Synthesis of AIE polyethylene glycol-block-polypeptide bioconjugates and cell uptake assessments of their self-assembled nanoparticles

Article abstract of DOI:10.1016/j.dyepig.2019.107640

A synthetic polypeptide-based bioconjugate bearing tetraphenylethylene (TPE) at the polypeptide side chains, named mPEG-PPLGgTPE, was synthesized by ring-opening polymerization (ROP) of α-amino acid-N-carboxyanhydride (NCA) along with post-polymerization

Full text of DOI:10.1016/j.dyepig.2019.107640

Accepted Manuscript
Synthesis of AIE polyethylene glycol-block-polypeptide bioconjugates and cell uptake
assessments of their self-assembled nanoparticles
Mingguang Yu, Songjing Zhong, Yusi Quan, Houhe Liu, Junyang Zhong, Xinlin Jiang,
Yisi Wang, Guodong Ye, Chunping Ma, Yugang Huang
PII:
S0143-7208(19)30916-7
DOI:
https://doi.org/10.1016/j.dyepig.2019.107640
Article Number: 107640
Reference:
DYPI 107640
To appear in: Dyes and Pigments
Received Date: 24 April 2019
Revised Date: 7 June 2019
Accepted Date: 11 June 2019
Please cite this article as: Yu M, Zhong S, Quan Y, Liu H, Zhong J, Jiang X, Wang Y, Ye G,
Ma C, Huang Y, Synthesis of AIE polyethylene glycol-block-polypeptide bioconjugates and cell
uptake assessments of their self-assembled nanoparticles, Dyes and Pigments (2019), doi: https://
doi.org/10.1016/j.dyepig.2019.107640.
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ACCEPTED MANUSCRIPT
Synthesis of AIE polyethylene glycol-block-polypeptide bioconjugates and cell
uptake assessments of their self-assembled nanoparticles
Mingguang Yu,1 Songjing Zhong,2 Yusi Quan,2 Houhe Liu,2 Junyang Zhong,2 Xinlin Jiang,2 Yisi Wang,2
Guodong Ye,2 Chunping Ma,3 Yugang Huang,2,*
1 School of Materials Science and Energy Engineering, Foshan University, Foshan 528000, P. R. China
2 Key Lab of Molecular Target & Clinical Pharmacology of Guangdong Province, School of Pharmaceutical
Sciences & the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou 511436, P. R. China
3 School of Materials and Metallurgical Engineering, Guizhou Institute of Technology, Guiyang 550003, P.
R. China
*Correspondence to: Dr. Yugang Huang (E-mail: hyug@gzhmu.edu.cn)
ABSTRACT
A synthetic polypeptide-based bioconjugate bearing tetraphenylethylene (TPE) at the polypeptide
side chains, named mPEG-PPLGgTPE, was synthesized by ring-opening polymerization (ROP) of α-amino
acid-N-carboxyanhydride (NCA) along with post-polymerization modification method. Interestingly, the
bioconjugate enabled both the aggregation induced emission (AIE) effect at 469 nm and the aggregation
caused quenching (ACQ) effect around 365 nm depending on water fraction in the tetrahydrofuran
/water (THF/H2O) mixed solution, and this feature was very different from the commonly used TPE-
based AIE polymers. The strong emission at the 365 nm was a rarely seen monomeric emission for the
TPE units. Further studies on the fluorescence spectra indicated that the monomeric emission was not
related with the secondary structure of polypeptides, but caused by the TPE unit itself. The mPEG-
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PPLGgTPE could self-assemble to AIE nanoparticles (NPs) in water in a mean size of 253 nm with
polyethylene glycol (PEG) as the shells in surface. In addition, HeLa cell uptake level of the NPs was
unsatisfactory according to the cell imaging results, which was independent of the polypeptides
conformation, the PEG length and the the size of NPs.
Keywords: Aggregation induced emission, synthetic polypeptide, aggregation caused quenching,
monomeric emission, cell uptake
1. Introduction
In recent years, many attentions have been paid to polypeptide-based bioconjugates derived from
ring-opening polymerization (ROP) of α-amino acid-N-carboxyanhydride (NCAs) because of their specific
applications in biomedical fields [1]. The attractive applications of these kinds of polymeric materials
benefit from continuous improvements in synthetic methods of side-chain functionalized polypeptides
[2]. Particularly in anticancer drug delivery, these improvements make these functionalized
bioconjugates sensitive to physiological conditions to tumor microenvironment [3-5], which enables the
nanocarriers to deliver the drugs into targeted sites in a fashion of passive targeting or active targeting.
To get desired biomedical values in use, generally there are two ways to obtain the side-chain modified
synthetic polypeptides. One is the functional monomer route, where side-chain modified (SCM) NCA
monomers are polymerized, and another one is the post-polymerization modification (PPM) route,
where functional molecules are chemically coupled with the reactive polypeptide side-chains [2]. In
contrast with the SCM method, using the PPM route to introduce functional groups onto polypeptide
side chains has some advantages because many functional groups, for example, the commonly used -
NH2, -OH and –COOH groups, will interfere the polymerization of the SCM monomers according to the
ROP mechanism so that they are difficult to incorporate into the SCM NCAs [6]. The PPM route depends
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on polymerizing first inert NCA monomers containing reactive groups to get the reactive side-chains.
