Fabrication of AIE-active amphiphilic fluorescent polymeric nanoparticles through host-guest interaction

Article abstract of DOI:10.1039/c6ra08677b

Fluorescent polymeric nanoparticles (FPNs) have obtained more and more attention in recent years due to their excellent performance in the fields of bioimaging, biosensing, theranostics and many other biomedical applications. In this work, we reported a novel method to fabricate amphiphilic fluorescent copolymers through host-guest interactions based on an aggregation-induced emission (AIE) active dye (named as Ad-PhNH₂) and β cyclodextrin (β-CD) contained polymers, which were synthesized by free radical polymerization and subsequent ring-opening reaction. These AIE active copolymers can self assemble into FPNs (named as PEGMA-IA-β-CD/Ad-PhNH₂) due to their amphiphilic properties. The hydrophobic dye was aggregated in the core and therefore can emit strong fluorescent intensity due to its AIE feature. However, the hydrophilic polymers that covered the hydrophobic core can endow good dispersibility in pure aqueous solution. Biological evaluation results demonstrated that PEGMA-IA-β-CD/Ad-PhNH₂ FPNs can be effectively internalized into cells and they have shown low cytotoxicity. More importantly, the molar ratio of β-CD to Ad-PhNH₂ can be facilely adjusted and the surplus β-CD can be used for carrying chemical anticancer agents. Furthermore, a large number of carboxyl groups were generated during the ring opening reaction. These negative carboxyl groups can be potentially used for further conjugation reactions and for biological delivery. The above described features of PEGMA-IA-β-CD/Ad-PhNH₂ FPNs make them a prospect in biological imaging and delivery applications.

Full text of DOI:10.1039/c6ra08677b

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Fabrication of AIE-active Amphiphilic Fluorescent Polymeric
Nanopartilces through Host-guest Interaction
Junyu Chena,#, Songsong Luoa,#, Dazhuang Xua, Yun Xuea, Hongye Huanga, Qing Wana, Meiying
Liua,*, Xiaoyong Zhanga,*, Yen Weib,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Fluorescent polymeric nanoparticles (FPNs) have obtained more and more attention in recent years for their excellent
performance in the fields of bioimaging, biosensing, theranostics and many other biomedical applications. In this work, we
reported a novel method to fabricate amphiphilic fluorescent copolymers through host-guest interaction based on an
aggregation-induced emission (AIE) active dye (named as Ad-PhNH2) and β cyclodextrin (β-CD) contained polymers, which
were synthesized by the free radical polymerization and subsequent ring-opening reaction. These AIE active copolymers
can self assemble into FPNs (named as PEGMA-IA-β-CD/Ad-PhNH2) due to their amphiphilic properties. The hydrophobic
dye was aggregated in the core and therefore can emit strong fluorescent intensity due to its AIE feature. However, the
hydrophilic polymers that covered on the hydrophobic core can endow the good dispersibility in pure aqueous solution.
Biological evaluation results demonstrated that PEGMA-IA-β-CD/Ad-PhNH2 FPNs can be effectively internalized into cells
and shown low cytotoxicity. More importantly, the molar ratio of β-CD to Ad-PhNH2 can be facilely adjusted and the
surplus β-CD can be used for carrying chemical anticancer agents. Furthermore, a large number of carboxyl groups were
generated during the ring opening reaction. These negative carboxyl groups can be potentially used for further
conjugation reaction and for biological delivery. The above described features of PEGMA-IA-β-CD/Ad-PhNH2 FPNs make
them prospect in biological imaging and delivery applications.
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important role in biological imaging utilization.25, 26 Varieties of
fluorescent nanoparticles with different components,
1. Introduction
Biological imaging in modern medicine is an important tool to
provide visual information on organs and biological processes.1ꢀ
6 A number of modern imaging techniques such as Xꢀray phase
tomography, singleꢀphoton emission computed tomography,
positron emission tomography, Raman imaging, magnetic
resonance imaging, ultrasound and fluorescence imaging, etc.
have been developed.7ꢀ10 All of them have been applied and
acted significant roles in variety of biological application like
stem cell transplantation, cancer detection, drug delivery,
therapeutic effect monitoring and so on.11ꢀ16 Among them, the
fluorescence imaging has attracted the most intensive attention
because the fluorescence used in cells could be an output signal
that provides obvious signal intensity and high resolution
images at subcellular levels.17ꢀ24 Moreover, the fluorescent
probes had many properties such as low cost, well designability
of dyes and final probes, desirable biocompatibility and so on.
