PH-responsive amphiphilic block copolymer prodrug conjugated near infrared fluorescence probe

Article abstract of DOI:10.1039/c4ra04423a

A novel amphiphilic multi-block copolymer conjugated with both a near infrared fluorescence probe and drug has been designed and prepared by means of ring-opening polymerization (ROP) of N-Carboxy Anhydride (NCA) monomers following a Reversible Addition-F

Full text of DOI:10.1039/c4ra04423a

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pH-responsive amphiphilic block copolymer
prodrug conjugated near infrared fluorescence
Cite this: RSC Adv., 2014, 4, 28186
probe
*
Tao Xing and Lifeng Yan
A novel amphiphilic multi-block copolymer conjugated with both a near infrared fluorescence probe and
drug has been designed and prepared by means of ring-opening polymerization (ROP) of N-Carboxy
Anhydride (NCA) monomers following a Reversible Addition-Fragmentation Chain Transfer (RAFT)
polymerization. At first, an amino group-containing RAFT agent was synthesized and it served as an
initiator for the sequential ROP of aspartic acid b-benzyl ester N-carboxy anhydride (Asp-NCA) and
3-carbobenzoxy-L-lysine NCA (ZLLys-NCA). Then the multi-block copolymer was prepared by
a
succeeding RAFT polymerization of poly(ethylene glycol) methyl ether acrylate (OGEA). At the end, both
anticancer drug doxorubicin and hydrophobic aminocyanine dye were chemical conjugated to the block
copolymer via a hydrazone or amide bond, respectively. The obtained NIRF copolymer and its micelles
were characterized by nuclear magnetic resonance (NMR), gel permeation chromatography (GPC),
dynamic light scattering (DLS), and UV-vis and fluorescence spectrophotometry. The prodrug has strong
fluorescence in the near infrared region and shows pH-responsive drug release behavior, and it has
potential application in the theranostics of cancer.
Received 12th May 2014
Accepted 9th June 2014
DOI: 10.1039/c4ra04423a
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nanocarriers provides a way to improve the dye stability because
it prevents the interaction of dye with either solvent or other dye
Introduction
molecules. Compared with physical encapsulation, chemical
conjugation of the dye molecules to the nanocarrier may be
more suitable in that it prevents the dye from leakage during
blood circulation. Thus prolonging the uorescent time of the
system.
Polymeric nanoparticles are potential theranostic systems
for future cancer treatment due to their versatilities in both
structure and functionality.11,14–16 Synthesis of polymeric mate-
rials with versatile structures and bioimaging groups are an
ongoing area due to their potential applications in future
biomedical areas.14 Amphiphilic polymers are known to self-
assemble into nano-sized micelle in aqueous solution, which
makes it a useful candidate for both drug delivery and cancer
diagnostics. The rationale for the above two applications lies in
the enhanced permeability and retention (EPR) effect of micelle
or nanogel.17
Chemotherapy is an effective method for tumor treatment,
but its application has been restricted owing to the lack of an
effective drug delivery method to tumor tissue. Small drug
molecules normally could not reach the tumor tissue efficiently
and nonspecic distribution of the administered drug in bio-
logical system not only results in unwanted drug waste, but also
leads to much more serious problems usually resulting from its
pronounced toxicity to normal tissue. Physically or chemically
loaded drug in nanoparticles could reduce drug loss during
blood circulation, thus increasing drug using efficiency.18,19
For cancer treatment, both diagnosis and subsequent therapy
are important, and theranosis has been attracted much atten-
tion, recently.1,2 A drug delivery system with bioimaging ability
also affords the possibility of a precise location of the drug
carrier, providing useful information both for the diagnosis of
disease and its subsequent treatment. Incorporating various
bioimaging agents such as quantum dots,3,4 iron oxide,5–7
radionuclide agents8 and organic dyes9–11 into nanocarrier have
performed for these purposes. Among the various kinds of
imaging methods developed, optical imaging is largely utilized
because of its non-invasive nature. However, strong absorption
from hemoglobin can be detected at wavelengths lower than
600 nm, and signicant background uorescence from endog-
enous biomolecules can be detected up to wavelengths of
680 nm.12 Imaging in the near infrared region (NIR, >700 nm)
has been widely used for live body imaging because it works in a
wavelength range where body tissue has the least interference.