Nowadays, complete functionalization of the side-chains in the next step of the PPM route is always
desired, so the most attractive idea for the PPM route is to make the side chains “clickable”. Hence,
various “clickable” groups, including alkyne, alkene and azide groups have been introduced into the
polypeptide side chains in the past 10 years [2]. Then, the functional molecules are chemically
conjugated to the side chains via copper-catalyzed azide-alkyne Huisgen 1, 3-dipolar cycloaddition
(CuAAc) [7-11], thiol-yne [12-13] and thiol-ene [14-18] click reactions during the post-polymerization
modification step.
Over the past decade, aggregation-induced emission (AIE) nanoparticles (NPs) formed by polymer-
based materials are found to be distinctive in biomedical applications because structure, composition,
self-assembly morphology and even biological functions of the polymers are tunable in a more ingenious
way than that of the traditional organic dyes and quantum dots (QDs) [19]. The AIE characteristic means
that dye molecules present strong emissions when they transform themselves from isolated states to
aggregated states, which was first reported by Tang in 2001 [20]. The AIE dyes are more popular than
the traditional organic dyes in bioimaging and drug delivery [21], since the molecular aggregation of the
latter often induces emission quenching effect, a phenomenon resulting from formation of non-emissive
energy transfer due to the aggregation of dye molecules [22]. Tetraphenylethylene (TPE), a model AIE
molecule, has been incorporated into various amphiphilic polymers to obtain self-assembled NPs to
visualize living cells, image and deliver drugs for the tumor therapy. But the AIE polymeric NPs
containing TPE are often prepared from polyacrylates and polystyrene-based amphiphilic polymers,
because the TPE unit can be easily introduced into the polymerizable acrylate and styrene monomers
[23-26]. Nevertheless, the NPs of synthetic polypeptide-based AIE bioconjugates bearing TPE units are
rarely seen in the previous reports. Hong et al. prepared a tetraphenylthiophene based AIE polypeptide
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of poly (γ-benzyl-L-glutamate) [27], but the tetraphenylthiophene located at chain terminal, and this
polymer was not amphiphilic. Scarcity of this synthetic polypeptide-based AIE polymer is normal,
because incorporating of the TPE molecules into inert SCM NCAs along with purification of NCA
monomers is harsh. Therefore, the PPM route depending on polymerizable NCA monomers having
reactive groups will be a better choice.
Scheme 1. Representative way to synthesize AIE polyethylene glycol-block-polypeptide containing TPE
In this work, a bioconjugate of polyethylene glycol-block-poly (γ-propargyl-L-glutamate) (mPEG-PPLG)
was synthesized by the ROP of γ-propargyl-L-glutamate NCA (PLG-NCA) with the methoxypolyethylene
glycol amine (mPEG-NH2) as a macroinitiator. Then, the PPLG side chains were modified with an azide of
(2-(4-(3-azidopropoxy)phenyl)ethene-1,1,2-triyl)tribenzene (TPE-N3) in a post-polymerization way
through the CuAAc reaction, which was illustrated in Scheme 1 with the targeted bioconjugate
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designated by mPEG-PPLGgTPE. The PPLG was selected as the post-polymerization precursor as it was
easy to access polypeptides with subsequent side chain thiol-yne and CuAAc modifications [7-8, 11-12,
28]. Then, the AIE characteristics of the mPEG-PPLGgTPE were studied in detail by fluorescence spectra.
In addition, features for the synthetic polypeptides are their unique α-helical and β-sheeted secondary
structure, which is very different from those commonly used polymers mentioned above. Hence, it will
be very interesting to understand the polypeptide “conformation–cell imaging” relationship and the
“nanoparticle structure–cell imaging” relationship by evaluating the cell uptake level of the obtained AIE
NPs.
2. Experimental section
2.1 Materials
Methoxypolyethylene glycol amine (mPEG-NH2, Mn = 5000 g/mol, 95%) and anhydrous N, N-dimethyl
formamide (DMF, 99.8%) were both purchased from J&K and used as received. Glutamic acid (99%),
CuSO4·5H2O (99.5%), sodium ascorbate (99.5%) and 1, 3-dibromopropane (99%) were all purchased
from Aladdin Reagent (China) and used without further purification. Propargyl alcohol (99%) and NaN3
(99%) was supplied by Xiya Reagent (China). Fetal bovine serum (FBS), DMEM and PBS buffer used for
cell culture were all purchased from Gibco (USA). 3-(4, 5-Dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) was supplied by Sigma-
Aldrich (USA). HeLa cells were kindly supplied by Professor. Yi Zhou at the School of Pharmaceutical
Sciences of Guangzhou Medical University.
