Folic acid targeted pH-responsive amphiphilic polymer nanoparticles conjugated with near infrared fluorescence probe for imaging-guided drug delivery

Article abstract of DOI:10.1039/c6ra05657a

A novel folic acid targeted pH-responsive amphiphilic polymer conjugated with near infrared (NIR) probe has been synthesized by the combination of reversible addition-fragmentation chain transfer (RAFT) polymerization of 2-(N-tert-butoxycarbonylamino)ethyl methacrylate (BEMA) and oligo(ethylene glycol)methacrylate (OEGMA), ring-opening polymerization of N-carboxy anhydride (NCA) and click reaction. After assembling to micelles, the polymeric nanoparticles has pH-responsive properties for delivery encapsulated drug into the tumor cell via a rapid process of expansion and subsequent disassembly. Folic acid linked to the amino groups on the branched chains would promote the endocytosis of the polymeric nanoparticles by the cells leading the improvement of the effect of doxorubicin (DOX). In vitro experiments on both MCF7 and HepG2 cells revealed the as-prepared polymer might be a potential candidate for future theranosic against cancer.

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Folic Acid Targeted pH-Responsive Amphiphilic Polymer
Nanoparticles Conjugated with Near Infrared Fluorescence Probe
for Imaging-guided Drug Delivery
Received 00th January 20xx,
Accepted 00th January 20xx
Liyi Fu, Le Liu, Zheng Ruan, Houbing Zhang and Lifeng Yan*
DOI: 10.1039/x0xx00000x
A novel folic acid targeted pH-responsive amphiphilic polymer conjugated with near infrared (NIR) probe has been
synthesized by the combination of reversible addition-fragmentation chain transfer (RAFT) polymerization of 2-(N-tert-
butoxycarbonylamino)ethyl methacrylate (BEMA) and oligo(ethylene glycol) methacrylate (OEGMA), ring-opening
polymerization of N-carboxy anhydride (NCA) and Click Reaction. After assembling to micelles, the polymeric nanoparticles
has pH-responsive properties for delivery encapsulated drug into the tumor cell via a rapid process of expansion and
subsequent disassembly. Folic acid linked to the amino groups on the branched chains would promote the endocytosis of
the polymeric nanoparticles by the cells leading the improvement of the effect of doxorubicin (DOX). In vitro experiments
on both MCF7 and HepG2 cells revealed the as-prepared polymer might be a potential candidate for future theranosic
against cancer.
www.rsc.org/
results. Here, we change our strategy to another target group
to make our design of multiblock polymer applicable for more
cancer cells (Scheme 1). Folic acid (FA), whose folate receptor
Introduction
Multifunctional nanomaterials, especially focused on targeting,
stimuli-response and bioimaging, attracted great attention and
have been widely studied recently.1-3 Plenty of polymers,
liposomes or some other nanoparticles were synthesized and
characterized to fulfill various demands of targeted delivery,4,5
stimuli-responsive imaging,6,7 targeted imaging,8,9 etc, for the
treatment of cancer. Multifunctional nanomaterials and
diversified methods improve our comprehension on cancer,
and the so-called “theranostic” indicates the importance of the
early stage diagnosis and the subsequent directed therapy. So
development of novel multifunctional nanomaterial will
overcome limitations with conventional diagnosis and therapy
and hold great promise for the future of cancer treatment.10,11
Targeted polymeric drug delivery system has attracted much
attention for it can enhance the enrichment of the polymeric
drug nanoparticles in tumor and endocytosis in cells. However,
the bioimaging of the target process during the drug delivery is
still scare. It is very interesting to develop new imaging-guided
targeted polymeric drug delivery system for new theranostics.
In our previous work, a galactose targeted pH-responsive
is overexpressed on the surface of many tumor cells, is
considered to be a satisfactory target group against breast
cancer, kidney cancer, liver cancer, and ovarian cancer
cells,13,14 while in normal tissues, these receptors have limited
distribution.15 The therapeutic efficacy of folate receptors has
been proven by selective accumulation into tumor cells by
folate receptor-mediated endocytosis.16
Scheme 1. Diagrammatic sketch ligand targeted polymer conjugated with NIR probe,
the micellization with DOX, selective accumulation in cancer cell, and the imaging of
the endocytosis of the micelle with subsequent pH triggered drug release
copolymer conjugated with
a cyanine dye had been
synthesized for bioimaging and drug delivery, which showed
efficient drug delivery behavior for the liver cancer cells
(HepG2).12 We used galactose to target and obtained satisfied
In addition, polypeptides have been studied for decades to
construct multiblock amphiphilic copolymers.17-20 Based on its
excellent characters of biocompatibility and biodegradability,
polypeptides are widely used in the field of nano-
pharmaceutical materials. Polypeptides synthesized by the
ring-opening polymerization (ROP) of α-amino acid N-
carboxyanhydrides (NCAs) have a satisfactory polydispersity
index (PDI) which could be controlled within 1.1 leading a
CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for
Physical Sciences at the Microscale,, iChEM, University of Science and Technology
of China. Jinzai Road 96#, Hefei, 230026, Anhui, P.R.China. Fax: (+86-551-
63606853); E-mail: (lfyan@ustc.edu.cn).