Cyanine dye is a type of the most widely used NIR probe as
long wavelength imaging agent with commercial availability
and FDA approval for clinical use.13 Physical encapsulation or
chemical conjugation cyanine dye into the polymeric
CAS Key Laboratory of So Matter Chemistry, Hefei National Laboratory for Physical
Sciences at the Microscale, Department of Chemical Physics, University of Science and
Technology of China, Hefei, 230026, P.R. China. E-mail: lfyan@ustc.edu.cn; Fax: +86-
551-3603748; Tel: +86-551-63606853
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Recently, polymeric prodrug nanogels or micelles have received tetrahydrofuran (THF) were rst reuxed with CaH2 overnight
much attention for they could offer several signicant advan- followed by distillation. N,N-Dimethylformamide (DMF) was
tages for cancer therapy, such as improved water solubility of dried over CaH2 at room temperature for 24 h before vacuum
drugs and enhanced drug bioavailability.20,21 In addition, poly- distillation. Triphosgene (99%), dichloromethane, doxorubicin
meric prodrugs are more stable in blood circulation since the hydrochloride (98%), triuoroacetic acid, sodium acetate,
drug is conjugated to the polymer with covalent bonds.22 The hydrazine dihydrochloride (98%), 3-bromopropionic acid,
development of intelligent nanoparticles23–26 which could N-hydroxysuccinimide (HOSu), N,N0-dicyclohexyl carbodiimide
release loaded drug under environmental stimuli at a desired (DCC), diisopropylethylamine (DIEA), di-tert-butyl dicarbonate,
site further optimizes the drug performance. Usually, the drug N-Boc-ethylenediamine, b-benzyl L-aspartate, and 3-carbo-
can be chemically bonded to non-responsive nanoparticles via a benzoxy-L-lysine were purchased from Aladdin Corporation,
responsive chemical bond,.27–29 pH-responsive nanoparticles China. Dialysis bag (cutoff Mw ¼ 8000) was obtained from
were developed on the basis of more acidic nature of tumor cell Bomei biotechnology corporation, China. mPEG (Mw ¼ 1900)
and its microenvironment.29,30 Usually, pH-responsive nano- were purchased from Aldrich and used as received without
particles are required to be stable at physiological pH (7.0), but further purication. Milli-Q water (18.2 MU) was prepared using
undergoes chemical bond cleavage and drug release activation a Milli-Q Synthesis System (Millipore, USA). Normal phase
under acid condition (pH ¼ 5.0–6.0 in endocytic vesicles or 6.5– column chromatography was carried out using 200–300 mesh
6.8 in tumor microenvironment).31 One meritorious example silica gel (Yantai institute of chemical engineering, China).
