Synthesis of polypeptide conjugated with near infrared fluorescence probe and doxorubicin for pH-responsive and image-guided drug delivery

Article abstract of DOI:10.1039/c2jm35627a

A near infrared fluorescent polymeric drug delivery system (NIRF DDS) with pH-responsive drug release properties has been designed and developed. This material was prepared by chemical conjugation of the anticancer drug doxorubicin and hydrophobic aminocyanine dye to triblock copolypeptide via hydrazone and amide bonds, respectively. Conjugation with aminocyanine shows almost no toxicity of the material, while conjugation with doxorubicin induces pronounced toxicity on the original biocompatible material. The pH sensitive drug release nature of the near infrared fluorescent polymeric drug (NIRF prodrug) was confirmed by accelerated drug release at pH of 5.0 via an in vitro drug release experiment and gradual drug cleavage from the NIRF prodrug during a confocal laser scanning microscopic (CLSM) experiment. The CLSM experiment also reveals that the released drug subsequently migrated to the nucleus, while the polymeric residue still remained in cytoplasm, indicating that the as-prepared polymer can be a promising candidate for theranosis of cancer.

Full text of DOI:10.1039/c2jm35627a

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PAPER
Synthesis of polypeptide conjugated with near infrared fluorescence probe and
doxorubicin for pH-responsive and image-guided drug delivery†
a
Tao Xing, Xianzhu Yang, Feng Wang, Bin Lai and Lifeng Yan
b
b
a
a
*
Received 19th August 2012, Accepted 5th September 2012
DOI: 10.1039/c2jm35627a
A near infrared fluorescent polymeric drug delivery system (NIRF DDS) with pH-responsive drug
release properties has been designed and developed. This material was prepared by chemical
conjugation of the anticancer drug doxorubicin and hydrophobic aminocyanine dye to triblock
copolypeptide via hydrazone and amide bonds, respectively. Conjugation with aminocyanine shows
almost no toxicity of the material, while conjugation with doxorubicin induces pronounced toxicity on
the original biocompatible material. The pH sensitive drug release nature of the near infrared
fluorescent polymeric drug (NIRF prodrug) was confirmed by accelerated drug release at pH of 5.0 via
an in vitro drug release experiment and gradual drug cleavage from the NIRF prodrug during a
confocal laser scanning microscopic (CLSM) experiment. The CLSM experiment also reveals that the
released drug subsequently migrated to the nucleus, while the polymeric residue still remained in
cytoplasm, indicating that the as-prepared polymer can be a promising candidate for theranosis of
cancer.
magnetic resonance imaging (MRI),8,9 radioisotopes for positron
Introduction
emission tomography (PET),10 or fluorescent chemical agents
such as quantum dots11–13 and organic dyes14–16 for optical
imaging. Optical imaging is a widely used method both for in vivo
and in vitro imaging, because of the commercial availability of
organic dyes and the relatively low cost of detection. However,
imaging with visible light suffers from a series of problems such
as low penetration (2–3 cm), pronounced background auto-
fluorescence, severe tissue absorption and scattering. Low depth
penetration of visible light reduces the detection availability and
greatly limits its use in living body imaging. Imaging in the near
infrared region (700–900 nm), on the other hand, eliminates
undesired background noises and thus becomes a more valid
method for in vivo imaging.17,18
For cancer treatment, both its early stage diagnosis and subse-
quent therapy go a long way to minimize the danger.1 The term
‘‘theranosis’’ has therefore been coined to describe a combination
of these two functionalities.2,3 Polymeric nanoparticles emerge as
potential theranostic systems for future cancer treatment due to
their versatility in both structure and functionality.4–6 The nano-
sized nature of these systems allows for a longer blood circulation
time and provides shelter for the loaded theranostic agents.
Besides, as a result of the highly permeable vascular structure of
tumor tissue induced by rapid and abnormal angiogenesis, as
well as a dys-functional lymphatic drainage, these nanoparticles
were found to preferentially accumulate at the position of the
tumor via an enhanced permeability and retention (EPR)
mechanism,7 thus reducing its non-specific accumulation, either
related to diagnosis or therapy.
Chemotherapy is a effective method for tumor treatment, but
its application has been restricted due to the lack of an effective
drug delivery method to tumor tissue. Small drug molecules
normally could not reach the tumor tissue efficiently, because of
the inherent physiological barriers such as blood component
interaction. Nonspecific distribution of the administrated drugs
in biological systems not only results in unwanted drug waste,
but also leads to much more serious problems, usually resulting
from its pronounced toxicity to normal tissue. The emergence of
nanoparticles offers new methods of drug administration;
physically or chemically loaded drugs in nanoparticles could
reduce drug loss during blood circulation, thus increasing drug
use efficiency.19,20 The development of intelligent nano-
particles,21–24 which could release a loaded drug under environ-
mental stimuli, further optimizes the drug performance in vivo.