† Footnotes relating to the title and/or authors should appear here.
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narrow size distribution. Also, the side chain of α-amino acid of modification.
(lysine, aspartic acid, etc) offers different sites for the demands
Scheme 2. Synthesis of the Folic acid Targeted, pH-Responsive and Near-
Infrared Triblock Amphiphilic Polymer via Polymerization and Click Reaction.
against the hydrophobic one according to our works
before12,21,22. Here, an azido modified RAFT agent was used as
the initiator for the radical polymerization. 22 units of
Oligo(ethylene glycol) methacrylate (OEGMA) were
polymerized to it with the same function as the traditional
hydrophilic Polyethylene glycol (PEG). Subsequently, 7 units of
t-butyloxy carbonyl (Boc) protected ethanolamine modified
methacrylate (BEMA) were inserted between the
trithiocarbonate and POEGMA to offer a binding site for the
folic acid and fluorescence probe. The number of folic acid
conjugated to the polymer should not be too much. Overmuch
Here,
a novel triblock amphiphilic polymer has been
designed and synthesized by a combination of Reversible
Addition-Fragmentation Chain Transfer Polymerization (RAFT
Polymerization), ROP and the Click Reaction (Scheme 2). The
polymer is divided into two parts (the hydrophilic part and the
hydrophobic one) to synthesize separately. To insure the
water-solubility, the molecular weight of the hydrophilic part
should be large enough with a ratio between 1:1 and 2:1
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folic acid would increase the hydrophobicity of the polymer
and insufficient FA would decrease the effect of targeting. In
another way, 10% molar ratio of fluorescence probe was
linked to the polymer to get a high quantum yields avoiding
the self-quenching22. For the hydrophobic part, since the
benzyloxy group could be aminolyzed by hydrazine or amine
for functionalization, β-benzyl-L-aspartate was selected to be
polymerized to the initiator of propargylamine. Although the
hydrazone bond is widely used for the pH-responsive drug
delivery of doxorubicin, this style of chemical bonding has
poorer ability of drug loading and is less sensitive to the stimuli
compared to the physical encapsulation. According to this, the
strategy of the response mechanism was changed from
chemical bonding to physical encapsulation to fulfil the
demands of rapid stimuli-response and drug release. So here,
N,N-diisopropylethylenediamine was used for aminolyse, and
the alkalinity of tertiary amino responded to the weak acidic
microenvironment at the pH of about 6.3 in the amphiphilic
system. 19 units of β-benzyl-L-aspartate were polymerized to
propargylamine to ensure the ability of stimuli-response. If the
number was less than 10, the micelles wouldn’t change a little
against the variation of the pH in the environment. After
conjugation with Click Reaction, activated folic acid and a
cyanine dye which is a near infrared (NIR) fluorescence probe
were linked to the amino group on the block of PBEMA by
covalent bonds following the de-protection of the Boc group.
The doxorubicin was encapsulated accompanied with the
formation of micelles. In normal neutral condition, micelles
could keep a tight and stable structure and thus protect the
drug in the micelles core. When in the tumoral acidic
condition, the ionization of the tertiary amino group would
contribute to the swell or collapse of micelles and the
encapsulated doxorubicin would be released rapidly.23-25
Three kinds of polymers with NIR probe, namely, targeting
polymers (TPs), nontargeting polymers (NPs) and polymers
without drugs (PWDs) were synthesized and characterized.
Two kinds of cancer cells, MCF7 and HepG2, were selected to
study the effect of target and uptake of folic acid in different
tumor environments.
Figure 1. 1H-NMRand 13C-NMR spectra of 2-(N-tert-butoxycarbonylamino)ethan-1-ol in
CDCl3 (a, b), and 2-(N-tert-butoxycarbonylamino)ethyl Methacrylate in CDCl3 (c, d).