2.2 Synthesis of propargyl-glutamate and NCA monomers
The γ-propargyl-L-glutamate (PLG) and γ-propargyl-D,L-glutamate (PDLG) was synthesized according
to an improved method reported in our previous work [12, 28]. Briefly, glutamic acid (10.0 g, 0.068 mol)
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was suspended in propargyl alcohol (24.8 g, 0.44 mol) in an ice-salt bath. Then sulfuric acid (8.2 g, 98%)
was added dropwise over 15 min. After that, the esterification reaction proceeded at room temperature
for 3 days. Triethylamine (12.0 g) was added into the mixture to precipitate the crude product, and then
isopropyl alcohol (200 mL) was added to dilute the mixture. The precipitate was filtrated and washed by
isopropyl alcohol. The wet precipitate was recrystallized in isopropyl alcohol/H2O (3:1, v/v, 10 mL of
isopropyl alcohol used for 1.0 g of wet precipitate). The γ-propargyl-glutamate was finally obtained as
white plates. Yields: 41%. 1H NMR (400 MHz, D2O, δ): 2.10 (dm, 2H, CH2CH2CO), 2.49 (t, 2H,CH2CH2CO),
2.80 (t, 1H, COOCH2C≡CH), 3.65 (t,1H, CHCH2CH2CO), 4.63 (d, 2H, COOCH2C≡CH). HR-MS: calcd. [M+K]
225.190, found. 225.196.
PLG-NCA and PDLG-NCA was synthesized according to an improved method in our previous work [12,
28]. Typically, PLG (3.0 g, 0.016 mol) was suspended in anhydrous ethyl acetate (120 mL) in a two-
necked flask with a reflux condenser and N2 bubbler. After heating to 70
°C, triphosgene (1.64 g, 55
mmol) was added and the mixture was heated for 45 min. The mixture was then heated to 85
°
C and
refluxed for 4 h under N2. The solution was cooled to room temperature, filtrated and washed with ice-
cold water (150 mL), ice-cold 0.5 % of NaHCO3 solution (150 mL) and ice-cold water (150 mL). The
organic phase was then dried by anhydrous MgSO4 and evaporated to obtain PLG-NCA as clear oil. Yields:
40-55 %. 1H NMR (400 MHz, CDCl3, δ): 2.18 (dm, 2H, CHCH2CH2COO), 2.52 (t, 1H, C≡CH), 2.60 (t, 2H,
CHCH2CH2COO), 4.44 (t, 1H, CHCH2CH2COO), 4.69 (d, 2H, COOCH2C≡CH), 6.98 (s, br, 1H, -NH- in ring).
NCAs monomers are well-known sensitive to moisture and alkali, so mass spectra analysis were not
made. The PLG-NCA and PDLG-NCA were directly used for the polymerization in the next step as soon as
possible.
2.3 Synthesis of polyethylene glycol-block-poly(γ-propargyl-L or DL-glutamate) (mPEG-PPLG and mPEG-
PPDLG)
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The ROP of PLG and PDLG-NCA monomers initiated by mPEG-NH2 was typically described as follows:
NCA monomers (0.53 g, 2.5 mmol) were dissolved into anhydrous DMF (1.0 mL) at room temperature,
and then the solution was slowly added into anhydrous DMF (4.0 mL) containing mPEG-NH2 (0.63 g,
0.126 mmol) with a syringe. The resulting solution was stirred for 3 days under dry N2. The resulting
solution was added into ether to precipitate the polymer. Yields: 70-85 %.
2.4 Synthesis of (2-(4-(3-azidopropoxy)phenyl)ethene-1,1,2-triyl)tribenzene (TPE-N3)
The synthetic route to TPE-N3 was described in the Scheme 1, and the reaction was carried out
according to a literature procedure [29-30]. The H NMR spectra were the same as the literature
reported.
First, 2-bromo-1,1,2-triphenylethene (4.0 g, 11.93 mmol), (4-hydroxyphenyl)boronic acid (2.0 g, 14.3
mmol), tetrabutylammonium bromide (TBAB, 0.15 g, 0.46 mmol), 2M of K2CO3 (15 mL) and dioxane (30
mL) were mixed together under N2, and then palladium (0) tetratriphenyl phosphate (50 mg) was added.
Under N2, the mixture was stirred for 24 h at 90 °C. After that, the mixture was poured into water and
extracted by ethyl acetate three times (60 mL × 3). The organic phase was collected and dried by Na2SO4.
The TPE-OH was purified by column chromatography. Yields: 72 %. 1H NMR (400 MHz, CDCl3, δ): 6.63
(2H, d, proton in aromatic ring adjacent to –OH), 6.90-7.10 (17H, m, protons in aromatic rings), 1.29 (s, -
OH); HR-MS: calcd. [M-1] 347.450, found. 347.144. IR (KBr, cm-1): ν = 3450 (-OH).
Next, TPE-OH (1.57 g, 4.51 mmol), K2CO3 powder (1.55 g, 11.2 mol) and 30 mL of acetone were mixed
together. Then 1,3-dibromopropane (3.64 g, 18 mmol) was slowly added, and the resulting mixture was
refluxed for 24 h at 60 °C. After that, the K2CO3 was filtrated and washed by 10 mL of acetone. The
collected solution was removed by rotary evaporation, and the residues were dissolved in ethyl acetate
(120 mL), washed twice by water (50 mL × 2) and then dried by MgSO4. The ethyl acetate was removed
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under rotary evaporation, and finally the crude (2-(4-(3-bromopropoxy)phenyl)ethene-1,1,2-
triyl)tribenzene (TPE-Br) was purified by column chromatography (cyclohexane/ethyl acetate, 50/1).