Usually, the drug can either be physically loaded into
Diagnosis of cancer in its early stage is of vital importance for
its subsequent treatment. Various tumor imaging methods based
on different imaging modalities have been developed for this
purpose: metallic or magnetic compounds such as iron oxide for
aDepartment of Chemical Physics, Hefei National Laboratory for Physical
Sciences at the Microscale, University of Science and Technology of China,
Hefei, 230026, P. R. China. E-mail: lfyan@ustc.edu.cn; Fax: +86-551-
3606853
bSchool of Life Science, University of Science and Technology of China,
Hefei, 230026, P. R. China
† Electronic supplementary information (ESI) available: Mass spectra of
compounds 4 and 5, CLSM images of free doxorubicin after 8 h. See
DOI: 10.1039/c2jm35627a
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environmental stimulus responsive nanoparticles,25,26 or chemi-
cally bonded to non-responsive nanoparticles via a responsive
chemical bond,27–29 while a combination of the above two
methods may be more optimal. pH25,29 and reduction30,31 are the
two most utilized stimuli for controlled drug delivery. The design
rationale of reduction sensitive nanocarriers lies in the fact that
glutathione, which acts as a reducing agent to cleave disulfide
linkage, was of a concentration several orders higher inside than
outside the cell.30 Cleavage of the disulfide bond stabilized
nanoparticle usually leads to partial or total disassembly of the
nanocarrier, activating the release of the embedded drug. pH-
responsive nanoparticles were developed on the basis of the more
acidic nature of the tumor cell. The lower local pH of the tumor
microenvironment was partially induced by the elevated rates of
glucose uptake but reduced rates of oxidative phosphorylation,
while insufficient blood supply and poor lymphatic drainage are
the other two factors contribute to this microenvironmental
acidity.32 pH-responsive nanoparticles thus developed are
required to be stable at physiological pH (7.0), but undergo
chemical bond cleavage and drug release activation under acid
conditions (pH ¼ 5.0). The pH-responsive nanoparticle can be
designed either as a pH-responsive nanoparticle with a physically
loaded drug, or a polymeric drug (prodrug) where the drug is
chemically attached to the nanoparticle via a pH sensitive bond.
A chemically bonded drug may be more optimal because of the
firmer connection between the nanoparticle and the drug, which
further reduces undesired drug leakage during delivery. One
notable example was the attachment of the anticancer drug
doxorubicin to hydrazine containing copolymers via the hydra-
zone bond; hydrolysis of the hydrazone linkage under acidic
conditions leads to activation of the originally dormant drug
delivery system.28,33
anticancer drug doxorubicin delivery (Scheme 1). Schematic
illustrations of the material synthesis were shown in Schemes 2
and 3. Triblock copolymer mPEG-PAsp-PZLLys 2 was prepared
in the first place by sequential ring opening polymerization of
aspartic acid b-benzyl ester N-carboxy anhydride (Asp-NCA)
and 3-carbobenzoxy-L-lysine NCA (ZLLys-NCA) by amino
terminated poly(ethylene glycol) methyl ether (mPEG-NH2).
Aminolysis of the side b-benzyl ester group with hydrazine
dihydrochloride leads to the formation of hydrazine function-
alized triblock copolymer 3. Doxorubicin was then attached to
the side chain of the copolymer via a pH sensitive hydrazone
bond to prepare prodrug 7. Heptamethine cyanine dye 5 was
attached to the prodrug 7 via an amide bond to prepare NIRF
prodrug 8. Micellization of the above NIRF prodrug into water
leads to the formation of NIRF DDS, with both drug delivery
and diagnostic abilities. Combining both the pH-responsive drug
release ability and the near infrared optical properties, the
material synthesized here can be a paradigm for future thera-
nostic agent synthesis, while the conjugation of nanoparticles
with hydrophobic cyanine dye can be a useful tip for future
fluorescent NIRF nanoparticle synthesis.
Results and discussion
Dye synthesis
Detailed synthetic procedures of cyanine 4 have been reported
elsewhere.36 Inserting a rigid cyclohexenyl ring into the poly-
methine chain of cyanine was found to be a valuable method for
its functionalization, in that it both increases the dye stability and
provides a useful site for further functionalization.39 A chlorine
atom located on the meso-position of the central ring could be
substituted by various nucleophilic regents such as amine,38,40
thiol41 and alcohol,36 while alcohol and thiol substitutions show
no significant influence on the optical properties. Amine substi-
tution leads to the formation of aminocyanine, which was found
to improve the dye stability and optical properties simulta-
neously.40 Both the absorption and emission of aminocyanine
shift to shorter wavelengths and a large Stokes shift was
observed. The large Stokes shift of the resulting aminocyanine
was assumed to be induced by intramolecular charge transfer
(ICT), which is also reflected by the break in the mirror image
relationship between the absorption and emitting curves. The
large Stokes shift minimizes self-quenching of these dye mole-
cules and helps to improve their quantum yield. The maximum
absorption and emission wavelengths of dye 4 are 780 and 815
nm in methanol, while they are 650 and 780 nm for dye 5. Blue
shifts of the absorption and emission could be attributed to the
ICT effect.
Combined NIRF cyanine dye with a responsive drug-conju-
gated nanoparticle can be a useful method for tumor theranosis.
With the aid of a NIRF cyanine dye, the accumulation behavior
of the nanoparticle could be conveniently monitored in vivo. Due
to the EPR effect directed nanoparticle accumulation, the
cyanine signal is also hoped to accumulate at the tumor site
during blood circulation. Tumor imaging could thus be realized
without using the usually complicated modified cyanine mole-
cules with tumor-targeting abilities.34–36 Cyanine dye can be
incorporated into the nanoparticle via physical encapsulation6 or
chemical conjugation,37,38 while chemical conjugation is a much
more suitable method since the separation of the two is no longer
possible because of a firmer linkage between them. Besides, non-
specific accumulation of free dye is also no longer possible and
information related with tumor diagnosis form the fluorescent
nanoparticle is much more reliable. Both hydrophobic and
hydrophilic cyanine molecules could be used for nanoparticle
conjugation; hydrophilic cyanine dye usually contains multi-
sulfonic acid groups to improve its water solubility, and the
purification of those high polar molecules was usually very
difficult while the yields remained low. On the other hand,
hydrophobic dyes are much easier to obtain with satisfactory
yields, usually via normal phase column chromatography. Thus,
labeling with hydrophobic cyanine molecules may be a more
promising method for tumor imaging.