This monomer was synthesized by two steps (Scheme 3).
Since the final product needed to be purified by gel
chromatography, the intermediate material, 2-(N-tert-
butoxycarbonylamino)ethan-1-ol, was simply processed after
reaction without further purification. Figure 1 shows the
typical 1H-NMR and 13C-NMR spectra of the intermediate 2-(N-
tert-butoxycarbonylamino)ethan-1-ol and the final monomer,
and the peaks representing each hydrogen atom could be
found in (a) and those for carbon atoms in (b). Due to the
different chemical environment, the peaks of the cis form and
the trans form hydrogen atoms on the double bond split to
different chemical shifts, which were represented by a and a’
(Fig.1c). It was found that all the hydrogen atoms could be
corresponded to the peaks in the spectrum and the integration
equalled the number of the atoms. The 13C-NMR spectrum also
indicated the correct structure of the final product (Fig.1d).
Synthesis of the Hydrophilic Part
Figure 2a showed the 1H-NMR spectrum of POEGMA22 in
CDCl3. Since there were various hydrogen atoms in different
chemical environments, many peaks corresponding to
hydrogen atoms overlapped and could not be integrated
respectively. Some characteristic peaks representing different
parts were chosen to identify the structure and the degree of
polymerization. The peak at the chemical shift of 4.17 ppm
represented the hydrogen atoms f on the methylene just next
to the oxygen atom, whose ratio of integration to the peak b,
and the middle 18 hydrogen atoms of the dodecyl, was about
44:18, illustrating that 22 units of OEGMA were polymerized to
the RAFT initiator on the average.
Results and Discussion
Synthesis of the Monomer 2-(N-tert-butoxycarbonylamino)ethyl
Methacrylate
Due to the fact that the reactive activity of the monomer, 2-
(N-tert-butoxycarbonylamino)ethyl Methacrylate, was lower
than the OEGMA monomer, more feed ratio or longer reaction
time was needed for the polymerization. 15 times of BEMA
was used as the reagent, as shown in Fig.2b, the integration of
peak e+h was 58 which indicated that totally 29 units of BEMA
and OEGMA were conjugated to the RAFT agent, indicates only
7 units were inserted between the trithiocarbonate and units
Scheme 3. Synthesis of the monomer, 2-(N-tert-butoxycarbonylamino)ethyl
Methacrylate.
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of OEGMA during 1 day, which gave the final structure with
PBEMA7-b-POEGMA22.
Figure 2. 1H-NMR spectra of POEGMA22 (a), PBEMA7-b-POEGMA22 (b), PBLA19 (c),
PBEMA7-b-POEGMA22-b-PBLA19 (d), PBEMA7-b-POEGMA22-b-P(Asp-DIEDA)19 (e), and
folic acid conjugated polymer (f): (a) and (b) were measured in CDCl3 while (c)-(f) were
measured in DMSO-d6.
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chain was less than the actual number of 66. And in Fig.2b, this
peak became wider and the integration did not increase much
after the polymerization of BEMA.
Figure 3. FT-IR spectrum of PBEMA7-b-POEGMA22 and PBEMA7-b-POEGMA22-b-PBLA19
.
Synthesis of the Hydrophobic Part
Figure 2c displayed the 1H-NMR spectrum of the
propargylamine initiated poly β-benzyl-L-aspartate (PBLA). The
peak at 3.8 ppm corresponding to the hydrogen atoms on the
methylene next to the triple bond was selected as the
standard for the integration. The area of peak h and g which
representing the number of the benzyl group indicated that
about 19 units of the protected aspartic acid were polymerized
to the initiator.
Synthesis of PBEMA7-b-POEGMA22-b-PBLA19 by Click Reaction
The hydrophilic and hydrophobic parts were efficiently
conjugated by Click reaction. As shown in Fig.2d, the
characteristic peaks of PBLA19 at 5.0 and 7.2 ppm and those of
PBEMAy-b-POEGMA22 at 3.2 and 4.0 ppm could be observed
and the ratio of the integration indicated the same proportion
of the hydrophilic and hydrophobic segments. Its symmetrical
size distribution and no existence of any shoulder peak which
was displayed in Figure 4a illustrated the complete reaction of
the reagents, and the disappearance of the peak at 2100 cm-1
in the FT-IR spectrum (Figure 3) also demonstrated the
thorough completion of the reaction.