1
Yields: 51 %. H NMR (400 MHz, CDCl3, δ): 6.60-7.20 (m, 19H, protons in aromatic rings), 3.55 (t, 2H,
BrCH2-), 2.28 (m, 2H, BrCH2CH2-), 4.13 (br, 2H, BrCH2CH2CH2O-). HR-MS: calcd. [M+1] 470.420, found.
470.121.
Finally, TPE-N3 was obtained by substitution reaction of TPE-Br with NaN3. Briefly, mixture of TPE-Br
(1.87 g, 4.0 mmol), NaN3 (0.90 g, 13.9 mmol) and 60 mL of DMF was refluxed for 24 h at 85 °C. After that,
the DMF was removed under rotary evaporation, and then 100 mL of ethyl acetate was added. The
organic phase was washed twice by water (30 mL × 2) and dried by MgSO4. The solvent was removed by
rotary evaporation, and finally the product was purified by column chromatography with
1
cyclohexane/ethyl acetate as eluent (50/1). Yields: 50 %. H NMR of TPE-N3, (400 MHz, CDCl3, δ: 6.40-
7.40 (m, 19H, protons in aromatic rings), 1.27 and 1.43 (br, 2H, N3CH2-), 2.06 (m, 2H, N3CH2CH2-), 3.53 (t,
2H, N3CH2CH2CH2O-). HR-MS: calcd. [M+1] 431.540, found. 431.204. IR (KBr, cm-1): ν = 2099 (-N3).
2.5 Grafting TPE-N3 onto the polypeptides
A typical grafting reaction of mPEG-b-PPLG was given as follows: mPEG-b-PPLG (0.20 g, 0.28 mmol of
alkyne), TPE-N3 (0.145 g, 0.34 mmol of azide) and CuSO4·5H2O (91 mg, 0.36 mmol) were mixed in DMSO
(6.0 mL). The mixture was bubbled with N2 for 20 min and then sodium ascorbate (78 mg, 0.39 mmol)
was added. After bubbling N2 for another 10 min, the reaction solution was sealed and stirred for 48 h at
50 °C. The resulting mixture was purified by dialysis (3500 cutoff) against water to remove the excess
sodium ascorbate. During the dialysis, EDTA was added to remove the copper salt. After lyophilization,
the obtained solid was dissolved in THF, passed through aluminium oxide column to remove copper salt
residues, and finally precipitated in cyclohexane to remove the unreacted TPE-N3. Yields: 32 %.
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2.6 Measurements
NMR spectra were recorded on a Bruke AVANCE III HD 400 MHz spectrometer and CF3COOD/CDCl3
(1/4, v/v) was used as solvent for the polymers containing polypeptide blocks; Size exclusion
chromatography (SEC) was performed on a Malvern Viscotek TDA305max with DMSO containing 20 mM
of LiBr as eluent. The measurement was carried out at 50 °C with a flow rate of 0.5 mL/min. The size
exclusion was made with an Iguard column and a connected I-Oligo column, Pullulan as standard sample.
The signals were collected with a refractive index detector; FTIR spectra were read on a Thermo Nicolet
6700 spectrophotometer and scanned 32 times with a resolution of 2 cm-1 by KBr disk method; UV-
visible absorption spectra were studied by Agilent 8453 spectrophotometer with THF/H2O as solvent.
The AIE effect was recorded by Fluorescence spectra (FL) on a Shimadzu RF-5301PC spectrometer with a
slit width of 3.0 nm and an excitation wavelength (λex) of 330 nm or 365 nm. The polymer was first
dissolved in the THF with a given concentration. Then, water was slowly added under a ultrasonic
clearer to make sure that the water content was increasing from 0 to 95 % (v %) with a polymer
concentration of 0.05 mg/mL; The AIE properties of the blocked polypeptides grafted by TPE was
studied by recording the FL of their micellar water solutions. The micellar solution was prepared by first
dissolving the AIE polymers in dimethyl sulfoxide (DMSO) with a concentration of 0.20 mg/mL and then
dialyzing the polymers against distilled water for 24 h. The water was exchanged every 4 h and finally
the nanoparticles (NPs) solution was diluted to 0.10 mg/mL before the test; Dynamic light scattering
(DLS) was used to determine size and zeta potential of the NPs on a Malvern Nano-ZS/ZEN-3600
Zetasizer. Three sets of measurements were performed for each sample. The micellar sample was
prepared as the same as the AIE determinations mentioned above; Transmission electron microscopy
(TEM) observation was performed on a JEM-2010 system. A drop of the micellar solution mentioned
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above was deposited on carbon coated copper grids for 1.5 min, and then the water was removed with
a piece of filter paper.