The obtained cyanine 4 and 5 were thoroughly characterized,
and mass spectra of 4 and 5 were shown in Fig. S1 and S2.† In
both cases the related molecular cation peak [MꢀBrꢀ]+ could be
observed.
Synthesis of hydrazine functionalized triblock copolymer
Triblock copolymer 2 was synthesized by sequential ring opening
polymerization of Asp-NCA and ZLLys-NCA with mPEG–
NH2, as shown in Scheme 2. To avoid side reactions associated
with NCA polymerization at elevated temperatures,42 the present
Here, we report the synthesis of a novel pH-responsive drug
delivery system with near infrared fluorescence (NIRF DDS) for
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Scheme 1 Endocytosis of the NIRF prodrug micelle and its subsequent pH triggered drug release.
signals corresponding to diblock copolymer 2, new chemical
shifts corresponding to the PZLLys moiety are observed.
Chemical shifts centered at 1.40 (–(CH2)3–Cbz) ppm correspond
to the side methylene chain of PZLLys; the carbobenzoxy group
of ZLLys could be observed at 5.02 (C6H5CH2–) and 7.25
(C6H5CH2–) ppm, which partially overlapped with the signals of
the PAsp moiety. The chemical composition of the triblock
1
copolymer could also be determined by H NMR. The integra-
tion ratio of peak a + b + c (corresponding to the side methylene
groups of PZLLys, Fig. 2) to peak f (corresponding to the
methylene group of the mPEG backbone) was calculated to be
170 : 60, which is also close to the theoretical value of 176 : 60,
indicating the formation of the targeted triblock copolymer.
GPC traces of both the diblock and triblock were shown in
Fig. 3, and a shift toward higher molecular weight (corre-
sponding to lower retention time) was observed after each
addition of monomer. Both the peaks were single and no
shoulder peak corresponding to the diblock copolymer could be
observed on the triblock copolymer trace, indicating that no
dead polymer chains were created during diblock copolymeri-
zation. The living nature of the polymerization could be attrib-
uted to the fact that the present polymerization was conducted at
low temperature (0 ꢁC). Side reactions were thus greatly
suppressed.42
Scheme 2 Synthesis of triblock copolymer 3 by sequential ring opening
polymerization.
polymerization was conducted at 0 ꢁC. Diblock copolymer
mPEG-PAsp 2 was obtained after stirring the solution at 0 ꢁC for
3 days. 1 mL solution was removed and the product was sepa-
1
rated from the solution for analysis. The H NMR spectrum of
the diblock copolymer (mPEG-PAsp) was shown in Fig. 1, and
chemical shifts corresponding to the mPEG moiety could be
observed at 3.23 ppm (CH3O–) and 3.50 (–CH2CH2O–) ppm.
Signals at 5.02 (C6H5CH2–) ppm and 7.28 (C6H5CH2–) ppm
could be assigned to the benzyl ester group of the PAsp moiety.
The protons of the side methylene group could be observed at
2.82 and 2.61 (–CH2–OBzl) ppm. The chemical composition of
the copolymer was determined by the integration ratio of the
respective proton peak. The integration ratio of peak c (methy-
lene group of mPEG backbone) to peak e (benzyl group of OBzl
group) was calculated to be 172 : 50, which is quite close to the
theoretical value 176 : 50, indicating the formation of the desired
diblock copolymer. To simplify the synthesis, ZLLys-NCA was
added after the consumption of Asp-NCA. The reaction was
stirred at 0 ꢁC for another 3 days. The 1H NMR spectrum of the
obtained triblock copolymer was shown in Fig. 2. Except for
Molecular weights and polydispersity indexes (PDI) of both
the diblock and triblock copolymer were evaluated by GPC, and
the results were listed in Table 1, entries 1 and 2, respectively.
Molecular weights calculated by 1H NMR integration were also
presented as references. In both cases a difference between the
two molecular weights could be observed, this is due to the
polystyrene standards used for molecular weight calibration.43
For the preparation of hydrazine functionalized triblock
copolymer 3, the side benzyl ester group of the PAsp moiety was
substituted by hydrazine dihydrochloride. Complete substitution
was confirmed by the 1H NMR spectrum shown in Fig. 4. Due to
the signal overlap of the OBzl group with the Cbz group of the
PZLLys moiety, direct observation of the disappearance of the
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Scheme 3 Synthesis of NIRF prodrug.
Fig. 1 1H NMR spectrum of diblock copolymer 1 in DMSO-d6.
Fig. 3 GPC traces of the diblock copolymer and triblock copolymer in
DMF; macroinitiator mPEG–NH2 was also presented as a reference.
Table 1 Molecular weights and polydispersity indexes of the obtained
diblock and triblock copolymer
Sample
MNn MRa
MGn PCb
Mw/MGn PC
mPEG44-PAsp10
mPEG-PAsp10-PZLLys10
3952
6572
5458
9459
1.04
1.10
a
b
Molecular weight determined by 1H NMR integration. Molecular
weight determined by GPC.
characteristic signals of the benzyl ester group is not possible.
However, the substitution could be evaluated by integration into
1
the H NMR spectrum. The integration ratio of peak h (benzyl
Fig. 2 1H NMR spectrum of triblock copolymer 2 in DMSO-d6.