Figure 4. (a) GPC traces of (1, black) PBLA19, (2, red) POEGMA22, (3, blue) PBEMA7-b-
POEGMA22 and (4, green) PBEMA7-b-POEGMA22-b-PBLA19 in DMF measured by the RI
detector. (b) GPC traces of the final Cy conjugated polymer (2) by fluorescence detector
(Excited at 650 nm and Emitted at 790 nm compared with that by RI detector (1).
Addition of Folic acid, Cyanine Dye and Doxorubicin
Figure 2e showed the 1H-NMR spectrum after the de-
protection of the Boc group and the addition of folic acid. The
disappearance of the peak at 1.45 ppm indicated the
successful leaving of Boc group, and the integration of peak p,
l, m and q illustrated that about 6 folic acids were conjugated
to the terminal amino groups leaving one for the linkage with
Cy.
Aminolysis of the Triblock Polymer
Due that little cyanine dye and doxorubicin were conjugated
and encapsulated respectively to the polymer, hardly any
peaks of them could be found in the 1H-NMR spectrum. In
order to demonstrate the conjugation of Cy, GPC with
fluorescence detector was used to characterize the product. As
shown in Figure 4b, the traces representing the cyanine dye
have the same elution time with the triblock polymer 4, which
indicated that Cy had been successfully conjugated to the final
polymer.
To ensure the completion of aminolysis, excess N,N-
diisopropylethylenediamine was used as the reagent to de-
protect the benzyl group. As shown in Fig.2d, the
disappearance of the peak at 5.0 and 7.2 ppm and the rise of
the peak at 0.97 ppm indicated the successful conversion from
benzyl group to terminal tertiary amino group. However, due
to some unknown reason for the macromolecular polymer, it
seemed that the integration of the methyl group was not very
accurate. The integration of peak k was only about 126, which
was less than the actual number of the hydrogen atoms. The
same phenomenon appeared in the 1H-NMR spectra of
POEGMA22 and PBEMA7-b-POEGMA22. In Fig. 2a, the
integration of peak e representing the methyl on the main
Polydispersity Index and Size Distribution
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Table 1. Molecular weights and polydispersity indexes of the polymers.
The polydispersity index and size distribution were measured
by GPC and DLS in Fig.4 and Fig.S1, respectively (Table 1). As
NMR
GPC
Sample
POEGMA22
Mn
Mn
Mw/Mn (PDI)
1.12
11485 12681
13089 14821
shown in Fig.4a, all the intermediate polymers had
a
PBEMA7-b-POEGMA22
PBLA19
1.16
satisfactory polydispersity index without any shoulder peaks
indicating that all of them had a suitable molecular weight
distribution, and larger molecular weight led a shift to lower
elution time. The valley at 11min was the solvent peak.
3950
4572
1.05
PBEMA7-b-POEGMA22-b-PBLA19 17039 19256
1.22
The results of DLS were displayed in Fig.S1 in the Supporting
Information. All of the TP, NP and PWD were in narrow size Optical Properties of the Polymer
distribution with a size diameter around 108, 106 and 115 nm,
The UV-vis and fluorescence spectra were measured to
characterize the optical properties of TP which was shown in
Fig.5. Two absorption peaks corresponding to doxorubicin and
Cy could be observed at the wavelength of 480 and 650nm,
and their emission peaks were at 590 and 790nm, respectively.
respectively. The zeta potential of the polymers was also
measured, which were 3.4, 7.3 and 8.4 mV for TP, NP and
PWD, respectively, indicating that they had positive charge
which was beneficial for the endocytosis by cells.
Micellce Stability
Since the zeta potentials of the particles were positive, the
measurements of stability were carried out. Fig.S3 and Table
S1 in the Supporting Information showed the stability of TP, NP
and PWD micells in 10% serum solution. For each sample, the
size distribution hardly changed in the experiment. In another
way, the diameter kept the same within 24 h, while at 36 h,
the value increased indicating the swell of the micelle. The
micelles might be unstable in the serum solution as the time
went on, however, they would keep a satisfactory stability
within a favorable time.
Figure 6. Drug delivery behavior of the targeted polymers under neutral (pH = 7.4),
weak acidic (pH = 6.0) and acidic (pH = 5.4) conditions.