These characteristics make fluorescent imaging play an
fluorescent properties and surface coating have already been
synthesized.27ꢀ34 As compared with small organic dyes,
fluorescent nanoparticles showed many advantages for
biological applications including good biocompatibility, better
chemical stability, enhanced photostability and multifunctional
potential.35ꢀ37 Among the fluorescent nanoparticles, fluorescent
polymeric nanoparticles (FPNs) have already attracted
increasing attention for their superiority in simple fabrication,
peculiar luminescence properties and surface functionalization
compared with fluorescence inorganic nanoparticles and
organic dyeꢀdoped inorganic nanoparticles. Inorganic
nanoparticles mainly contained the semiconductor quantum
dots with great luminescent properties and rare earthꢀdoped
upconversion nanoparticles which can emit visible emission
under excitation with a nearꢀinfrared laser.38 Although many
successes have been made in the research of fluorescent
inorganic nanoparticles, some shortcomings cannot be ignored.
For example, most of fluorescent inorganic nanoparticles are
difficult to be biodegradable, and exhibited obvious toxicity to
living organisms.39 The basic principle for fabrication of FPNs
is relied on the self assembly of amphiphilic dyes contained
polymers in aqueous solution.40ꢀ45 The hydrophobic
components such as dyes were encapsulated in the core, which
were covered by the hydrophilic segments. Therefore, these self
a.Department of Chemistry and Jiangxi Provincial Key Laboratory of New Energy
Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031, China.
b.Department of Chemistry and the Tsinghua Center for Frontier Polymer Research,
Tsinghua University, Beijing, 100084, P. R. China.
c. # These authors contributed equally to this work
† Footnotes relaꢀng to the ꢀtle and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [The Synthetic routes of
amino-terminated β-CD and Ad-PhNH2 and 1H NMR spectrum of Ad-PhNH2 were
provided in supplementary information]. See DOI: 10.1039/x0xx00000x
assembly
nanostructures
displayed
excellent
water
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47
dispersibility in pure aqueous solution.46,
However, many
FPNs based on the self assembly procedure still possess some
problems. One of the most issues is the significant decrease of
fluorescent intensity of FPNs based on conventional organic
dyes. This phenomenon was known as aggregation caused
quenching (ACQ) effect. The phenomenon is even more serious
for dyes with emission in the farꢀred/nearꢀinfrared(FR/NIR)
tissue transparency window.48 A novel type of organic dyes that
can emit stronger luminescence in the aggregation state was
found by Tang et al. in 2001, which called aggregationꢀinduced
emission (AIE) dyes.49 It provided
a useful route for
overcoming the ACQ effect and good chooses to fabricate
FPNs with desirable luminescent performance. From then on, a
huge number of dyes with AIE properties were synthesized and
utilized for fabrication of luminescent polymeric nanoprobes.
Supermolecular chemistry has got much attention in the field
of molecular recognition, supermolecular hydrogels and
varieties of other functional materials by self assembly.50ꢀ53 The
process that gained the supermolecular polymers with typical
dynamic polymeric structure was orthogonal assembly of
monomers through supermolecular interactions at chain ends
such as metalꢀligand bonding, πꢀstacking, Hꢀbonding and hostꢀ
guest interaction. So far, the molecular recognition mechanism
of cyclodextrin (CD) and its derivatives as an important part of
supermolecular chemistry have gained widely concern.37, 54ꢀ59
Because CD owned many advantages such as good stability,
excellent biodegradability, low toxicity, which was the ideal
host molecules similar to enzymes and had the properties of
enzymes’ models. Among them, βꢀCD was commonly used for
its better rigidity. The ester of organic molecules (host) with
suitable size and shape would enter its cavity, formed as hostꢀ
guest inclusion complexes by hostꢀguest interaction. Although
different strategies have been set up for fabrication of
multifunctional AIE active polymeric nanoparticles, the
fabrication of AIE active FPNs through host guest interaction
has received rarely attention. The fabrication of AIEꢀactive
FPNs based on the βꢀCD contained copolymers and
Adamantane (Ad) terminated AIE dye has rarely been reported
thus far.
In this contribution, we report for the first time that
amphiphilic AIEꢀactive supermolecules could be facilely
formed taking advantage of host guest interaction between βꢀ
CD pendant copolymers (PEGMAꢀIAꢀβꢀCD) and Ad contained
AIE dye (AdꢀPhNH2). As shown in Scheme 1, βꢀCD pendant
copolymers can be synthesized by simple ringꢀopening reaction
between amino groups of βꢀCD and anhydride group of
poly(PEGMAꢀIA), which can be prepared by the free radical
polymerization using itaconic anhydride (IA) and poly(ethylene
glycol) methyl ether methacrylate (PEGMA) as the monomers.