2.7 Cytotoxicity assay
The cytotoxicity of the AIE-NPs for HeLa cells was assayed by using MTS cell viability test. Cells were
seeded in 96-wells plates at 6000 cells/well and grown for 24 h at 37 °C, 5% CO2 in DMEM medium
containing 10% FBS, 1% penicillin and streptomycin. After removing the medium, the AIE NP solution
was added into the wells, and then DMEM was added to dilute the solution into desired concentration.
After that, the cells were incubated at 37 °C for 24 h again. Then, 20 μl of MTS was added into the cells
in each well and the plates were incubated for additional 2 h. The OD values were then recorded at the
wavelength of 490 nm using a microplate reader (Biotek). For each sample, 4 times were repeated at
each concentration.
2.8 Cell uptake
The cell imaging ability of the AIE NPs for the HeLa cells was evaluated with a Nikon ECLIPSE 80i
confocal laser scanning microscopy (CLSM) with an excited wavelength of 405 nm and a Leica DMi8
fluorescence microscope. Cells were seeded in culture dishes and grown for 24 h. After that, the given
solutions of AIE NPs in DMEM culture were added into each dish. After incubation for 4 h, the cells with
internalized AIE NPs were washed by PBS three times before the microscope observation. Flow
cytometry (Beckman) determination was also performed to investigate the cell uptake of the AIE NPs,
and the cells were cultured similar to the CLSM observations.
3. Results and discussion
3.1 Synthesis and characterization
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Figure 1.1 H NMR spectra of mPEG-PPLG in CDCl3/CF3COOD (4/1, v/v) and mPEG-PPLGgTPE in CDCl3.
The synthesis of AIE bioconjugate of mPEG-b-PPLGgTPE was given in the Scheme 1. First, mPEG-block-
poly(propargyl-L-glutamate) naming mPEG-PPLG was synthesized by the ROP of the PLG-NCA monomer
with mPEG-NH2 as an initiator. Then, TPE was grafted onto the polypeptide blocks by the CuAAc
coupling reaction, and the obtained polymer was denoted by mPEG-PPLGgTPE. Structures of all the
1
resulting polymers were characterized by the H NMR in Figure 1, where peak assignments were also
made. Because length of the mPEG (Mn = 5000 g/mol) blocks had been known, the length of PPLG with a
degree of polymerization (DP) of 9.0 was calculated according to area ratio of peak 2 and peak 6 in the
Figure 1. Thus, the obtained copolymer was denoted by mPEG-PPLG9. The structure of the mPEG-b-
1
PPLGgTPE in the final step was also confirmed by the H NMR spectra. The peak assignments showed
that the side-chain –CH2– protons of peak 2 was shifted from 4.70 to 5.10 ppm (peak 7) after the CuAAc
coupling reaction, and no residual signal of the peak 2 at 4.70 ppm was observed after the grafting
reaction. These results indicated that the TPE was successfully grafted onto the polypeptide side chains
with a nearly complete grafting efficiency. A distinct peak at 7.75 ppm (peak 8) assigned to resulting
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triazole ring also proved to be the high grafting efficiency of the CuAAc reaction, so the obtained
polymer was denoted by mPEG-PPLG9gTPE.
Figure 2. (A) SEC traces of the mPEG-PPLG9 and the mPEG-PPLG9gTPE in DMSO; (B) FTIR spectra of the mPEG-PPLG9 and the
mPEG-PPLG9gTPE.
The polymers as prepared were further characterized by SEC in DMSO, which was given in Figure 2A.
It can be seen that molecular weight (Mw) of the mPEG-PPLG9gTPE was bigger than that of the mPEG-
PPLG9, indicating that the TPE molecules were grafted onto the polypeptides successfully. In addition,
determined polydispersity index (PDI) was 1.24 for the mPEG-PPLG9 and 1.51 for the mPEG-PPLG9gTPE.
FTIR spectra of the mPEG-PPLG9 and the mPEG-PPLG9gTPE in solid were shown in Figure 2B, where the
mPEG-PPLG9 showed typical absorption of the alkynes at the 2123 and 3297 cm-1. After the CuAAc
reaction, the obtained mPEG-PPLG9gTPE the two absorption bands were absent in the Figure 2B, also
indicating that the grafting efficiency was high.
3.2 Absorption and fluorescence properties
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Figure 3. (A) UV absorption spectra of the mPEG-PPLG9gTPE (0.05 mg/mL) in THF/H2O; (B) FL spectra of the mPEG-PPLG9gTPE
(0.05 mg/mL) with increasing water fraction in the THF/H2O, λex = 330 nm; (C) Intensity of maximum emission wavelength (λem
)
with increasing water fraction, λex = 330 nm; (D) Digital photos of the mPEG-PPLG9gTPE in THF/H2O under a hand-hold UV-lamp
(λmax = 302 nm).