was the attachment of doxorubicin (DOX) to hydrazine con-
taining copolymers via hydrazone bond, hydrolysis of the
hydrazone linkage under acidic condition leads to activation of
the originally dormant drug delivery system.28,32
Synthesis of LLys-NCA
Combined a NIR dye with a pH-responsive drug-conjugated LLys-NCA was prepared taking a literature method.42 In brief,
nanoparticle can be a useful method for tumor theranosis. With Nepsilon-carbobenzoxy-L-lysine (2.0 g, 7.1 mmol) was sus-
the aid of a NIR dye, the accumulation behavior of the nano- pended in 50 mL dry THF, followed by the addition of a THF
particle could be conveniently monitored in vivo. Due to the EPR solution of triphosgene (2.1 g, 7.1 mmol). Next, the obtained
effect directed nanoparticle accumulation, NIR uorescence suspension was stirred at 45 ꢀC for 2 h, followed by ltration to
signal is also hoped to accumulate at the tumor site during remove traces unreacted Nepsilon-carbobenzoxy-L-lysine, and
blood circulation. Tumor imaging could thus be realized the ltrate was collected and crystallized three times from a
without using the usual complicated modied cyanine mole- mixture of THF and hexane to give the anhydride as white
cules with tumor-targeting ability.33–35 Cyanine dyes can be crystals (1.6 g, 73% yields). 1H NMR (300 MHz, CDCl3, d, ppm) d
incorporated into nanoparticle via chemical conjugation.36,37 7.48 (s, 5H), 6.75 (s, 1H), 5.42–5.13 (m, 2H), 5.01 (s, 1H), 4.40 (s,
Both hydrophobic and hydrophilic cyanine molecules could be 1H), 3.32 (d, J ¼ 5.5 Hz, 2H), 2.23–1.40 (m, 6H). 13C NMR (300
used for nanoparticle conjugation.
MHz, DMSO, d, ppm): d 171.8, 156.1, 152.0, 137.3, 128.4, 127.8,
In our previous studies, polypeptide nanogels or micelles 65.1, 57.0, 41.3, 31.1, 30.6, 28.7. FT-IR (KBr, thin lm, cmꢁ1):
with conjugated NIR probe had been synthesized for imaging 1785, 1854.
the drug delivery in both in vitro and in vivo, and a long NIR
image-guided drug delivery had been achieved.20,38–41
Here, we reported the synthesis of a novel amphiphilic multi-
block copolymer prodrug with both near infrared uorescence
Synthesis of RAFT agent 2 and 3
and pH responsive property. Both ring-opening polymerization 2.0 g (5.5 mmol) of RAFT agent 1 was dissolved into 50 mL of
(ROP) of N-Carboxy Anhydride (NCA) monomers and reversible dried CH2Cl2, and the obtained yellow solution was cooled in an
addition-fragmentation chain transfer (RAFT) polymerization ice bath. Then, 20 mL CH2Cl2 containing of 1.50 g (7.3 mmol) of
have been involved, and both anticancer drug doxorubicin and DCC and 0.76 g (6.6 mmol) of HOSu were added and the mixture
hydrophobic aminocyanine dye were chemical conjugated to was stirred for 36 h, and a white suspension was obtained (RAFT
the block copolymer (Scheme 1). Micellization of the above agent 2). Aer removing the precipitate, the solution was cooled
NIRF prodrug into water leads to the formation of NIRF poly- down to ꢁ10 ꢀC, and 0.73 g (5.5 mmol) of N-Boc-ethylenedi-
meric prodrug with both drug delivering and diagnostic abili- amine was added, and the mixture was kept 0 ꢀC overnight.
ties. Combined both pH-responsive drug release ability and Aer the solvent was removed under vacuum, the obtained oil
near infrared optical property, the material synthesized here residue was puried by column chromatography using a
can be a paradigm for future theronostic application in cancer mixture of petroleum ether and ethyl acetate (5 : 1) as eluent. At
treatment.
the end, 2.1 g of a yellow oil-like product was obtained (78%
yields). H NMR (400 MHz, CDCl3, d, ppm): 0.88 (t, 3H, CH3),
1
1.25–1.67 (m, 20H, CH2), 1.43 (s, 9H, CH3), 1.72 (s, 6H, CH3),
3.23–3.34 (m, 6H, CH2). 13C NMR (400 MHz, CDCl3, d, ppm)
14.06, 22.61, 25.70, 27.62, 28.31, 28.90, 29.01, 29.26, 29.36,
Experimental section
Materials
All chemical agents with AR purity except specically indicated 29.46, 29.54, 31.83, 37.02, 39.86, 40.98, 56.95, 79.38, 156.40,
were purchased from Sinoreagent Corporation. n-Hexane and 173.02, 220.71.