Then AdꢀPhNH2 is mixed with PEGMAꢀIAꢀβꢀCD to form the
final products (PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs). The
Scheme
1
Schematic showing the preparation of PEGMA-IA-β-CD/Ad-PhNH2
copolymers via host guest interaction.
2. Experiment
2.1 measurements and materials
All of the chemical agents and solvents were obtained from
commercial sources and used as received. All aqueous solutions
were prepared with distilled water. βꢀCD (M
Tosyl chloride (TsCl M : 190.65 Da, 99% ), ethylenediamine
:60.10 Da, 99% ), itaconic anhydride (IA, M
Da, 96%), poly(ethylene glycol) methyl ether
950 Da, 98%), 2,2ꢀ
164.21 Da, 98%),
: 199.28 Da, 98%,), 4ꢀAminobenzyl cyanide
: 132.16 Da, 98%) and et al were purchased from Aladdin
W: 1135 Da, 99%),
W
(EDA, M
112.19
W
W:
methacrylate (PEGMA,
azodiisobutyronitrile (AIBN,
phenothiazine (M
(M
MW:
MW:
W
W
(Shanghai, China). Anhydrous ethyl acetate and DMF were
provided from Heowns (Tianjin, China). H Nuclear magnetic
1
resonance (NMR) spectra were recorded on a Bruker Avanceꢀ
400 spectrometer with D2O, TMS and DMSO as the solvents.
The synthetic materials were characterized by Fourier
transform infrared spectroscopy (FTꢀIR) using KBr pellets. FTꢀ
IR spectra were obtained using a Nicolet 5700 (Thermo Nicolet
Corporation). Fluorescence spectra were measured on a PE LSꢀ
55 spectrometer with a slit width of 3 nm for both excitation
and emission. UV–Vis absorption spectra were recorded on
UV/Vis/NIR PerkinElmer Lambda750 spectrometer (Waltham,
MA, USA) using quartz cuvettes of 1 cm path length.
Transmission electron microscopy (TEM) images were
recorded on a JEMꢀ1200 EX microscope operated at 100 kV.
The TEM specimens were made by placing a drop of the
nanoparticles suspension on a carbon coated copper grid. The
hydrodynamic size distribution of the final products PEGMAꢀ
IAꢀβꢀCD/AdꢀPhNH2 FPNs in water was examined using
dynamic laser scattering (DLS).
2.2 Preparation of monoꢀ6ꢀdeoxyꢀ6ꢀ(pꢀtolysulfonyl)ꢀβꢀCD
PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 are characterized by a number of
techniques and examined for biological imaging applications.
30 g βꢀCD (26.4 mM) and 20 g NaOH were dissolved in 666
mL water. The solution was immersed into an iceꢀwater bath,
and then 10 g TsCl was added slowly, causing the formation of
white precipitates. After further stirring for 5 h at 0°C, the
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unreacted TsCl was removed by suction filtration, then using pure product 2. The intermediate product 2 (1.22 g, 3.2 mM)
the 10% hydrochloric acid solution adjusting the filtrate’s pH at and 2ꢀ(4ꢀamino phenyl) acetonitrile (1.68 g, 6.4 mM) were
about 7. The suspension was refrigerated overnight at 0 °C in added into 40 mL ethanol solution and the mixture was stirring
the refrigerator. The resulting precipitate was recovered by at room temperature. The terabutyl ammonium hydroxide
suction filtration and recrystallized from hot water 3 times solution (0.8 M, 10 drops) was added and mixture was refluxed
getting the monoꢀ6ꢀdeoxyꢀ6ꢀ(pꢀtolysulfonyl)ꢀβꢀCD (monoꢀ6ꢀ for 2 h until a red solid was precipitated. The obtained red
OTsꢀβꢀCD). The product was dried under vacuum at 60 °C for solids were washed with ethanol for several times until dark red
12 h.