Drug Release
The in vitro drug delivery behavior were studied under normal
neutral condition (pH 7.4), intracellular weak acidic
environment (pH 6.0) and the environment in endosomes of
tumor cells (pH 5.4), respectively. As shown in Fig.6, the
polymeric nanoparticles was very sensitive against the acidic
environment. Doxorubicin encapsulated in the micelles were
released rapidly and subsequently accessed into a balance
within 8 h. In order to better understand the behavior of the
release, the size transformation of the micelles was measured
by means of DLS, as shown in Fig.7. Under normal cellular
condition, the micelles were kept in a stable status with the
diameter around 100nm (Fig.7a), and little doxorubicin might
leak out from them. This was why 30% of the drug would be
delivered to the neutral environment with a little longer time.
As the condition became weak acidic, the micelle expanded to
a larger diameter of 115nm, 137nm, 328nm and 514nm with
wider size distribution at pH 6.8, 6.6, 6.2 and 5.8, respectively
.
Figure 5. Absorption (black line) and emission (red line) curves of the TP (a) excited at
480 nm and (b) excited at 650 nm. The excitation and emission slit widths were 10 and
10 nm.
So the gaps of the micelles became larger, and the drug was
easier to seep which showed a better behavior in delivery. At
the pH of about 5.4, the original micelles began to collapse and
irregular accumulation appeared. This is why a large quantity
of doxorubicin can be released to the environment and rapidly
reached the balance.
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Figure 7. The transformation of the size and size distribution of targeted polymers with
the decrease of pH.
Cytotoxicity
The MTT method was utilized to study the cytotoxicity of TP,
NP and PWD against the MCF7 and HepG2 cells, respectively.
Keeping the concentration of doxorubicin encapsulated in the
micelle consistent, the cell survival ratio was measured under
gradient concentration, and the concentration of PWD was
kept the same with that of TP. The results were shown in Fig.
8. In both MCF7 and HepG2 cells, more than 80% cells
cultivated with polymers without drug still alive, indicating the
low toxicity of the polymer itself. Since these two kinds of cells
both had overexpressed folate receptors on their surface, cells
incubated with TPs had lower survival ratio than those with
NPs under different concentration. Beyond that, another two
phenomena should be paid attention. Firstly, at the same level
of concentration, polymers with drug had better performance
than the free doxorubicin. This might be not only contributed
to the effect of folic acid, but also because that the polymers
with a positive charge on their surface were easier to be
endocytosed by cells leading a better effect of the drug.
Secondly, this diversity and the differences between TP and NP
decreased as the concentration increased. It could be
illustrated that the function of folic acid and the polymer was
just synergic. At a low concentration or in a short time, they
would improve the endocytosis of the cells and the effect of
drugs, while the concentration increased to a particular level,
the drug would be easily endocytosed and the function of
them would not express obviously. The values of IC50 were
Figure 8. Cytotoxicity of TP (navy), NP (dark cyan), PWD (black) and free doxorubicin
(red) in MCF7 (a) and HepG2 (b) cells.
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also calculated, which were 33 μg/mL and 47 μg/mL for TP and
NP when MCF7 cells were used and were 42 μg/mL and 52
μg/mL for TP and NP when HepG2 cells were used.
Cellular Uptake Study
Both the MCF7 and HepG2 tumor cells were cultivated with
polymers of consistent concentration of doxorubicin for 24 h.
At predetermined time (2 h, 6 h, 12 h, 24 h), the cells were
observed by the fluorescence microscope after staining the
cell nuclei with DAPI. The images of the fluorescence
microscope were shown in Fig.9 and Fig.S2. For all the cells
cultivated for 12 h and 24 h, the images were captured with
the exposure time of 200 ms. While for the cells of 6 h, images
of 200 ms were too dim to recognize, which might because
nanoparticles accumulated as time went on and there were
fewer micelles at 6 h. In the same way, the exposure time for
cells of 2h should be extended to 930 ms to obtain pictures
bright enough. It was apparent that images of 24 h were
brighter than those of 12 h indicating higher concentration of
polymeric nanoparticles. So it could be concluded that as the
extension of the cultivated time, more polymers were
endocytosed by the cells. The trace of the polymers without
drug could be observed in Fig.9c and Fig.S2c. It could be found
that hardly any macromolecule gathered in the nuclei, while
they just dispersed all around in a random condition.