The UV-vis absorption spectra (Figure 3A) of the mPEG-PPLG9gTPE in THF/H2O mixture were
performed to reveal the aggregate emergence when the water fraction (fw, V %) was increaing from
0 to 95 %. As seen in the Figure 3A, the mPEG-PPLG9gTPE showed a maximum of absorption peak
(λmax) around 280 nm and a shoulder peak at 310 nm in the pure THF. As the fw exceeded 40 %, the
absorption spectra was broader in a red shift, indicating that a Mie effect of light scattering was
caused by the nanosized aggregation formation (as shown in following self-assembly analysis) at
higher fw [31]. Under higher water content, inner cores of the formed NPs would be more and more
compact, leading to the absorption of the mPEG-PPLG9gTPE in 70-80 % of H2O/THF higher than that of
90-95 %. FL spectra (Figure 3B) were further used to investigate the AIE feature resullting from the
aggregation of the mPEG-PPLG9gTPE. As indicated by the Figure 3B, a fluorescence in the range of 350
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to 400 nm centered with λem of 365 nm was observed when the fw was below 70 %. However, this
emission vanished and a new emission peak was detected at the λem of 469 nm when the fw was above
70 %, showing a typical AIE property as a result of the aggregation of the mPEG-PPLG9gTPE. Further,
the emission intensity at the λem of 365 and 468 nm was plot versus increasing fw in Figure 3C, where
the results clearly indicated that both an aggregation-caused quenching (ACQ) effect and an AIE effect
could be seen for this polymer. Actually, a covered blue emission at 416 nm could be revealed when the
λex was changed to be 365 nm (Figure S1). According to the above FL spectra, the mPEG-PPLG9gTPE
could emit different color lights from purple to cyan depending on the fw. Thus, visual inspection of
the emission colors was presented in the Figure 3D, where a color transfer of purple-blue-cyan was
recorded with increasing fw when the sample was exposed under a UV-lamp. This visual result was
consistent with the PL spectra result.
The AIE effect was believed to connect with restriction of intramolecular rotation (RIR ) of TPE
units, which inhibited the nonradiative decay and thus led to the aggregation-enhanced emissions
[32]. In this work, interestingly, the monomeric emission showing ACQ effect at 365 nm was hard to
see in previous literatures. The increasing fw would enhance the local concentration of TPE units,
which means that the concentrated TPE speices enabled the fluorescence quenching as a result of
the formation of π-π interaction between phenyl rings. The concentration dependent fluorescence
quenching of the monomeric emission for the mPEG-PPLG9gTPE was similar to the typical
luminophore of pyrene, which was summarized by Birks [33] in his classical photophysics book. The
monomeric emission peak of pyrene at 390 nm was decreasing with an enhancing concentration in
the concentration region above 10-5 M, while the peak disappeared until a small and broad emisison
without fine bands appeared at the 477 nm in the redder spectral region when the concentration
was up to 10-1 M, a indicator of the emission change from monomeric spieces to excimers [33].
However, the FL spectra of the mPEG-PPLG9gTPE here showed fine emisisons around 365 nm
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without any shift upon local concentration increase of TPE units, indicating that this fluorescence
came from radiative decay of TPE monomeric spieces, not from the emissions due to the formation
of excimers and exciplex [22,33]. As indicated by the Figure 3B, the mPEG-PPLG9gTPE showed the
broad bump at 469 nm when the fw was increased to 90-95%, even though this AIE emisison was not
caused by the formation of excimers and exciplex. The reason is that the ACQ effect of TPE
monomeric spieces are hardly seen in so many previous literatures until now, and the RIR mechnism
proposed by Tang et al. [32] can well explain the AIE features of the TPE-based materials at the 469
nm. Hence, further studies on the fluorescence properties of the TPE-N3 will be necessary in the
future.
Figure 4. (A) FL spectra of the TPE-N3 (0.12 mM) in THF/H2O, λex = 330 nm; (B) Intensity of maximum emission wavelength (λem
)
with increasing water fraction, λex = 330 nm.
Particularly, some recent reports indicated that the conformation of the synthetic polypeptides
would determine physical and biological properties of these kinds of biomaterials [34]. Thus, there was
an interesting speculation whether the secondary structure of the polypeptides determined the
unique luminescence property of the mPEG-PPLG9gTPE or not. To testify this, an AIE bioconjugate
with racemic polypeptide blocks, denoted by mPEG-PPDLGgTPE, was prepared by the ROP of
racemic NCA monomer (PDLG-NCA) ) at the same time. The FL spectra (Figure S2) of this polymer
showed the same tendency like the mPEG-PPLG9gTPE when the fw was rising, suggesting that the
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fluorescence of the TPE units was not related with the secondary structure of polypeptides. To gain
more insights into the caused monomeric emission of the mPEG-PPLGgTPE, the FL spectra of the
TPE-N3 in dilute solution (0.05 mg/mL, 0.12 mM) was scanned with the increasing fw, which was
displayed in Figure 4A. Clearly, the FL spectra of TPE-N3 showed one fine monomeric emission at
365 and 385 nm along with a broad bump in the red shift region when the fw was below 70%. After
that, the monomeric emission became stronger and headed towards shorter wavelength reigon
when the fw was high. Additionally, a new bump from 400 to 450 nm appeared as the fw was
increasing. After the fw exceeded 70%, another broad emission with stronger intensity could be
detected at the 469 nm, showing the typical AIE behavior of the TPE again. Figure 4B showed the
fluorescence intensity variation as the fw was increasing. It was found that both the emissions at the
365 nm and 469 nm altered little when the fw was less than 70%, but the intensity was sharply
enhanced after that. Interestingly, the monomeric emission was reduced while the fw was up to 95%,
which means that the fluorescence properties of the TPE-N3 are very similar to that of the pyrene:
the increasing local concentration of TPE-N3 can enhance the monomeric emission in the low
concentration range, whereas the ACQ effect will happen in concentrated solution if the fw is close
to 100%. To our best knowledges, the strong monomeric emission of the TPE was reported in only
three literatures. He et al. [35] reported an amphiphilic TPE derivative containing secondary amines
to detect wide-range pH alteration in the THF/H2O mixture. This TPE derivative could show strong
monomeric emission at 377 nm at the low pH range, because the resulting electrostatic repulsion
between the protonated amines let one molecule not be close to another one to form the π-π
interaction between phenyl rings. Zheng et al. [36] reported synthesis of a 4-armed star
oligo(ethylene glycol) (OEG) with the TPE unit as the core. Once this TPE was included in the cavity
of γ-cyclodextrin (γ-CD), the formed complex could show an enhanced monomeric emission at 388
nm. The big cavity of the γ-CD confined the rotation of the phenyl rings of the TPE, which stopped
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the formation of π-π interaction and then favored the emissive decay of the excited TPE monomer.