solids were obtained. The PhꢀNH2 was obtained after dried for
24 h under vacuum oven. The adamantine terminated PhNH2
(AdꢀPhNH2) was facilely obtained via amidation reaction
2.3 Preparation of monoꢀ6ꢀdeoxyꢀ6ꢀethylenediamineꢀβꢀCD
The monoꢀ6ꢀdeoxyꢀ6ꢀethylenediamineꢀβꢀCD (EDAꢀβꢀCD) was between amino groups contained AdꢀPhNH2 and adamantine
synthesized according to the previous report.60 In brief, 12 g of chloride. The purified PhNH2 (1.13 g, 2.0 mM) and 1ꢀ
monoꢀ6ꢀOTSꢀβꢀCD was reacted with an excess amount of adamantoyl chloride (0.796 g, 4.0 mM) were dissolved in dry
ethylenediamine (EDA) at 80 °C for 4 h. The solution was THF (60 mL). Meanwhile, two drops of triethylamine were
cooled down and most of the unreacted EDA was removed by added in follow. The reaction mixture was reacted at room
rotary evaporation. The rest of the liquor was dissolved in temperature overnight. AdꢀPhNH2 was obtained via filtration
waterꢀmethanol mixture and precipitated in acetone 3 times. and further purified washing with water. The product was dried
The product was dried at 50 °C under vacuum for 24 h.
at 50 °C under vacuum for 24h.
2.4 Synthesis of poly(PEGMAꢀIA)
2.7 Preparation of PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2
The poly(PEGMAꢀIA) was synthesized according to the The obtained PEGMAꢀIAꢀβꢀCD (500 mg) and AdꢀPhNH2 (150
previous report.61 The poly(PEGMAꢀIA) was prepared via mg) were blended in 50 mL anhydrous DMF solution and
living free radical polymerization using PEGMA and IA ultrasonically treated for 10 min. The reaction system was
monomers. In brief, PEGMA (3.2 mM, 3.04 g) and IA (8 mM, stirred continuously at room temperature overnight without any
960 mg) were mixed and dissolved in an anhydrous ethyl other treatment. Then anhydrous ether was added into the
acetate solution (60 mL) at 80 °C for 10 min. AIBN (8 mM, solutions. White precipitates were formed after refrigeration
1.32 g) in 30 mL anhydrous ethyl acetate solution was quickly overnight at ꢀ20 °C. The PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 could
added into the reaction bottle and stirred at 80 °C for 24 h after be obtained via centrifugation and washing three times to
the mixture was completely dissolved. The system was always remove the unreacted reagents. The product was dried under
maintained under a N2 atmosphere before the reaction was vacuum oven at 40 °C overnight.
accomplished. Then the crude product was purified by dialysis
against an anhydrous ethyl acetate solution for 24 h .The
product was dried under vacuum oven at 40 °C overnight.
2.7 Cytotoxicity evaluation of PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2
FPNs
The cell viability of PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs
towards HUVEC cells was determined by cell counting kitꢀ8
2.5 Preparation of PEGMAꢀIAꢀβ–CD
PEGMAꢀIAꢀβ–CD was prepared by the ringꢀopening reaction (CCKꢀ8) assay based on our previous reports.63 The
between amine and anhydride in an anhydrous DMF solution. experimental procedure could be demonstrated by follows:
The preparation procedure could be summarized as follows. HUVEC cells were put into 96ꢀwell microplates at a density of
The previously obtained poly(PEGMAꢀIA)(1 g) was dissolved 5×104 cells mL–1 in 160 ꢁL of respective media containing 10%
in anhydrous DMF (20 mL) and mixed with EDAꢀβꢀCD (8 g) fetal bovin serum (FBS). After 24 h of cell attachment, the cells
in anhydrous DMF (40 mL). The system was stirred were incubated with different concentrations of FPNs (20ꢀ120
continuously at 40 °C for 10 h. The product was gotten through µg mL–1) for 12 and 24 h. Then these uninternalized FPNs were
dialysis treatment with fresh water for 24 h and dried under removed by repeatedly washing with Phosphate buffered saline
vacuum oven at 40 °C for 24 h.
(PBS) three times. 10 ꢁL of CCKꢀ8 dye and 100 ꢁL of
Dulbecco's Modified Eagle's medium (DMEM) cell culture
medium were added to each well and incubated for 2 h at room
2.6 Preparation of PhꢀNH2 and AdꢀPhNH2
The synthetic of AdꢀPhꢀNH2 was shown in Scheme S1 in temperature. Afterward, plates were analyzed using
a
detail. The intermediate product 1 was synthesized according to microplate reader (Victor Ш, PerkinꢀElmer). Measurements of
the previous report using bromohexadecane and phenothiazine formazan dye absorbance were carried out at 450 nm, with the
as templets.62 In 1000 mL bottles, POCl3(36 g, 0.24 mol) and reference wavelength at 620 nm. The values were proportional
DMF(11.7 g, 0.159 mol) was added to 30 mL ClCH2CH2Cl at 0 to the number of live cells. The percentage reduction of CCKꢀ8
°C mixing for 30 min. The intermediate product 1 (7.59 g, 18 dye was compared to controls, which represented 100% CCKꢀ8
mM) was subsequently added and reacted at temperature of 90 reduction. Three replicate wells were used per microplate, and
°C overnight. The crude product was slowly added into cool the experiment was operated for three times. Cell survival was
water and extracted with ethyl acetate for three times. Then expressed as absorbance relative to that of untreated controls.