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Scheme 1 Synthesis of NIRF pH-responsive polymeric prodrug via a combination of ROP and RAFT polymerization.
CH2), 1.69 (s, 6H, CH3), 3.13 (s, 2H, CH2), 3.28 (t, 2H, CH2), 3.49
(s, 2H, CH2). 13C NMR (100 MHz, CDCl3, d, ppm) 14.58, 23.14,
25.77, 28.08, 29.44, 29.57, 29.80, 29.92, 30.02, 30.08, 32.36,
37.74, 39.02, 40.49, 57.29, 175.75, 221.92.
Synthesis of RAFT agent 4
0.5 g of the as-prepared RAFT agent 3 was dissolved in 2.0 mL of
triuoroacetic acid, and it was stirred for 1 h at room temper-
ature. Then the solvent was removed under vacuum, and the
obtained oil residue was puried by column chromatography
using a mixture of ethyl acetate and methanol (10 : 1) as eluent.
Aer the solvent was removed under vacuum, 0.4 g of yellow
powder of RAFT agent 4 was obtained (80% yields). 1H NMR
(400 MHz, CDCl3, d, ppm) 0.88 (t, 3H, CH3), 1.25–1.69 (m, 20H,
Synthesis of RAFT-PZLLys-PAsp
To a ame dried and argon purged schlenk tube, 0.12 g (0.23
mmol) RAFT agent 3 was added under the protection of argon,
followed by the addition of 10.0 mL of DMF, and the system was
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cooled down to 0 ꢀC, and then 40 mL (0.23 mmol) DIEA was
added, and the mixture was stirred for 20 min. Next, 0.68 g
(2.2 mmol) ZLLys-NCA was added using its DMF solution, and
Preparation of NIR polymeric prodrug 10 micelle
The micelle was prepared by dissolving 4 mg polymer into 1 mL
DMF, the obtained solution was sealed into dialysis bag and
dialyzed against water to remove DMF and allow the formation
of micelle.
ꢀ
the reaction was carried out for 3 days under 0 C under mild
stirring. Aer the NCA monomer was polymerized completed
(FT-IR tracing), 0.1 g (0.46 mmol) of di-tert-butyl dicarbonate
was added and the reaction was continually carried out over-
night to protect the terminal amine group. Then, the product
was added into 100 mL cool ethyl ether to get precipitate, and it
was removed again in DMF for dialysis. At the end, 0.9 g product
with light yellow was obtained, and the yield is 83%.
Characterization
1H NMR spectra were to measured on a Bruker AC 300 or 400
spectrometer as indicated. Deuterated dimethyl sulfoxide
(DMSO) or deuterated chloroform containing 0.03 v/v % tetra-
methylsilane (TMS) was used as the solvent. FT-IR spectra were
measured on a Bruker EQUINOX 55 Fourier transform infrared
spectrometer using the KBr disk method. Size and size distri-
bution of the nanogels were determined by dynamic light
scattering (DLS) carried out on a Malvern Zetasizer Nano ZS90
with a He–Ne laser (633 nm) and 90ꢀ collecting optics.
Measurements were performed at room temperature and the
data were analyzed by Malvern Dispersion Technology Soware
4.20. Fluorescence measurements were carried out on a Shi-
madzu RF-5301 PC Fluorescence spectrophotometer with an
excitation and emission slit width of 5 and 10 nm, respectively.
UV-vis spectra were obtained on a Shimadzu UV-2401PC Ultra-
violet spectrophotometer. Molecular weights of the samples
were determined by Gel Permeation Chromatography (GPC)
equipped with two columns (one Shodex GPC KD-804 column
and one guard column), a refractive index detector (RID-10A),
DMF was used as the mobile phase and the measurement was
Synthesis of POGEA-PZZLys-PAsp
1.95 g OGEA (4.06 mmol) and 0.5 g (0.1 mmol) PZZLys-PAsp were
dissolved into 2 mL of DMF in a Schlenk tube under stir, and
100 mmol DMF containing of 3.3 mg AIBN was added, then the
mixture was subjected to three frozen–pump–thaw circles to
remove any dissolved oxygen, sealed and heated to 80 ꢀC under
stirring for 12 h. Aer the polymerization, the solution was
cooled to terminate the reaction. Purication of the sample was
done by dialysis against water. 1.9 g products were obtained
aer removing water by freeze-drying, and the yield is 78%.