This journal is ª The Royal Society of Chemistry 2012
group of the PZLLys moiety) to peak d (methylene group
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Fig. 4 1H NMR spectrum of hydrazine functionalized triblock copoly-
mer 3 in DMSO-d6.
adjacent to Cbz protected amine group of PZLLys) was 5 : 2
after reaction, which corresponds to the composition of PZLLys,
indicating the full removal of OBzl from the side chain of the
triblock copolymer.
Fig. 6 Absorption (solid line) and emission (dashed line) curves of the
NIRF prodrug micelle solution (0.8 mg mLꢀ1). (a) Excited at 480 nm. (b)
Excited at 650 nm; the excitation and emission slit widths were 5 and
15 nm, respectively. The insets of b are images of the NIRF prodrug
micelle solution and its fluorescent image upon excitation at 670 nm.
Synthesis of prodrug 7
Doxorubicin was attached to the side chain of the above
hydrazine functionalized triblock copolymer by a condensation
reaction between the two, shown in Scheme 3. The condensation
of the carbonyl group with the hydrazine group yields drug–
and this may be due to the fact that a low drug content (8.6%,
weight ratio) was conjugated. Quantification of the drug conju-
gation ratio was thus evaluated by fluorospectrophotometry
after treating the NIRF prodrug solution with 2 M concentrated
hydrochloride aqueous solution after 1 h. The drug conjugated to
polymer conjugates via
a pH sensitive hydrazone bond.
However, the 1H NMR spectrum of the prodrug is poorly
resolved, and proton signals corresponding to the doxorubicin
moiety are not visible in the 1H NMR spectrum of the prodrug,
even at a sample concentration of 10 mg mLꢀ1 (data not shown),
Fig. 5 1H NMR spectrum of NIRF prodrug 8 with 10% and 50% dye substitution in DMSO-d6.
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Table 2 Quantum yields of the NIRF prodrug at different degrees of
aminocyanine substitution
Substitution
10%
25%
50%
Ff
0.0250
0.0199
0.0074
Fig. 9 Toxicity of copolymer 3, prodrug 7 and NIRF prodrug 8 against
A549cells. A549cells wereincubatedwith the respective materialsfor 72 h.
Synthesis of NIRF prodrug 8
For the labeling of macromolecules with cyanine, several
methods have been reported in the literature. Direct nucleophilic
substitution of the meso-position chlorine atom on the central
ring with the amine group of the macromolecules is a straight-
forward method,37,38 but the validity of this method depends not
only on the steric factors resulting from the bulky size of cyanine
molecules and macromolecules but also on the nucleophilicity of
the amine group. Here in this work, in order to facilitate the
labeling, the obtained cyanine dye 5 was first reacted with HOSu
to form an activated ester, then prodrug was added to prepare
1
the NIRF prodrug.44 The H NMR spectrum of the obtained
fluorescent prodrug was shown in Fig. 5. At a low dye attach-
ment (10% molar ratio), signals corresponding to the dye moiety
are barely visible, while when a higher amount (50% molar ratio)
of dye is used for labeling, signals of the dye molecules are
partially discernable. Signals corresponding to the benzyl group
of the dye could be observed at 8.0 ppm, while signals corre-
sponding to the polymethine chain could be observed at 5.9 and
7.5 ppm (overlap with signals of the dye benzyl group).
Fig. 7 Size and size distribution of the NIRF prodrug micelle solution
(1.0 mg mLꢀ1) determined by (a) DLS and (b) TEM. The micelle solution
was prepared by first dissolving the material and then dialysis against
water.
Optical properties
UV-Vis and fluorescent spectra of the NIRF prodrug aqueous
solution were shown in Fig. 6. Characteristic absorption peaks
corresponding to the drug and dye moieties could be observed at
488 and 662 nm, while emission peaks corresponding to the drug
and the dye were observed at 587 and 725 nm in Fig. 6a and b,
respectively. The insets of Fig. 6b show images of the NIRF
prodrug micelle solution and its fluorescence image upon exci-
tation at 670 nm. The original light purple solution has strong
fluorescence in the near infrared range, confirming its potential
applications in the area of bioimaging. Cyanine dye is known to
undergo aggregation in both organic and aqueous solutions.
Aggregation of the dye molecules either leads to a blue or a red
shift of the absorption, depending on H or J form aggregates.45
In Fig. 6, no absorption other than 662 nm and 488 nm could be
observed, indicating that no J and H form aggregation took
place. This may be due to a lower number of dye molecules being
used for labeling, and the restriction of these molecules to the
polymer backbone by chemical bonds, and the chances of
Fig. 8 Drug release behavior of the NIRF prodrug under neutral
(pH ¼ 7.2) and acidic (pH ¼ 5.0) conditions.
the polymeric chain was determined to be 8.6% (weight ratio),
indicating that each polymeric chain contains one doxorubicin
molecule on average. We also found that overfeeding of the drug
does not markedly improve drug conjugation but complicates the
final product purification, while when a 1 : 1 feed ratio was used,
the reaction was qualitative and the purification could be simply
accomplished by dialysis.
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Fig. 10 In vitro cellular uptake studies of (a) NIRF prodrug, (b) free doxorubicin at 1 h, 4 h and 8 h. (c) In vitro cellular uptake study of the NIRF
prodrug at 24, 48 and 72 h. Nuclei: stained blue with DAPI; green: F-actin; red: doxorubicin; yellow: aminocyanine (the original red fluorescence of
cyanine was changed to yellow to differ from the fluorescence of doxorubicin).
aggregation were thus greatly reduced. Aminocyanine 5 is
hydrophobic, and it is worth noticing that the excitation wave-
length of the NIRF prodrug is 662 nm and not in the near
infrared range (700–900 nm) in the strictest sense. However, this
does not imply that the bioimaging ability of the NIRF prodrug
will be markedly reduced, because commercially available NIR
dye Cy 5.5, which is widely used for bioimaging, has maximum
absorption and emission values of 673 and 692 nm in water.