flash column chromatography using 1:10 mixture of ethyl Results are presented as mean ± standard deviation (SD).
acetate and petroleum ether as eluent was done and obtained the
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2.8 Confocal microscopic imaging
HUVEC cells were cultured in DMEM supplemented with 10%
heatꢀinactivated fetal bovine serum (FBS), mM
2
Glucantamine, 100 U mL–1 penicillin, and 100 ꢁg mL–1 of
streptomycin. Cell culture was controlled at 37 °C in a similar
humidified condition of 95% air and 5% CO2 in culture
medium. Culture medium should be updated every three days
for maintaining the exponential growth of the cells. Before
treatment, cells were seeded in a glass bottom dish with a
density of 1×105 cells per dish. On the day of treatment, the
cells were incubated with PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs
at a final concentration of 40 µg mL–1 for 3 h at 37 °C.
Afterward, the cells were washed three times with PBS to
remove the PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs and then fixed
with 4% paraformaldehyde for 10 min at room temperature.
Cell images were obtained using a confocal laser scanning
microscope (CLSM) Zesis 710 3ꢀchannel (Zesis, Germany)
with the excitation wavelength of 405 nm.
Fig. 1 1H NMR spectra of mono-6-OTS-β-CD and EDA-β-CD.
For preparation of PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2, the
hydrophilic copolymers (PEGMAꢀIA) were synthesized using
PEGMA and IA as the monomers and AIBN as the initiator via
free radical living polymerization. EDAꢀβꢀCD was facilely
linked with the IA of PEGMAꢀIA via ringꢀopening reaction
with EDAꢀβꢀCD at 40 °C. Then PEGMAꢀIAꢀβ–CD and Adꢀ
PhNH2 were reacted via hostꢀguest interactions. The successful
preparation of PEGMA–IA, PEGMAꢀIAꢀβꢀCD and PEGMAꢀ
IAꢀβꢀCD/AdꢀPhNH2 could be confirmed by the 1H NMR
spectra (Fig. 2). The structure of PEGMAꢀIA could be
3. Results and discussion
Due to the sixth hydroxyl of βꢀCD strongest alkaline and
nucleophilicity, we can easily get the sixth mono replacement
of the derivatization for the βꢀCD. And the AdꢀPhNH2 was
facilely obtained via an amidation reaction. The structure
information of these obtained products were first characterized
and confirmed by 1H NMR spectroscopy. The structure of
monoꢀ6ꢀOTSꢀβꢀCD could be confirmed from 1H NMR
1
(DMSO) spectra. The result of H NMR spectra from Fig. 1 is
1
confirmed. The data from H NMR spectra are described as
described as follows. δ = 2.41 ppm (3H, ꢀCH3), δ = 3.18ꢀ3.42
ppm (14H, Hꢀ2,4), δ = 3.44ꢀ3.78 ppm (28H, Hꢀ3,5,6), δ=4.14ꢀ
4.48 ppm (6H,OHꢀ6), δ=4.72 ppm (3H, Hꢀ1), δ = 4.81ppm(4H,
Hꢀ1), δ = 5.44ꢀ5.90 ppm (14H, OHꢀ2,3), δ =7.40 ppm (2H,
aromatic protons), δ = 7.71 (2H, aromatic protons) demonstrate
the successful preparation of monoꢀ6ꢀOTSꢀβꢀCD. The resulting
follows (DMSO). The peak at δ = 1.1ꢀ1.3 ppm (ꢀCH3), δ = 1.7ꢀ
1.8 ppm (ꢀCH2ꢀCH2), δ = 3.20 ppm (ꢀCOꢀCH2), δ = 3.60 ppm (ꢀ
OꢀCH2), δ = 4.21 ppm (ꢀCOOꢀCH2) demonstrate the successful
preparation of PEGMAꢀIA by free radical living
polymerization. For the structure of PEGMAꢀIAꢀβꢀCD,
different peaks are summarized as follows (DMSO). The peak
at δ = 1.15ꢀ1.30 ppm (ꢀCH3), δ = 1.75 ppm (ꢀCH2ꢀCH2), δ = 2.9
ppm (ꢀCOꢀCH2), δ = 3.2 ppm (ꢀOꢀCH2ꢀ), δ = 3.34 ppm (NHꢀ
CH2), δ = 4.05 ppm (COOꢀCH2), δ = 5.60 ppm (OꢀCHꢀO), δ =
7.92 ppm (ꢀCOꢀNH). Furthermore, we can also know that the
PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 was successfully synthesized.