Synthesis of polymer 8
1.5 g (75.4 mmol) POGEA-PZZLys-PAsp was dissolved in 5.0 mL
DMF, then 0.48 g (4.53 mmol) hydrazine dihydrochloride and
1.55 mL N,N-diisopropylethylamine were added sequentially,
next the mixture was stirred at 45 ꢀC for 36 h till the suspension
became clear. Purication of the sample was performed by
dialysis against water. 1.2 g products were obtained aer
removing water by freeze-drying, and the yield is 83%.
ꢁ1
ꢀ
performed at 30 C at a sample concentration of 3 mg mL
.
Monodispersed polystyrene standards were used for the cali-
bration of Mn, Mw and Mw/Mn.
Molar extinction coefficient of the sample was determined by
Beer's law based on three separate measurements. The relative
uorescence quantum yield was determined by using the
following equation:43
Synthesis of polymeric prodrug 9
FF(x) ¼ (As/Ax)(Fx/Fs)(nx/ns)2FF(s)
1.0 g (5.2 mmol) of polymer 8 and 30 mg doxorubicin hydro-
chloride were added into a Ar gas protected dried round bottom
ask (25 mL), and 5.0 mL DMF was added as solvent, and then
where F, A and n refer to the quantum yield, absorption and
refractive index of the solvents used. F(x) stands for the area
under the emission curve. Subscripts x and s represent sample
and stander, respectively. Indocyanine green (ICG) was used as
a reference standard with a quantum yield of 0.078 in MeOH.44
ꢀ
the mixture was stirred at 45 C for 36 h. Aer reaction, puri-
cation of the sample was performed by dialysis against water.
0.61 g (60% yields) product was obtained aer removing water
by freeze-drying, and the context of DOX in the product is 2.9%
measured by means of uorescence spectrophotometry.
In vitro drug release
Drug release behavior of the NIR polymeric prodrug was eval-
uated under both acidic and neutral condition. For the neutral
Synthesis of NIR polymeric prodrug 10
2.5 mg (2.5 mmol) aminocyanine was dissolved in 1 mL DMF, a condition, 400 mL NIRF prodrug solution (1 mg mLꢁ1) was
DMF solution (1 mL) containing 0.40 mg (3.0 mmol) HOSu and sealed into a dialysis bag, which was then immersed into 50 mL
0.75 mg (3.8 mmol) DCC were added. The combined solution 0.2 M phosphate buffered saline (PBS) solution, release exper-
was stirred at dark for 24 h, followed by adding into 2 mL DMF iment was conducted at 37 ꢀC on a shaking bath. 2 mL samples
containing of 300 mg (1.5 mmol) of the as-prepared polymeric were removed at predetermined time points and 2 mL fresh PBS
prodrug 8 under strong stir, and the mixture was mildly stirred was added at the same time. Drug released was quantied by
overnight at dark. Aer reaction, purication of the sample was using uorospectrophotometer at an emission wavelength of
performed by dialysis against water for 48 h. 242 mg (81% 557 nm with an excitation wavelength of 480 nm. For the acidic
yields) product was obtained aer removing water by freeze- condition, a similar method was employed except for 0.2 M
drying.
acetate buffer was employed as the release media.