Quantum yields of NIRF prodrugs with different degrees of
dye substitution were listed in Table 2. With the increasing
percentage of dye attached, the quantum yield decreases. This
may be due to the self-quenching of dye molecules via FRET at a
higher degree of dye attachment.46
The results were shown in Fig. 8. Under neutral conditions, slow
drug release was observed in the first 30 h, with no further release
during the following 50 h. The total drug release was less than
40% (37%) in the 80 h period, while under acidic conditions an
obvious acid promoted drug release behavior was observed. Fast
and sustained drug release could be observed during the whole
time range, with 80% drug release during the 80 h period.
Accelerated drug release may be due to the cleavage of the
hydrazone bond under acidic conditions, leading to the release of
the conjugated drug.
In vitro cytotoxicity studies
In vitro toxicity of NIRF prodrug 8 was evaluated by a MTT
method against the A549 cell. The cell toxicity of copolymer 3
and prodrug 7 were also evaluated as references (Fig. 9). For
copolymer 3, its toxicity upon the A549 cell is low; 80% of the
cells were alive after 72 h incubation at a concentration of 0.25
mg mLꢀ1, while for prodrug 7 and NIRF prodrug 8 the toxicity
is much more pronounced and increases steadily with concen-
tration of the materials. Only 11% of the cells were alive after
72 h incubation at a concentration of 0.25 mg mLꢀ1 for both the
NIRF prodrug and the prodrug. Since the difference between
copolymer 3 and prodrug 7 is the conjugated doxorubicin, the
toxicity of the prodrug could be attributed to conjugated doxo-
rubicin. For the NIRF prodrug and the prodrug, their toxicity
Size and size distribution of the obtained nanogel
Size and size distribution of the NIRF prodrug micelle was
determined by a combination of TEM and DLS. The results were
shown in Fig. 7a and b, respectively. The diameter of the NIRF
nanoparticle had an average value of 238 nm, while in the TEM
image the diameter of the micelle is an approximate size of
around 250 nm.
Drug release behavior of the NIRF prodrug
In vitro drug release behavior of the NIRF prodrug was evalu-
ated under acidic (pH ¼ 5.0) and neutral (pH ¼ 7.2) conditions.
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upon the A549 cell was similar, indicating that the amino-
cyanine’s toxicity on the A549 cell is negligible.
drug cleavage from the NIRF prodrug was confirmed by in vitro
drug release, and further evaluation of the material on A549 cell
reveals a gradual drug cleavage and its subsequent migration into
the nucleus. With the aid of the NIRF label, the drug–polymer
separation process could be conveniently and clearly observed
during the 72 h incubation. Due to the excellent drug release
behavior of the NIRF prodrug and its unique near-infrared
fluorescence properties, the present material has potential
applications in biomedical areas as a theranostic agent.
Cellular uptake studies
To analyze the internalization and subcellular localization of the
NIRF prodrug, cultured A549 cells was treated with the NIRF
prodrug (46 mg mLꢀ1) or free doxorubicin (3.3 mg mLꢀ1) of
equivalent doxorubicin concentration for 72 h, followed by
observation of the cell under confocal laser scanning microscope
at different time points (1 h, 4 h, 8 h, 24 h, 48 h and 72 h). To
localize the NIRF nanoparticle, the cytoskeleton F-actin was
labeled using Alexa 488-phalloidin and the cell nuclei were
stained with DAPI. The results were shown in Fig. 10. For cells
incubated with free doxorubicin, the fluorescence of doxorubicin
at the nucleus was evident even after 1 h of incubation, while
almost no signal from the cytoplasm could be observed
(Fig. 10b). For the NIRF prodrug, quite distinctive drug trans-
portation and distribution phenomena were observed. The drug
signal was dispersed throughout the cell and the signal intensity
increases gradually from 1 h to 8 h (Fig. 10a). Since the amino-
cyanine was conjugated to the NIRF prodrug, the location of the
polymeric matrix could be determined by the location of the dye
molecules. The gradual increase in the fluorescence intensity of
cyanine in the cytoplasm and its overlap with the signal from
doxorubicin indicate the gradual endocytosis of the NIRF pro-
drug. No cyanine fluorescence could be detected in the nucleus,
indicating that the polymeric matrix could not enter the nucleus.
In order to testify the pH-responsive nature of the NIRF prodrug
and confirm its gradual drug release ability via pH regulation, the
cell culture media containing the NIRF prodrug was replaced
with blank culture media after 8 h (in order to eliminate inter-
ference from the continued endocytosis NIRF prodrug). The
results after 8 h are shown in Fig. 10c. After 24 h incubation, the
primarily dispersed doxorubicin fluorescence intensity becomes
centralized around the nucleus; the lower fluorescence intensity
from the nucleus indicates the migration of the released drug
from the NIRF prodrug to the nucleus, while the signal from
aminocyanine could only be observed around the nucleus, con-
firming the drug release properties of the NIRF prodrug.