1
data of EDAꢀβꢀCD from H NMR (D2O) spectra: δ=2.69 ppm
(2H, NH2CH2ꢀCH2), δ = 2.90 ppm (2H,ꢀCH2NHꢀβꢀCD), δ =
3.30ꢀ3.51 ppm (14H, Hꢀ2,4), δ = 3.64ꢀ3.84 ppm (28H, Hꢀ3,5,6),
δ = 4.92 ppm (7H, Hꢀ1) demonstrate the successful preparation
of EDAꢀβꢀCD. The chemical structure of AdꢀPhNH2 was also
confirmed by 1H NMR spectroscopy. 1H NMR spectrum of Adꢀ
PhNH2 in CDCl3 is shown in Fig. S1. The shifts ranged from
1.70 to 2.1 ppm were consistent with Ad protons. The peaks at
about 6.80 ꢀ 7.80 ppm were found due to the protons of benzyl.
The peak at 1.25 ppm represents the methylene group of the
side chain (ꢀCH2ꢀCH2ꢀ). Furthermore, the shift at 3.93 ppm was
observed due to the methylene protons connected with
heterocyclic ring. All the above results confirmed the
successful synthesis of AdꢀPhNH2.
1
The results of H NMR for the PEGMAꢀIAꢀβꢀCD/AdꢀPhꢀNH2
are described as follows (DMSO). The peak at δ = 1.15ꢀ1.30
ppm (ꢀCH3), δ=1.75 ppm (ꢀCH2ꢀCH2), δ = 2.86 ppm (ꢀCOꢀCH2ꢀ
), δ = 3.16 ppm (ꢀOꢀCH2ꢀ), δ = 3.32 ppm (NHꢀCH2), δ = 4.09
ppm (COOꢀCH2) , δ = 5.65 ppm (OꢀCHꢀO) , δ = 7.10 ppm (Sꢀ
CꢀCH=), 7.60 ppm (Phꢀ) , 7.70ꢀ7.86 ppm (NꢀCꢀCH=),7.95 ppm
(ꢀCOꢀNHꢀ).
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Fig. 3 FT-IR spectra of PEGMA-IA, PEGMA-IA-β-CD and PEGMA-IA-β-CD/Ad-PhNH2.
The optical properties of PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 were
examined by UVꢀVis and FL spectroscopy. The UVꢀVis
spectrum of PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 was displayed in
Fig. 4A, the maximum absorption peaks of PEGMAꢀIAꢀβꢀ
CD/AdꢀPhNH2 dispersed in pure water was located at about
206 nm, which can be attributed to the π→π* transition. On the
other hand, a shoulder peak at about 230 nm was also found in
the UVꢀVis spectrum. This peak can be assigned to the band П
of benzene amino derivatives. Furthermore, because PEGMAꢀ
IAꢀβꢀCD is absent of adsorption between 200ꢀ400 nm,
therefore we could further conclude that AdꢀPhNH2 has
incorporated into PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 successfully
through hostꢀguest interaction. The final copolymers (PEGMAꢀ
IAꢀβꢀCD/AdꢀPhNH2) are tended to self assembly due to their
amphiphilic properties. The hydrophobic AdꢀPhNH2 will be
encapsulated in the core while the hydrophilic segments such as
PEGMA and IA will expanded into water. It is therefore the
self assembly nanostructure will emit strong luminescence and
good water dispersibility for the AIE active properties of Adꢀ
PhNH2 and hydrophilic PEGMA and IA. The AIE properties of
AdꢀPhNH2 were displayed in Fig. S2. It can be seen that the
fluorescent intensity of AdꢀPhNH2 was changed corresponding
with the fraction of water to THF. When the water fraction is
60%, the fluorescent intensity was reached to the lowest value.