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Cytotoxicity
Cytotoxicity of both the NIR polymeric prodrug was evaluated
with a methyl tetrazolium (MTT) viability assay against Hela
cell. Hela cells were seeded in a 96-well plate with a cell number
of 5000 cells per well. A 1640 solution containing 10% fetal
bovine serum (FꢀBS) was used as the cell culture media. Aer
incubated at 37 C for 24 h under a 5% CO2 atmosphere, the
culture media was replaced with a series of nanogel solution (in
1640 media containing 10% FBS) ranging from 0 to 0.25 mg
mLꢁ1. Then the cells were incubated at 37 ꢀC for 2 days, and the
culture media was removed and 100 mL fresh 1640 (10% FBS)
was added to each well, followed by the addition of 25 mL MTT
stock solution (5 mg mLꢁ1). 100 mL extraction buffer (20% SDS
in 50% DMF, pH 4.7, prepared at 37 ꢀC) was added to each well
aer an additional incubation of 2 h. The obtained solution
were incubated overnight, the absorbance of the solution was
measured at 490 nm using a Bio-Rad 680 microplate reader. The
cell viability was normalized to of Hela cells cultured in
complete culture medium.
Fig. 1 1H NMR spectrum of RAFT-PZLLys.
(–(CH2)3–Cbz) can be assigned to the protons of two methylene
groups adjacent to the Cbz group, while chemical shis at
2.9 ppm (–CbzNH–CH2–) could be assigned to protons of
methylene groups adjacent to amino nitrogen atom connected
to Cbz group, respectively. Chemical shis corresponding to the
methyl and methylene groups of RAFT agent could be observed
at 0.8 ppm (CH3(CH2)11–) and 1.2 ppm (CH3(CH2)11–), respec-
tively. Molecular weight (Mw) of the product was determined by
the integration ratio of peak b to k is 3 : 19, close to the theo-
retical value, indicating the polymer degree is 10.
Results and discussion
The combine of RAFT and ROP techniques provides many
changes to design and synthesis of novel copolymers, especially
multi-functional block copolymers. Here, the target copolymer
was prepared as a route described in Scheme 1. RAFT agent 1
can be obtained commercially and directly used as the feed
stock, and aer it was activated with HOSu and DCC in CH2Cl2,
it can reacts easily with N-Boc-ethylenediamine to form RAFT
agent 3, aer it was deprotection by triuoroacetic acid, an
amide terminal RAFT agent 4 was formed, where the amide
group is the efficient initiator for ROP of NCA monomers while
the tri-thioester structure used for the controlled polymeriza-
tion of acrylate monomers.
Next, RAFT agent 4 was used to initiate the ROP polymeri-
zation of ZLLy-NCA at rst, and then the Asp-NCA sequentially
to form a diblock linear polypeptide. The choice of ZLLy-NCA
and Asp-NCA mainly depends on the side chains reactive groups
for the potential reaction to both NIR probe or pH-responsive
linkage. Both the ROP of NCA monomers were carried out at
0 ꢀC to avoid the active monomer mechanism (AMM) that result
in the bad controlling of the polymerization.
Synthesis of RAFT-PZLLys10-PAsp10
Diblock copolymer RAFT-PZLLys-PAsp was synthesized by
sequential ring-opening polymerization of Asp-NCA by the
RAFT-PZLLys. The 1H NMR spectrum of the diblock copolymer
was shown in Fig. 2. Except for those chemical shis corre-
sponding to RAFT agent 4 and RAFT-PZLLys, new chemical
shis relevant to the methylene groups in the side chain of Asp
(–CH2–OBzl) could be observed at 2.8 ppm. Chemical shis
corresponding to the OBzl group could be observed at 7.3 ppm
(C6H5CH2–) and 5.1 ppm (C6H5CH2–), and part of them overlap
with the signals of Cba group. The polymeric degree of PAsp can
be determined by integration ratio of relevant peaks in 1H NMR.
In Fig. 2, the integration ratio of peak j + m to k + n is 40 : 41,
The as-prepared diblock of polypeptide with a RAFT segment
was used as the chain transfer agent for the RAFT polymeriza-
tion of a macro-monomer OGEA using AIBN as initiator to
induce PEG chains into the copolymer. Then, the copolymer
conjugated with NIR probe (Cy 7,7) and DOX by covalent, while
the linkage between DOX and copolymer is the pH-sensitive
hydrazine group.