Cells cultivated with TP and NP showed the similar
phenomena in the aggregation of doxorubicin. As shown in all
Fig.9a, 9b, and Fig.S2a and S2b, almost all of the fluorescence
emitted by doxorubicin which increased as the cultivated time
extended appeared in the region of nuclei (displayed in line
Merge 1), while a little presented nearby. Considering the
treatment mechanism of doxorubicin, the released DOX finally
gathered in the nuclei, and the undelivered ones would
coincide with the macromolecule which could be well implied
by the images. From another aspect, it marched with the
results of drug delivery. Due to the great sensitivity of physical
wrapping compared with chemical connection, the micelle
would rapidly collapse after entering the intracellular
environment of tumor cells at the pH of about 5.4 and the
doxorubicin encapsulated could be released immediately.
Figure 9. Images of fluorescence microscope of MCF7 cells cultivated with (a) TP, (b) NP
and (c)PWD for 2 h, 6 h, 12 h and 24 h, respectively.
Both for MCF7 and HepG2 cells, treating with TPs displayed
stronger fluorescence at each time point compared with those
treated with NPs. This phenomenon is consistent with the
results of cytotoxicity. In a short time of cultivation, the
interaction between folic acid and folate receptor would
observably improve the endocytosis of the polymers by cells
leading a better utilization of drugs.
Flow cytometry
In order to further prove the uptake of TP was much higher
than that of TP, measurements of flow cytometry were carried
out. As shown in Fig.10, higher mean fluorescence intensity
(MFI) could be observed in the MCF7 cells cultivated with TP
indicating a more population of DOX-positive cells for TP than
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those of NP. The result corresponded with those of cytotoxity This material was prepared according to another work which
and cellular uptake study, collectively illustrating that the folic was done by ourself.12
acid would improve the endocytosis of the polymers by tumor
cells.
Synthesis of APA-RAFT-POEGMA22
225mg (0.464 mmol) RAFT-APA, 19.0mg AIBN (0.116 mmol
0.25 equiv) and 6.96g OEGMA (Mw=500, 13.9 mmol 30 equiv)
were dissolved in 2 mL DMF in a Schlenk tube under an argon
gas atmosphere. The mixture was stirred at 80oC for 24 h and
then purified against ultrapure by dialysis. Followed by freeze-
drying, 4.85g yellow solid (91.1% yields) was obtained.
Synthesis of 2-(N-tert-butoxycarbonylamino)ethan-1-ol
In a 250 mL round-bottom flask, 2.24g (36.7 mmol) 2-
aminoethan-1-ol was dissolved in 20mL DCM, followed with
the addition of 8.0g Boc2O (36.7mol, 1 equiv) in 10 mL DCM
dropwise at 0oC. The mixture was reacted at room
temperature for 4 h. After filtration, the solvent was removed
by reduced pressure distillation. 5.38g (91.0% yields) of
product was obtained as transparent liquid. 1H-NMR (300
MHz, CDCl3) δ 3.64 (t, J = 5.1 Hz, 2H), 3.23 (t, J = 5.1 Hz, 2H),
1.41 (s, 9H). 13C-NMR (75 MHz, CDCl3) δ 156.77 (s), 78.12-75.48
(m), 62.03 (s), 43.19 (s), 28.36 (s).
Figure 10. DOX-positive cell populations measured by flow cytometry of MCF7 cells
cultivated with TP and NP at 37oC for 2 h (blue), 6 h (wine) and 12 h (dark cyan).
Synthesis of 2-(N-tert-butoxycarbonylamino)ethyl Methacrylate
3.0g 2-(N-tert-butoxycarbonylamino)ethan-1-ol (18.6 mmol)
and 2.82g triethylamine (27.7 mmol, 1.5 equiv) were dissolved
in 20 mL dry DCM in a 100 mL round-bottom flask. 3.1g
methacryloylchloride (29.8 mmol, 1.6 equiv) was dropped into
the solution in an ice bath and the mixture was stirred at 0oC
for 24 h. After filtration and removing the solvent by reduced
pressure distillation, the raw product was purified via column
chromatography (petroleum ether/ethyl acetate, 10:1 v/v).
Removing solvent and vacuum drying gave 3.7g white solid,
Experimental Section
Materials
Unless specifically indicated, the chemical reagents used in the
experiments were purchased from Sinoreagent Corporation.
