon polymerization
A series of resins were made up using different formula with SR610, BSA
and HAGM as monomers, T1-T3 as initiators, respectively, to investigate
their potentials in 2PP. Blank monomers and monomers plus Eosin were
both used as contrasts. Using line scanning method, the 2PP threshold
energy (Eth) and the laser-induced breakdown threshold energy (Ebr) of
these resins under illumination of an 820 nm fs laser could be observed
by CCD on live. Here, Eth is defined as the lowest laser power at focus
point that can fabricate a solid line and Ebr is defined as lowest laser
power at focus point that can induce intense damage to materials under
the same scan speed. The power between Eth and Ebr is defined as the
fabrication window (FW). [33] The obtained FW (Eth and Ebr) data for PIs
in all formulas are listed in Table 1.
It is pursued for a formula to have a broad FW and lower Eth in
application, which can ensure a high quality and efficiency in mass
production. In Table 1, it is shown that T1-T3 all exhibit lower 2PP Eth
than that of Eosin when a same monomer is used. Furthermore, it is
obvious that T2 performs an overwhelmingly excellent fabrication
capacity in SR610. The Eth of resin R3 (containing T2 as PI) is much
lower than those of other SR610 resins, while the corresponding
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differences among BSA or HAGM resins are limited. It should be due to
the lack of enough reactive groups in BSA or HAGM monomers so that
they are not sensitive to the amounts of free radicals or other active
species generated by PIs.
For R1-R4, both of 2D and 3D nanopatterns can be fabricated by 2PP. A
representative line array obtained using R2 (T1 as PI) by changing the
incident energies with a fixed line scan speed (10µm/s) and a carbon
nano-tube from R3 (T2 as PI) with 0.72mW power and 110 µm/s scan
speed are shown in Figure 5a and 5b, respectively. Due to the poor
rigidity of protein BSA, only 2D nanopatterns were obtained using
R6-R9 by 2PC. Figure 5c displays the designed model (left) and real
pattern (right) of the 2D logo of our laboratory made using R8.
All 3D structures fabricated using R11-R14 based on HAGM partially
collapsed. A typical result is shown in Figure 6a. It should be due to their
poor mechanical strength. As mentioned above, the reactive groups in
both BSA and HAGM are limited, which may result in low degree of
crosslinking or polymerization. Moreover, the water contents of R5-R9
and R11-R14 are rather high, which will decrease the rigidity of the final
structures. Through adding some PEGDA into R11-R14, a new formula
(10 wt% HAGM, 5 wt% PEGDA, 0.16 wt% PI and 84.84 wt% H2O) was
studied. A well-developed 3D carbon nano-tube was obtained, as shown
in Figure 6b. This indicates that HAGM may have potentials in cells
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adhesion and encapsulation through improving its mechanical property,
the real performance will be further investigated.
3.5 Cytotoxicity Studies
Biocompatibility is a vitally important factor for the application of
water-soluble PIs. Figure 7 displays the cell viability after 24 h
cultivation in substrates containing these PIs with a concentration of 3
mM, which approximates to the concentration of T1-T3 used in the 2PP.
It is shown that Y2 and Y3 have severe cytotoxicity and over 95% of
HepG2 cells are dead, while nearly 60% of HepG2 cells keep alive
incubated with T1-T3 for 24 h. The differences among Y2, Y3 and T2
are mainly the number of sodium carboxylate groups. Obviously,
introducing more sodium carboxylate groups is beneficial to achieve a
better biological safety for these compounds, which should be due to the
improved water solubility and lowered cellular uptake of compounds by
[27-28]
modifying more water-soluble groups as we reported.
With the
increased size of the middle ring from T1 to T3, there is a gradual
enhancement of the cell viability, which could be owing to their different
molecular structures or different cellular uptakes. Overall, T3 enjoys the
best biocompatibility at this concentration.
4．Conclusion
Three water-soluble benzylidene cyclanone dyes T1, T2 and T3 were
designed and synthesized by introducing four sodium carboxylate groups
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into different benzylidene cyclanone moieties (cyclobutanone,
cyclopentanone and cyclohexanone, respectively) through simple and
high-efficient Michael addition reaction and Aldol reaction. Due to their
different middle ring, these compounds exhibited different photo-physical
and photo-chemical properties. Compared with commercial water-soluble
PI Eosin, all of these compounds had larger σ2 values and showed better
potentials to initiate 2PP or 2PC of water-soluble acrylate (SR610),
bovine serum albumin (BSA) and hyaluronic acid derivative (HAGM).