Note that this OEGylated TPE was also an amphiphilic TPE derivative. Recently, Lin et al. [37]
reported a novel photo-switchable polymer bearing TPE moieties within the backbones by the
CuAAc reaction. Upon UV-irradiation, the strong monomeric emission of the TPE was found at 385
nm in the THF/H2O mixture becase of the photo-cyclization happed in the polymer backbone.
Chemical structure of all the amphiphilic TPE derivatives mentioned above was complex, however,
the hydrophobic TPE-N3 we reported here was the frst TPE-based AIE molecule showing strong
monomeric emission in a simpler structure. To further study the effect of functional group on the
fluorescence properties of the TPE units, the FL spectra (Figure S3) of the TPE-Br in THF/H2O were
aslo measured. As seen in Figure S3, although the TPE-Br could show the monomeric emission of
TPE units in the THF/H2O when the fw was less than 70%, its intensity was very weak. The AIE
emission of the TPE-Br was very strong when the fw was high. This result indicated that the TPE-Br is
a typical AIE molecule, very different from the TPE-N3. More importantly, this result verfied that the
unique monomeric emission of the TPE-N3 was determined by its azide group. Overally, both the
mPEG-PPLG9gTPE and the TPE-N3 could display the AIE and the ACQ phenomenon depending on
water fraction, and the polypeptide conformation had no effect on the fluorescence properties of
the obtained AIE polymers. Thus, it can be concluded that the unique fluorescence behaviors of the
mPEG-PPLG9gTPE result only from the TPE units. However, note that the mPEG-PPLG9gTPE showed
better AIE ability than that of the TPE-N3, because the effective concentration of TPE molecules in this
polymer was much less than that in the TPE-N3. It means that the intramolecular rotation of the TPE
phenyl rings was strongly restricted by the polypeptide chains within core of the self-assembled NPs.
3.3 Self-assembly behaviors
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Figure 5. (A) DLS results of the mPEG-PPLG9gTPE (0.05 mg/mL) with increasing water fraction in THF/H2O; (B) DLS results of the
mPEG-PPLG9gTPE micelles (0.20 mg/mL) in water; (C) TEM image of the mPEG-PPLG9gTPE micelles, and the inset was local
amplification of the micelles with a scale bar of 500 nm.
Upon adding water into the THF solution of the mPEG-PPLG9gTPE, aggregation will be formed with
increasing water content. Thus, the formation of aggregations and their size changes in the THF/H2O
mixture were measured by DLS, which was displayed in Figure 5A. Interestingly, it was found that the
mPEG-PPLG9gTPE can form particles in pure THF with a mean size of 27.3 nm. These particles are
probably unimers of the mPEG-PPLG9gTPE because of the large bulk of the grafted TPE units at the side
chains and the rigid conformation of the polypeptide blocks. However, the DLS did not give any
repeatable and reliable results of the formed particles with the fw of 10 and 20 %. Above 20%, nano-
sized aggregations were formed with decreasing size when the water content was increasing, indicating
that the inner cores of the NPs were more and more compact because of closer aggregation of the
hydrophobic blocks in the mPEG-PPLG9gTPE.
The mPEG-PPLG9gTPE could self-assemble into NPs in water because of its amphiphilic nature. DLS
was still used to determine size of the formed NPs after dialysis, which was seen in Figure 5B. The
obtained NPs were in size region of 100 to 600 nm, with an average value of 253 nm and a PDI of 0.121.
Additionally, the determined zeta potential was -0.16 mV, close to zero as it was expected, because the
coated PEG blocks onto the NP surface were electroneutral. Morphology of the self-assembled NPs was
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detected by TEM, and the results in the Figure 5C indicated that the particles were spherical micelles
with a slightly broad size distribution, which was identical with the DLS results. The mPEG-PPLG9gTPE
NPs could emit bright cyan lights visually in water under a hand-hold UV lamp, and the AIE persistence
time was long because more than 60 % of initial emission intensity was remaining after 10 days (Figure
S4).