Azodiisobutyronitrile
methacrylate (OEGMA, Mw=500), methacryloyl chloride,
propargylamine, β-benzyl-L-aspartate, triphosgene,
N,N,N’,N’,N’-pentamethyldiethylene triamine (PMDETA), folic
acid, N,N’-dicyclohexyl carbodiimide (DCC), N-
(AIBN),
oligo(ethylene
glycol)
1
yielding 86.7%. H-NMR (300 MHz, CDCl3) δ 6.06 (s, 1H), 5.53
(s, 1H), 4.72 (s, 1H), 4.14 (t, J = 5.3 Hz, 2H), 3.38 (t, J = 5.3 Hz,
2H), 1.88 (s, 3H), 1.38 (s, 9H). 13C-NMR (75 MHz, CDCl3) δ
167.28 (s), 155.80 (s), 136.00 (s), 125.92 (s), 78.29-75.64 (m),
63.93 (s), 39.67 (s), 28.33 (s), 18.28 (s).
hydroxysuccinimide (HOSu), doxorubicin hydrochloride,
diisopropylethylamine (DIEA) were purchased from Aladdin
Corporation, China. N,N-diisopropylethylenediamine was
purchased from Tokyo Chemical Industry (TCI) Corporation,
Shanghai, China. Roswell Park Memorial Institute (RPMI), 4’6-
diamidino-2-phenylindole (DAPI) was purchased from
Synthesis of PBEMA7-b-POEGMA22
In a Schlenk tube, 800mg POEGMA22 (0.070 mmol), 2.9mg
AIBN (0.018 mmol, 0,25 equiv) and 239mg BEMA (1.04 mmol,
15 equiv) were dissolved in 4 mL DMF. After reacted at 80oC
for 24 h, the solution was sealed in dialysis bag and dialyzed
against water. Removing the water by freeze-drying gave
720mg white solid, yielding 78.9%.
Invitrogen, Carlsbad.
Dulbecco modified eagle medium
(DMEM), fetal bovine serum (FBS) and methyl thiazolyl
tetrazolium (MTT) were obtained from Sangon Corporation,
China. The normal phase column chromatography process
used 200-300 mesh silica gel (Yantai Institute of Chemical
Engineering, China). The dialysis bags (cut off Mw = 1000, 3000
and 7000) were purchased from Jinsui Bio-Technology
Corporation, Shanghai, China. Tetrahydrofuran (THF) and n-
hexane were dried by with CaH2 under reflux overnight and
distilled. Anhydrous N,N-dimethylformamide (DMF) was
obtained by stirring with CaH2 at room temperature for 2 days
followed by vacuum distillation. The ultrapure water was
prepared using a Milli-Q Synthesis System (Millipore).
Synthesis of BLA-NCA
This proceed was the same as our another work.12
Synthesis of PA-PBLA19
To a flame-dried Schlenk tube purged with argon, 11.1mg
propargylamine (0.201 mmol) was mixed with 5 mL dry DMF.
After cooled to 0oC in an ice bath, 1.0g BLA-NCA (4.02 mmol,
20 equiv) dissolved in 10 mL DMF was added into the tube.
Synthesis of the RAFT-APA
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The solution was reacted at 0oC for 3 days and at room mixture was sealed in a dialysis bag and was dialyzed against
temperature for 12h. After dialysis against ultrapure water, the water for 1 day. The micelle was obtained.
white precipitation was filtered and washed with water.
Freeze-drying gave 710 mg powder, yielding 89.2%.
NP was synthesized without the conjugation of Folic acid and
PWD was obtained without the encapsulation of DOX.
The drug loading rate was measured by using UV
spectrophotometer at the wavelength of 480nm. After
compared with a UV standard curve made by ourselves, the
Synthesis of PBEMA7-b-POEGMA22-b-PBLA19 by Click Reaction
A 4 mL DMF solution containing 497mg PBEMA7-b-POEGMA22 ratio of drug weight to polymer weight was 3.0% for TP and
(38 μmol) and 150mg PBLA19 (38 μmol, 1 equiv) was added to 2.7% for the NP.
a Schlenk tube. After adding 5.5mg CuBr (38 μmol, 1 equiv)
and 6.6mg PMDETA (38 μmol, 1 equiv) into the system, the
All the reactions in this part were carried out in the dark.
reaction was carried out under argon gas atmosphere at 30oC Characterization
for 2 days. The solution was purified by dialysis against an
Deuterated chloroform and deuterated dimethyl sulfoxide
aqueous solution containing EDTA. After removing the water
by freeze-drying, 512mg yellow solid (79.1% yields) was
obtained.
containing 0.03 vol% tetramethylsilane (TMS) were used as the
solvent for the measurements of 1H-NMR and 13C-NMR spectra
by using a Bruker AC 300 spectrometer. A Shimadzu UV-2401
PC Ultraviolet was used to measure the UV-vis spectra.