Much lower threshold energies and wider fabrication windows were
obtained in the formulations containing these PIs, especially for T2. The
cytotoxicity of T1-T3 was investigated by MTT assay using HepG2 cells.
Nearly 60% of cells kept alive after 24 h incubation with these PIs when
the concentration of them reached the same level used in 2PP or 2PC,
indicating T1-T3 would have potential application prospects in 2D and
3D nanostructures fabrication based on biomaterials under aqueous
environments.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgement
This work was supported by the National Natural Science Foundation of
China (61575208) and the Austrian-Chinese Cooperative R&D Projects
(FFG and CAS, KYSB20160049).
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Figure captions
Figure 1. Structures of Y2 and Y3
Figure 2. Normalized one-photon absorption spectra (left) and
florescence emission spectra (right) excited at 510 nm of PIs in deionized
water at concentration of about 10-6 M.
Figure 3. Two-photon absorption cross-sections of compounds T1-T3 in
deionized water.
Figure 4. DBC versus time of resins R2 (containing T1 as PI), R3 (T2)
and R4 (T3).
Figure 5. (a) 2D micropattern fabricated in R2 by changing the incident
energy with a fixed line scan speed of 10 µm/s; (b) 3D carbon nano-tube
fabricated in R3 with 0.72mW power and 110 µm/s scan speed; (c) 2D
logo of our laboratory made using R8, the designed model (left) and real
pattern (right).
Figure 6. 3D carbon nano-tube structures fabricated using (a) R14 and (b)
R14 plus PEGDA with 0.85 mW power and 110 µm/s scan.
Figure 7. Cell viability after 24 h cultivation in substrates containing
different PIs with a concentration of 3 mM.
Scheme 1. Structures and synthetic routes for T1, T2 and T3.
Table 1. The formulas and fabrication window of all resins.
Table 2. One- and two-photon photo-physical properties of PIs in
deionized water.
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Table 1. The formulas and fabrication window of all resins.
Resin SR610(wt%) BSA(wt%) HAGM(wt%) H2O(wt%) Eosin(wt%) T1(wt%) T2(wt%) T3(wt%) Eth(mW) Ebr(mW)
R1
R2
79.87
0
0
0
0
19.97
19.97
19.97
19.97
20
0.16
0
0
0
0.51
0.37
0.08
0.43
-
5.24
5.93
6.94
5.65
7.79
5.12
5.78
6.22
5.57
7.43
5.54
7.08
7.37
8.05
10.16
79.87
0
0.16
0
0
R3
79.87
0
0
0
0
0.16
0
R4
79.87
0
0
0
0
0
0.16
R5
80
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
R6
15
15
15
15
15
0
0
84.84
84.84
84.84
84.84
85
0.16
0
0
0
3.31
2.14
1.87
1.97
-
R7
0
0
0.16
0
0
R8
0
0
0
0.16
0
R9
0
0
0
0
0.16
R10
R11
R12
R13
R14
R15
0
0
0
0
0
10
10
10
10
10
89.84
89.84
89.84
89.84
90
0.16
0
0.16
0
0
0
0
0
1.07
0.84
0.59
0.38
-
0
0
0
0
0
0
0.16
0
0
0
0
0.16
0
0
0
0
ꢀ-ꢁmeans no polymerization was found during the experiment.
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Table 2. One- and two-photon photo-physical properties of PIs in
deionized water.
εꢀꢁꢂ
ꢁꢃꢄ
ꢅꢆ
Φf
ΦΔ
Δν
σꢇ
(GM)
λ
λ
ꢀꢁꢂ
(104 M−1
cm−1)
2.87
ꢀꢁꢂ
Comp
(nm)
(nm)
(10-2)
(10-2)
(cm-1)
T1
T2
T3
522
513
478
661
644
636
1.28
1.10
0.52
1.72
1.40
1.18
4028
3965
5197
567
808
231
5.50
4.94
λꢁꢀꢃꢁꢄꢂ is the maximum absorption wavelength in diluted solutions; εꢀꢁꢂ is the molar
ꢁꢃꢄ
ꢅꢆ
absorption coefficient at λ
in diluted solutions; λ
is the maximum
ꢀꢁꢂ
ꢀꢁꢂ
fluorescence emission wavelength at the excitation of 510 nm; ꢀν is stoke’s shift; Φf
is the fluorescence quantum yield in water; Φꢀ is the singlet oxygen quantum yield in
methanol; σꢇ is the maximum 2PA cross-section within the wavelength range of
750 840 nm measured in diluted solutions; 1 GM = 10-50 cm4s/photon.
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