Synthesis of RAFT-PZLLys10
RAFT-PZLLys was synthesized by the ring-opening polymeriza-
tion of ZLLys-NCA using the RAFT agent 4 as the initiator. Fig. 1
1
shows the H NMR spectrum of the product. Chemical shi at
7.3 ppm (C6CH5CH2–) and (C6CH5CH2–) could be attributed to
the protons of the Cbz group, chemical shi at 1.5 ppm Fig. 2 1H NMR spectrum of RAFT-PZLLys-PAsp.
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Table 1 Molecular weights and polydispersity indexs of the obtained
homopolymer, diblock and multi-block copolymer
Sample
MGwPC
MNwMR
PDI
PZLLys10
PZLLys10-PAsp10
POGEA31-PZLLys10-PAsp10
4214
5179
25 009
3026
4976
19 856
1.01
1.09
1.16
weight shis to lower elution time, indicating the propagation
of the polymer chain from homogeneous polymer to multi-
block copolymer. Molecular weights of the obtained samples
were listed in Table 1. Polydispersity indexes of all the sample
remain low (<1.2), indicating the living nature of the all poly-
merizations. Mw of all the samples were larger than calculated
Mw form GPC, while may be due to linear polystyrenes were
Fig. 3 1H NMR spectrum of RAFT-PZLLys-PAsp.
indicating that every diblock copolymer chain contains 10 Asp used for the Mw calibration.
repeating units.
Hydrazinolysis of the copolymer
Synthesis of POGEA31-PZLLys10-PAsp10 copolymer
Hydrazine dihydrochloride was used to hydrazinolysis the PAsp
segment in the multi-block copolymer. 1H NMR spectrum of the
product was shown in Fig. 5. Characteristic peak of the Bzl
groups locate at 7.3 ppm was decreased aer deprotection, and
the integration ratio of peak f to k is 1 : 1, indicating the signal
at 7.3 ppm all come from the Cbz groups in PZLLys segment,
and it reveals that the hydrazinolysis is complete.
Triblock copolymer POGEA31-PZLLys10-PAsp10 was synthesized
by RAFT polymerization of OGEA monomer using AIBN as
initiator and RAFT-PZLLys10-PAsp10 copolymer as the chain
transfer agent, and the polymerization was conducted for 12 h
in DMF, then the unreacted monomer was removed by dialysis
against water. Since the terminal amino group of RAFT-
PZLLys10-PAsp10 may degrade the RAFT agent at 80 ꢀC, so it was
1
protected by the Boc group before reaction. H NMR spectrum
Preparation of NIR probe and DOX conjugated polymer
prodrug
of the product was shown in Fig. 3. Characteristic chemical
shis of the POGEA block could be observed at 3.6 ppm
(–CH2CH2O–) and 3.4 ppm (CH3OCH2–). As described above,
repeating unit of the POGEA could be determined by integration
ratio of the relevant peak k and p, integration ratio of the two
peaks is 1 : 73, indicating the polymeric degree of OGEA is 31.
Molecular weights and weight distributions of the obtained
polymers were determined by GPC, and the results were shown
in Fig. 4. With each addition of the monomers, the molecular
The conjugation of DOX molecule to the copolymer was carried
out in DMF by the hydrazine group, the molar ratio of DOX to
the copolymer is 1 : 10 to insure the efficient reaction of DOX.
The content of DOX in the copolymer can be obtained by
measuring the uorescence intensity at 557 nm (lex ¼ 480 nm),
and the result reveals that the context of DOX in the copolymer
is 2.8 wt%.
The conjugation of NIR probe to the copolymer was also
carried out by the reaction of aminocyanine-OSu with the
hydrazine group. Different substitution degrees (10%, 25%, and
Fig. 4 GPC traces of the homopolymer, diblock copolymer and tri- Fig. 5 1H NMR spectrum of hydrazine functionalized triblock copol-
block copolymer in DMF.
ymer in DMSO-d6.