Doxorubicin fluorescence in the nucleus became stronger as time
went by. After 72 h incubation, almost no drug signal could be
detected in the cytoplasm. Full drug cleavage from the NIRF
prodrug had taken place and almost all the drug molecules had
migrated into the nucleus, while the polymer matrix remained in
the cytoplasm, as indicated by the yellow color from amino-
cyanine around the nucleus. For free doxorubicin its distribution
does not change much after 8 h, and the drug was mainly
detected in the nucleus (Fig. S3†). It is worth noticing that the
near infrared fluorescence properties of the NIRF polymer do
not change too much after 72 h incubation, indicating the
potential application of this material as a theranostic agent.
Experimental
Materials
All the chemicals with AR purity, unless specifically indicated,
were purchase from Sinoreagent corporation. n-Hexane and
tetrahydrofuran (THF) were first refluxed with CaH2 overnight
followed by distillation. N,N-Dimethylformamide (DMF) was
dried over CaH2 at room temperature for 24 h before vacuum
distillation. Hydrazine dihydrochloride, triphosgene, doxoru-
bicin hydrochloride, N-hydroxysuccinimide (HOSu), N,N0-dicy-
clohexyl carbodiimide (DCC), diisopropylethylamine (DIEA),
2,3,3-trimethylbenzoindolenine, 3-bromopropionic acid, b-
benzyl L-aspartate, and 3-carbobenzoxy-L-lysine were purchased
from Aladdin Corporation, China. Dialysis bag (cutoff Mw
¼
8000) was obtained from Bomei biotechnology corporation,
China. mPEG (Mw ¼ 1900) were purchased from Aldrich and
used as received without further purification. Milli-Q water (18.2
MU) was prepared using a Milli-Q Synthesis System (Millipore,
USA). Normal phase column chromatography was performed
using 200–300 mesh silica gel (Yantai institute of chemical
engineering, China).
Characterization
1H NMR spectra were measured on a Bruker AC 300 spec-
trometer. Deuterated dimethyl sulfoxide (DMSO) or deuterated
chloroform containing 0.03 vol% tetramethylsilane (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. Molecular weights of the samples were
determined by Gel Permeation Chromatography (GPC) equip-
ped with two columns (one Shodex GPC KD-804 column and
one guard column) and a refractive index detector (RID-10A).
DMF was used as the mobile phase and the measurement was
performed at 60 ꢁC at a sample concentration of 3 mg mLꢀ1
.
Monodispersed polystyrene standards were used for the cali-
bration of Mn, Mw and Mw/Mn. Transmission electron micros-
copy measurements were performed on a JEOL-2010 microscope
with an accelerating voltage of 200 kV. Size and size distribution
of the micelle solution were determined by dynamic light scat-
tering (DLS) carried out on a Malvern Zetasizer Nano ZS90 with
a He–Ne laser (633 nm) and 90ꢁ collecting optics. Measurements
were performed at room temperature and the data was analyzed
by Malvern Dispersion Technology Software 4.20. Fluorescence
measurements were carried out on a Shimadzu RF-5301PC
Fluorescence spectrophotometer with excitation and emission
slit widths of 5 and 15 nm, respectively. UV-Vis spectra were
Conclusions
A near infrared fluorescent prodrug with pH-responsive drug
release ability was designed and developed. The obtained NIRF
prodrug has strong fluorescence in the near infrared range and
could release the conjugated drug via pH regulation. Sustained
obtained on
a
Shimadzu UV-2401 PC Ultraviolet
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60 000
spectrophotometer. ESI mass spectra were obtained on a LCQ
Advantage MAX Spectrometer using methanol solutions of the
sample.
C58H72N3O6, 906.4; found, 906.5 [M–Brꢀ]+.
3
¼
M
ꢀ1$cmꢀ1, FF ¼ 0.64. Rf ¼ 0.83 (MeOH : EtOAc ¼ 1 : 6).
The molar extinction coefficient of the sample was determined
by Beer’s law, based on three separate measurements. The rela-
tive fluorescence quantum yield was determined using the
following equation:47
Synthesis of LLys-NCA
LLys-NCA was prepared following a literature method.49 In
brief, nepsilon-carbobenzoxy-L-lysine (2.0 g, 7.1 mmol) was
suspended in 50 mL dry THF, followed by the addition of a THF
solution of triphosgene (2.1 g, 7.1 mmol). The obtained suspen-
sion was stirred at 45 ꢁC for 2 h, followed by filtration to remove
traces unreacted nepsilon-carbobenzoxy-L-lysine, and the filtrate
was collected and crystallized three times from a mixture of THF
and hexane to give the anhydride as white crystals (1.6 g, 73%
yields).
1H NMR (300 MHz, CDCl3, d, ppm): 7.48 (s, 5H), 6.75 (s,
1H), 5.42–5.13 (m, 2H), 5.01 (s, 1H), 4.40 (s, 1H), 3.32 (d, J ¼ 5.5
Hz, 2H), 2.23–1.40 (m, 6H).
13C NMR (300 MHz, DMSO, d, ppm): 171.8, 156.1, 152.0,
137.3, 128.4, 127.8, 65.1, 57.0, 41.3, 31.1, 30.6, 28.7. FT-IR (KBr,
thin film, cmꢀ1): 1785, 1854.
FF(x) ¼ (As/Ax)(Fx/Fs)(nx/ns)2FF(s)
Where F, A and n refer to the quantum yield, absorption and
refractive index of the solvents used. Fx stands for the area under
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.48
Synthesis of parent cyanine dye 4
This material was prepared in 68% yield according to a literature
method.36 In brief, 1-hydroxycarbonylethyl-2,3,3-trime-
thylbenzoindoleninium bromide (0.42 g, 1.16 mmol) and 2-
chloro-1-formyl-3-(hydroxymethylene)cyclohex-1-ene (0.10 g,
0.58 mmol) were dissolved into a mixed solvent of benzene
(15 mL) and n-butanol (35 mL) in a two-necked flask fitted with a
Dean–Stark trap and a condenser. The mixture was heated to
110 ꢁC under a nitrogen atmosphere for 16 h, after which the
solvent was removed under vacuum. The obtained green solid
was washed with ether and purified by normal phase column
chromatography, with a gradient elution from EtOAc to EtOAc/
methanol (5 : 1). Removing the solvent after column chroma-
tography yields 0.35 g dye 3 (68% yields) as a deep green solid.