The fluorescent intensity was rapidly increased when further
increase the fraction of water. The enhanced fold is greater than
1.5. The above results clearly showed that AdꢀPhNH2 is an AIE
active dye. Furthermore, the fluorescent intensity of the final
FPNs in the solution with different ratios of water and DMF
Fig. 2 1H NMR spectra of PEGMA-IA, PEGMA-IA-β-CD and PEGMA-IA-β-CD/Ad-PhNH2
The functional groups on PEGMAꢀIAꢀβꢀCD/AdꢀPhꢀNH2 were
evaluated by FTꢀIR spectroscopy. As displayed in Fig. 3, a
strong peak at about 2925 cm−1 was observed in all the samples,
which can be attributed to the stretching vibration of CH2 and
CH3. However, the intensity of the peak was obviously
decreased in the sample of PEGMAꢀIAꢀβꢀCD and PEGMAꢀIAꢀ
βꢀCD/AdꢀPhNH2 as compared with PEGMAꢀIA. On the other
hand, there are some tangled peaks appearing in the range of
650 to780 cm−1, which means the fingerprint region of βꢀCD.
Furthermore, the peak at 3397 cmꢀ1 can be assigned to
stretching vibration of hydroxyl group. As compared with
PEGMAꢀIA, the peak intensity of at 3397 cmꢀ1 was
significantly enhanced in the samples of PEGMAꢀIAꢀβꢀCD and
PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2. The obvious stretching vibration
of CꢀN at 1027 cmꢀ1 also proved that AdꢀPhNH2 and βꢀCD had
being linked on the polymer. Furthermore, compared with the
sample of PEGMAꢀIAꢀβꢀCD, a new peak that appeared at 1158
cm−1 can be ascribed to the fingerprint region of CꢀS. All of
these results confirmed the successful formation of PEGMAꢀ
IAꢀβꢀCD/AdꢀPhꢀNH2 through hostꢀguest interactions.
was also examined by PL spectroscopy. As shown in Fig. S3
,
the fluorescent intensity change of PEGMAꢀIAꢀβꢀCD/Adꢀ
PhNH2 showed similar trend like the AdꢀPhNH2. The
maximum fluorescent intensity was achieved when the water
fraction was 80%. The intensity value is about 3 folds of
minimal fluorescent intensity. All of the above results
suggested that both AdꢀPhNH2 and PEGMAꢀIAꢀβꢀCD/Adꢀ
PhNH2 possess desirable AIE properties. This is very important
for fabrication of FPNs with desirable fluorescent properties.
As evidenced by insets of Fig. 4A, good light transmittance was
found after PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs were dispersed
in water (left cuvette in Fig. 4Aꢀa). After the PEGMAꢀIAꢀβꢀ
CD/AdꢀPhNH2 water suspension was irradiated by UV lamp at
365 nm, uniform red fluorescence was observed (right cuvette
in Fig. 4Aꢀb). The above results clearly demonstrated that the
successful formation amphiphilic AIE active copolymers
through supermolecular interactions. Fig. 4B showed the
detailed information about the fluorescence of PEGMAꢀIAꢀβꢀ
CD/AdꢀPhNH2. The maximum emission wavelength was
located at about 573 nm, while the fluorescence excitation
wavelength of FPNs was appeared at 432 nm when the
emission wavelength was set at 573 nm. The photostability is
another very important factor for the FPNs for bioimaging
applications. A number of previous reports have demonstrated
that AIE active FPNs could display better photostability both in
solution and internalization into cells.11, 64
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Fig. 5 TEM image of PEGMA-IA-β-CD/Ad-PhNH2 FPNs. The size of PEGMA-IA-β-CD/Ad-
PhNH2 FPNs is ranged from 300 to 500 nm.
Considered the high water dispersibility and desirable AIE
active fluorescent properties, the potential applications of these
PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs in biomedical fields were
evaluated. First, the cytotoxicity of PEGMAꢀIAꢀβꢀCD/Adꢀ
PhNH2 FPNs was determined for exploring their potential
biomedical applications.
A
preliminary biocompatibility
evaluation was conducted using the CCKꢀ8 assay to evaluate
Fig. 4 (A) UV-Vis spectrum of PEGMA-IA-β-CD/Ad-PhNH2 FPNs, insets were the visible the effect of the biomaterials on the cell viability. As shown in
image of such FPNs in water; “a” and “b” are represented the optical images of
Fig. 6, it showed no significantly decreased after HUVEC cells
were incubated with distinct concentrations of PEGMAꢀIAꢀβꢀ
CD/AdꢀPhNH2 FPNs (0ꢀ120 ꢁg mL−1) for 12 and 24 h.