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50%) of the NIR probe were synthesized, which are indicated as
NIRF P1, NIRF P2 and NIRF P3, respectively.
1H NMR spectrum of the as-prepared DOX and NIR probe
conjugated copolymer is shown in Fig. 6. The signal of copol-
ymer still preserves, while the signals for NIR probe and DOX
are almost invisible, indicating the low context of them. For
NIRF P1, P2 and P3, the context of NIR probe are 0.47%, 1.17%
and 2.3%, respectively, which were calculated by means of
uorescence.
The as-prepared NIRF copolymers can be assembled into the
micelle structure. Size and size distribution of the NIRF micelle
was characterized by DLS. The results were shown in Fig. 7. The
NIRF micelle has a size distribution from 60 nm to 400 nm, with
an average diameter of 120 nm.
Optical property of the NIRF micelle was characterized by a
the combination of UV-vis spectrophotometer and uorescence
spectrophotometer. Adsorption and emission spectra of the
NIRF micelle were shown in Fig. 8. The micelle solution has two
typical adsorptions at 480 nm and 675 nm, corresponding to
that of DOX and NIR probe, while their emission upon
Fig. 8 Absorption (solid line) and emission (dash line) curve of NIRF P1
micelle solution (0.8 mg mLꢁ1). (a) Excited at 480 nm. (b) Excited at 650
nm, the excitation and emission slit widths were 5 and 15 nm,
respectively.
excitation are 557 and 730 nm, respectively. It is worth noting
that no adsorption besides 635 nm could be observed, indi-
cating no aminocyanine aggregation took place under this
condition. This may be due to the aminocyanine molecules
were xed on the polymer backbone, thus eliminated the dyes'
tendency to aggregate.
Quantum yields of the NIRF micelles with different
percentage of NIR dyes were determined by a standard method
using ICG as reference, and the results were listed in Table 2.
Fig. 6 1H NMR spectrum of NIR polymer 8 with different substitutions With increasing amount of dye molecules attached to the
of NIR dye in DMSO-d6.
polymer chain, quantum yield decreases, and this could be
attributed to the uorescence resonance energy transfer (FRET)
between dye molecules at higher amount of dyes.45
In vitro drug release behavior of the NIRF prodrug was
evaluated under both acidic (pH ¼ 5.0) and neutral (pH ¼ 7.2)
condition, respectively. The results were shown in Fig. 9. Under
neutral condition, a slow drug release was observed in the rst
5 h, with no further release during the following 60 h, the total
drug release was less than 20% in the 60 h period, while under
acidic condition, an obvious acid promoted drug release
behavior was observed, fast and sustained drug release could be
observed during the whole time range, with near 90% drug
release during the 60 h period. Accelerated drug release may be
Table 2 Quantum yields of NIRF P1, P2 and P3
Fig. 7 Size and size distribution of NIRF polymeric prodrug 10 micelle Substitution
NIRF P1
0.25
NIRF P2
0.058
NIRF P3
0.055
solution (1.0 mg mLꢁ1) determined by DLS, Micelle solution was
FF
prepared by first dissolving the material and then dialysis against water.
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infrared range and could release the conjugated drug via pH
regulation. Sustained drug cleavage from the NIRF prodrug was
conrmed by in vitro drug release. The application of the NIRF
produrg as a theranostic agent for cell and animal experiments
will be carried out in the next step.
Acknowledgements
This work is supported by the National Basic Research Program
of China (no. 2011CB921403 and 2010CB923302), and the
National Natural Science Foundation of China (no. 51373162).
The authors would like to thank Bin Lai for his help with the
MTT measurement.
Fig. 9 Drug release behavior of the NIRF P1 at neutral (pH ¼ 7.2) and
acid (pH ¼ 5.0) condition.
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