3.4 Cell uptake assessments
Figure 6. (A) Cytotoxicity of the AIE NPs assessed by the MTS assays. (B) Cell uptake of the AIE NPs revealed by flow cytometry.
Dot line: HeLa cells uptake of the AIE NPs. Solid line: without any NPs as the control; (C) The CLSM images and (D) fluorescence
microscope images of the HeLa cells with the internalized mPEG-PPLG9gTPE NPs.
Cell imaging is a fundamental aspect of the AIE-polymers for use in the biomedical field. Even though
various AIE-polymer NPs have been widely used for cell imaging because of their good luminous ability,
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many attentions were paid on the acrylate-based and PS-based polymers [19]. However, the synthetic
polypeptides are very different from those common polymers because the polypeptide chains are rigid
and can adopt α-helical and β-sheeted conformation [34]. Actually, rare synthetic polypeptide-based AIE
polymers having TPE units were reported in former researches. As we all know, the strong
intramolecular and intermolecular hydrogen bond interactions favor formation of the α-helical and the
β-sheeted conformations, and thus studying the cell imaging ability of the mPEG-PPLG9gTPE NPs will be
interesting and helpful to understand the structure-function relationship of the synthetic polypeptide-
based materials. Herein, the cell uptake ability of the mPEG-PPLG9gTPE NPs was evaluated by the flow
cytometry analysis, the fluorescence microscope as well as the CLSM observations. In order to exclude
the cytotoxicity effect of the mPEG-PPLG9gTPE, the MTS viability assay was carried out at the same time.
Figure 6A showed the HeLa cell viability under different polymer concentration. It can be seen that the
mPEG-PPLG9gTPE was non-cytotoxic with the concentration as high as 100 μg/mL. In Figure 6B, the flow
cytometry analysis showed that there was nearly no difference between the cells cultured with and
without mPEG-PPLG9gTPE NPs, implying that the cell uptake level of the AIE NPs was quite low.
Fluorescence microscope was further used to observe the cells internalized with AIE NPs, which was
given in Figure 6C. Unfortunately, no clear images of the HeLa cells were gained by the microscope
observation, which means that the cell uptake level of the AIE NPs was not high. In Figure 6D, CLSM
observation was also made to confirm this, because the CLSM could supply clearer images. As expected,
the CLSM made the same result as the fluorescence microscope. Thus, concordant results made by both
the flow cytometry analysis and the microscope observations confirmed that the mPEG-PPLG9gTPE NPs
were hard to internalize into the cells. Even though reasons leading to that poor cell uptake level are
complicated, the influence of structure parameters, including the conformation of the polypeptide and
the chain length of the PEG, were studied by the CLSM observations. In this work, the mPEG5k-
PPDLGgTPE NPs with racemic polypeptide blocks mentioned above and the mPEG2k-PPLGgTPE NPs
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with shorter PEG chains were both prepared, and their CLSM images of the formed NPs were
presented in Figure S5. Similarly, clear images could not be obtained again, which indicated that the
conformation of the polypeptides and the PEG chain length could not affect the cell uptake level of the
AIE NPs. Particularly, the assembled NPs of mPEG5k-PPDLGgTPE was smaller in size than that of the
mPEG-PPLG9gTPE NPs (198.9 ± 5.4 nm, DLS results), still showed the poor cell uptake level, indicating
that the size of NPs could not determine the cell internalization process, either.
4. Conclusions
In summary, a synthetic polypeptide-based AIE bioconjugate consisting of TPE at the polypeptide side
chains were synthesized by the post-polymerization modification method. The self-assembled NPs of
the amphiphilic bioconjugates could show strong AIE performance. Interestingly, the TPE units in the
synthetic polypeptide-based AIE copolymer could show a strong monomeric emission in the THF/water
mixture, and thus a color transformation of purple-blue-cyan was seen with the naked eyes. The
monomeric emission was independent of the polypeptide conformation. The self-assembled NPs
showed poor cell uptake level, which was not determined by the polypeptide conformation, the PEG
chain length and the size of the NPs. This work will be helpful to understand the fluorescence
characteristic of the TPE units in the synthetic polypeptide-based AIE polymers.
Acknowledgement
This work is financially supported by the National Natural Science Foundation of China (51403043),
the municipal college research program of Guangzhou education bureau (1201610202) and the High-
Level Talent Start-Up Research Project of Foshan University (gg040945).
Notes
The authors declare no competing financial interest.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at: xxxx
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Highlights
•
•
•
•
A
synthetic
polypeptide-based
amphiphilic
bioconjugate
bearing
tetraphenylethylene (TPE) is synthesized
The obtained amphiphilic bioconjugate shows both the aggregation-induced
emission and the monomer emission of the TPE in the THF/H2O mixture.
Conformation of the polypeptide blocks within the bioconjugate is not
responsible for the monomer emission of the TPE
Self-assembled nanoparticles (NPs) of the amphiphilic bioconjugate showing the
AIE feature are hard to internalize into HeLa cells