PEGMA-IA-β-CD/Ad-PhNH2 FPNs without and with UV lamp irradiation. It can be seen
that the symbol of cuvettecan be observed from the inset “a”, indicating good water
dispersibility of PEGMA-IA-β-CD/Ad-PhNH2 FPNs. The inset of “b” is the optical image of
Moreover, no significant cell viability decrease was detected
PEGMA-IA-β-CD/Ad-PhNH2 FPNs after irradiation with UV lamp at 365 nm. (B)
fluorescence excitation (Ex) and emission (Em) spectra of PEGMA-IA-β-CD/Ad-PhNH2
after 24 h incubation even the concentration of PEGMAꢀIAꢀβꢀ
CD/AdꢀPhNH2 FPNs was as high as 120 ꢁg mLꢀ1. The cell
FPN(λEx = 432 nm, λEm = 573 nm).
viability values at all experimental concentrations are obviously
The morphology of micelles appeared as spheres with suitable
greater than 90%. The IC50 values based on cell viability
examination is greater than 500 ꢁg mLꢀ1. Based on the cell
size ranged from 300 to 500 nm. Based on the TEM images, we
calculated the size distribution of PEGMA-IA-β-CD/Ad-PhNH2
viability evaluation, we could conclude that PEGMAꢀIAꢀβꢀ
and suggested that the size distribution is about 385.8 ± 90.2
CD/AdꢀPhNH2 FPNs show negative toxic effect on HUVEC
nm, which suggested that the resulting amphiphilic
cells. The excellent biocompatibility of PEGMAꢀIAꢀβꢀCD/Adꢀ
copolymers were self-assembled into nanoparticles in aqueous
PhNH2 FPNs makes them promising candidates for biomedical
solution. The probable mechanism forming spherical PEGMA-
applications.
IA-β-CD/Ad-PhNH2 could explain that the Ad-PhNH2 was
hydrophobic and curled into a sphere in water, while the
hydrophilic PEGMA-IA-β-CD were covered on the surface of
the hydrophobic core and contact with water, causing the
good water dispersibility. All the results showed the FPNs had
equal size and excellent dispersibility in aqueous solution,
good prospect for biomedical applications. Furthermore, the
hydrodynamic size distribution of PEGMA-IA-β-CD/Ad-PhNH2
FPNs in water was determined to be 453.59
± 144.4 nm with
relative broad polydispersity index of 0.235 (Fig. S4). As
compared with the TEM characterization, the size distribution
is relative large, which may be ascribed to the expand
polymers in water.
Fig. 6 The biocompatibility evaluation of HUVEC cells after incubating with distinct
concentrations (0–120 μg mL−1) of PEGMA-IA-β-CD/Ad-PhNH2 FPNs.
The interaction between PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs
and HUVEC cells was determined using CLSM. As shown in
Fig. 7, strong fluorescence could be observed from the HUVEC
cells after they were incubated with 40 ꢁg mLꢀ1 of PEGMAꢀIAꢀ
βꢀCD/AdꢀPhNH2 FPNs for 3 h (Fig. 7B). Because the free
PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs in cell culture medium
were almost completely removed by repeatedly washing, the
fluorescent signal in CLSM should be the AIE active FPNs that
were internalized by cells. Moreover, we could also found that
the location of fluorescent signal is covered on the location of
cells (Fig. 7C). Therefore, we can confirm that PEGMAꢀIAꢀβꢀ
CD/AdꢀPhNH2 FPNs can be used internalized by cells and the
fluorescence intensity is strong enough for biological imaging.
6 | J. Name., 2012, 00, 1-3
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Furthermore, many dark areas were surrounded by the areas
with strong fluorescent signal. The dark areas should be the
location of cell nucleus. Due to the absent of targeting agents
on the surface of PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs, the
internalization of PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs is mainly
ascribed to the nonspecific endocytosis, which has been
demonstrated by Liu et al.65 It is well known that the cell
nucleus pore is less than 30 nm, which is much smaller than the
size of PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs. Therefore, we
think that PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs are difficult to
enter cell nucleus directly and mainly distributed in the
cytoplasm. Considered the properties included their high water
dispersibility, low toxicity and desirable AIE active optical
properties, PEGMAꢀIAꢀβꢀCD/AdꢀPhNH2 FPNs are expected to
be promising candidates for biomedical applications.
Acknowledgements
This research was supported by the National Science
Foundation of China (Nos. 51363016, 21474057, 21564006,
21561022), and the National 973 Project (Nos.
2011CB935700).
Notes and references
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Novel polymeric luminescent nanoprobes with aggregation induced emission (AIE) properties were
fabricated via hostꢀguest interaction between the βꢀCD pendant copolymers and
Adamantaneꢀterminated AIE dye