1H NMR (300 MHz, DMSO, d, ppm): 8.34 (dd, J ¼ 22.4, 11.1
Hz, 4H), 8.09 (t, J ¼ 8.2 Hz, 4H), 7.77 (d, J ¼ 8.7 Hz, 2H), 7.58
(ddd, J ¼ 23.5, 15.4, 7.8 Hz, 4H), 6.40 (t, J ¼ 20.7 Hz, 2H), 4.63
(s, 4H), 3.96 (t, J ¼ 6.1 Hz, 4H), 2.84 (d, J ¼ 38.6 Hz, 8H), 1.95 (s,
16H), 1.57–1.32 (m, 4H), 1.20 (dd, J ¼ 14.2, 6.9 Hz, 4H), 0.76 (t,
J ¼ 7.1 Hz, 6H). MS (m/z, ESI): calcd for C52H60ClN2O4, 811.3;
found, 811.4 [MꢀBrꢀ]+. 3 ¼ 183 333 Mꢀ1$cmꢀ1, FF ¼ 0.082.
Rf ¼ 0.75 (MeOH : EtOAc ¼ 1 : 6).
Synthesis of Asp-NCA
This material was synthesized according to a similar method with
ZLLys-NCA of 75% yield.
1H NMR (300 MHz, CDCl3, d, ppm): 7.45–7.3 (5H, m, Ar–H),
6.2 (1H, s, NH), 5.2 (2H, s, CH2), 4.61(1H, t, CH) and 2.85 (2H,
t, CH2). FT-IR (KBr, thin film, cmꢀ1): 1782, 1850.
Synthesis of triblock copolymer 2
To a flame dried and argon purged Schlenk tube, 0.68 g (0.36
mmol) mPEG–NH2 was added under argon, followed by the
addition of 10 mL dry DMF to solubilize the macroinitiator. The
obtained solution was precooled in an ice-water bath. A DMF
solution (5 mL) containing 0.90 g (3.60 mmol) Asp-NCA was
added to theꢁmacroinitiator solution. The combined solution was
stirred at 0 C for 3 days. Periodical removal of the generated
carbon dioxide was realized by connection of the flask to a
vacuum line. 1 mL reaction solution was removed at the end of
polymerization for analysis, and a DMF solution containing 1.03
g (3.36 mmol) ZLLys-NCA was subsequently added. The
obtained mixture was stirred for another 3 days at 0 ꢁC, followed
by precipitation into 100 mL cold ether to separate the product.
The obtained white solid was redissolved in 10 mL DMF and
further purification was performed by extensive dialysis against
water. Removing water by freeze-drying gives 2.0 g product as a
white solid (90% yield).
Synthesis of aminocyanine dye 5
Cyanine dye 4 (150 mg, 0.17 mmol) and 6-aminocaproic acid (30
mg, 0.23 mmol) were added to 5 mL dry DMF, followed by the
addition of 29 mL DIEA (22 mg, 0.17 mmol). The obtained
solution was heated to 70 ꢁC and stirred in the dark for 8 h. The
solvent was then removed under vacuum, and the obtained deep
blue solid was washed with ether and further purified by a
column chromatography method with gradient elution from
EtOAc to EtOAc/methanol (5 : 1). 100 mg (59% yield) deep blue
solid was obtained after removing the solvent under vacuum.
1H NMR (300 MHz, DMSO, d, ppm): 8.13 (t, J ¼ 13.0 Hz,
2H), 7.96 (dd, J ¼ 8.8, 6.1 Hz, 4H), 7.70 (d, J ¼ 12.1 Hz, 2H),
7.56 (dd, J ¼ 17.2, 8.5 Hz, 4H), 7.38 (dd, J ¼ 16.3, 9.1 Hz, 2H),
5.84 (d, J ¼ 12.9 Hz, 2H), 4.37 (s, 4H), 3.94 (t, J ¼ 6.6 Hz, 4H),
3.76 (s, 2H), 2.78 (t, J ¼ 6.9 Hz, 4H), 2.56 (4H, overlap with the
solvent signal), 2.27 (t, J ¼ 7.2 Hz, 2H), 1.95–1.71 (m, 18H),
1.70–1.55 (m, 2H), 1.47–1.36 (m, 4H), 1.26–1.14 (m, 4H), 0.94–
0.83 (m, 2H), 0.76 (q, J ¼ 7.3 Hz, 6H). MS (m/z, ESI): calcd for
Synthesis of hydrazine functionalized triblock copolymer 3
For the preparation of hydrazine functionalized triblock copol-
ymer 3, 0.25 g (0.037 mmol) triblock copolymer 2 was dissolved
into 5 mL dry DMF, followed by the addition of 0.15 g (1.43
mmol) hydrazine dichloride and 0.50 mL (2.90 mmol) DIEA.
ꢁ
The obtained suspension was stirred at 45 C for 36 h to get a
clear solution, and purification was performed by extensive
dialysis of the DMF solution against 250 mL water for 48 h. The
water was changed every 12 h. Removal of the water by
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freeze-drying gives 0.21 g (0.035 mmol) product as a white solid
(94% yield).
The prodrug and NIRF prodrug micelle solutions were prepared
by first dissolving the material into DMF and then removing the
organic solvent by dialysis. A549 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 (FBS) was used as the cell
Synthesis of prodrug 7
ꢁ
For the preparation of prodrug 7, 50 mg (8.4 mmol) hydrazine
functionalized triblock copolymer 3 was dissolved into 5 mL
DMF. 4.8 mg doxorubicin hydrochloride (8.3 mmol) was then
added to the solution. The obtained red solution was stirred at
45 ꢁC in the dark for 36 h. The obtained DMF solution was
sealed into a dialysis bag and further purification was achieved
by dialysis. Removal of the water after dialysis by freeze-drying
gives 40 mg product as a red powder (75% yield). For the
determination of the drug conjugation content, 150 mL (1 mg
mLꢀ1) prodrug solution was added to 10 mL concentrated 2 M
hydrochloride aqueous solution, and the obtained solution was
stirred at room temperature for 1 h to cleave the hydrozone
bond. The drug concentration was determined using a fluo-
rospectrophotometer at an emission wavelength of 557 nm, with
an excitation wavelength of 480 nm and excitation and emission
slit widths of 5 and 10 nm. The drug conjugation content (defined
as the ratio of drug weight to prodrug weight) was calculated to
be 8.6%.
culture medium. After incubating at 37 C for 24 h under a 5%
CO2 atmosphere, the culture medium was replaced with a series
of material solutions (in 1640 media containing 10% FBS),
ranging from 0 to 0.25 mg mLꢀ1. Then the cells were incubated at
37 ꢁC for 3 days, and the culture medium 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 mLꢀ1). 100 mL
extraction buffer (20% SDS in 50% DMF, pH 4.7, prepared at
ꢁ
37 C) was added to each well after an additional incubation of
2 h. The obtained solution was incubated overnight, and the
absorbance of the solution was measured at 490 nm using a Bio-
Rad 680 microplate reader. The cell viability was normalized to
A549 cells cultured in the complete culture medium.
Cellular uptake study
A549 cells (7 ꢂ 104 cells per well) were seeded on coverslips in 24-
well plates and incubated for 24 h. The NIRF prodrug was added
to a final concentration of 46 mg mLꢀ1 and incubated with the
cells for 8 h, after which the culture medium with the NIRF
prodrug was replaced with a blank culture medium without the
NIRF prodrug, and the incubation was continued for another
64 h. Free doxorubicin was added to a concentration of 3.3 mg
mLꢀ1 (equivalent to the drug concentration of the NIRF pro-
drug) and treated by the same method. After removing the
medium, the cells were washed twice with cold phosphate buff-
ered saline (PBS, pH 7.4, 0.01 M) and fixed with 4% formalde-
hyde (Sigma Aldrich, St. Louis, USA). To label the cytoskeleton,
the cells were incubated with Alexa Fluor 488-phalloidin (Invi-
trogen, Carlsbad, USA) at 25 ꢁC for 20 min (after incubation
with 1 mg mLꢀ1 bovine serum albumin (BSA) in PBS to decrease
nonspecific interactions) followed by rinsing with PBS. To label
the nucleus, the cells were incubated with 1 mg mLꢀ1 DAPI
(Beyotime, Haimen, China) for 5 min in PBS, followed by
extensive rinsing with PBS. The slides were mounted with one
drop of Fluoromount Aqueous Mounting Medium (Invitrogen,
Carlsbad, USA) and observed using a Zeiss LSM 710 laser
confocal scanning microscope imaging system (Heidenheim,
Germany) with an upright confocal microscope and an excita-
tion wavelength of 633 nm.
Synthesis of NIRF prodrug 8
Aminocyanine dye 5 (5.0 mg, 5.1 mmol) was dissolved into 1 mL
dry DMF solution, followed by the addition of 1 mL DMF
solution containing 0.69 mg HOSu (6.0 mmol) and 1.5 mg DCC
(7.3 mmol). The obtained mixture was stirred in the dark for 24 h
at room temperature. 90 mL of the above dye solution was
removed and added dropwise to a stirred solution of prodrug 7
(30 mg, 4.6 mmol) in DMF. The combined solution was stirred
overnight, followed by precipitation into cold ether to separate
the NIRF prodrug. Further purification was performed by
dialysis of a DMF solution of the NIRF prodrug in the dark.
Removal of the water after dialysis by freeze-drying gives 28 mg
product as a grey solid (81% yield). The NIRF prodrug with 25%
and 50% dye substitution (molar ratio) was prepared in a similar
manner.
In vitro drug release
Drug release behavior of the NIRF prodrug was evaluated under
both acidic and neutral conditions. For the neutral conditions,
400 mL NIRF prodrug solution (1 mg mLꢀ1) was sealed into a
dialysis bag, which was then immersed into 50 mL 0.2 M phos-
phate buffered saline (PBS) solution; release experiment was
conducted at 37 ꢁC on a shaking bath. 2 mL sample was removed
at predetermined time points, and 2 mL fresh PBS was added at
the same time. Drug release was quantified by using a fluo-
rospectrophotometer at an emission wavelength of 557 nm, with
an excitation wavelength of 480 nm. For acidic conditions, a
similar method was employed, except that 0.2 M acetate buffer
was employed as the release media.
Acknowledgements
We would like to thank the National Natural Science foundation
of China (no. 20874095 and no. 50173147) for the financial
support. Professor Jun Wang of USTC is greatly appreciated for
both his beneficial discussion with us and his generous help.
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