Shimadazu RF-5301PC fluorescence spectrophotometer was
used to measure the fluorescence intensity with the excitation
and emission slit widths of 10 and 10nm. FT-IR spectra were
obtained by using a Bruker EQUINOX 55 Fourier transform-
infrared spectrometer with the KBr disk method. The
molecular weights and PDI of the polymers were measured by
gel permeation chromatography (GPC, LC-20AD, Shimadzu)
which was equipped with a refractive index detector (RID-
10A), a Shodex GPC KD-804 column, and a spectrofluorometric
detector (RF-20Axs, Shimadzu). DMF was used as the mobile
Aminolysis of the Triblock Polymer
In a flame-dried Schlenk tube, 440mg PBEMA7-b-POEGMA22-b-
PBLA19 (25.8 mmol) was dissolved in 3 mL anhydrous DMF.
149mg N,N-diisopropylethylenediamine (1.03 mol, 40 equiv)
was added to the solution under argon atmosphere. The
mixture was heated to 45oC and stirred for 3 days. Following
dialysis, freeze-drying gave 380mg yellow solid (83.0% yields).
Activation of Folic Acid
300mg folic acid (680 mmol) and 78.2mg HOSu (680 mmol, 1 phase and monodispersed standard polystyrene samples to
equiv) was dissolved in 5 mL DMSO. After cooled to 0oC, 1 mL calibrate the value of Mn, Mw and Mw/Mn. Dynamic light
DMSO solution containing 156mg DCC (814 mmol, 1.2 equiv) scattering (DLS, Malvern Zetasizer Nano ZS90 with He-Ne laser
was added. The mixture was stirred for 24 h and prepared for and 90o collecting optics) was used to measure the size and
the next step without any further treatment.
size distribution of the micelle solution. The absorbance
for MTT cytotoxicity was measured using the Bio-rad iMak
Depretection of Boc and Conjugation of Folic Acid
microplate
reader.
The
fluorescence
microscope
measurements were carried out on Olympus U-HGLGPS. The
results of the flow cytometry were measured on a FACSCalibur
flow cytometer (BD Biosciences) and then analyzed with BD
FACSCalibur flow cytometer.
350mg PBEMA7-b-POEGMA22-b-P(Asp-DIEDA)19 (19.7 mmol)
was dissolved in a mixed solvent of 1 mL TFA and 2 mL DCM.
After stirred for 6 h, the solvent was removed by reduced
pressure distillation. The obtained oily solid was dissolved into
2
mL DMF, followed by the addition of 66mg N,N-
Micellce Stability
diisopropylethylamine (512 mmol, 26 equiv) and 1.22 mL of
the FA-NHS (138 mmol 7equiv) solution prepared above. It was 200 μL of the three micelle solutions (10 mg/mL) were added
stirred at room temperature in the dark for 12 h. The solution to 1 mL 10% serum aqueous solutions and were kept for 2 h,
was purified by dialysis against water. After freeze-drying, 12 h, 24 h, 36 h, respectively. Then 0.1 mL of the solution was
312mg yellow solid (80.3% yields) was obtained.
taken out for the measurement of DLS.
Conjugation of Cyanine Dye and Encapsulating Doxorubicin
In vitro Drug Delivery
Amino modified cyanine dye was synthesized as it mentioned
in our previous work.12 5mg aminocyaine dye (5.73 μmol), The performance of drug delivery was measured both in
0.8mg HOSu (6.95 μmol, 1.2 equiv) and 1.4mg DCC (6.79 μmol, neutral and acidic microenvironments. For the neutral
1.18 equiv) were dissolved in 1 mL dry DMF. After stirred for condition, 500 μL micelle solution (10 mg/mL) was sealed in a
24 h, 300 μL of it was added to 2 mL DMF containing 200mg dialysis bag and then immersed in 50 mL PBS solution (pH 7.4)
polymer 6. The solution was reacted for 12 h.
at 37oC on a shaking bath. At each predetermined time point,
15mg doxorubincin hydrochloride (25 mmol) was dissolved in 2 mL of the PBS solution was removed and isopyknic fresh PBS
1 mL DMF and neutralized by 3.8mg triethylamine (TEA) solution was added. The delivery quantity of doxorubicin was
(37.5mmol, 1.5 equiv). It was stirred for 5 min and then added calculated by testing the fluorescence intensity of emission
to the solution prepared above. After mixing uniformly, the wavelength at 577nm with an excitation wavelength at 480nm.
10 | J. Name., 2012, 00, 1-3
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For the acidic condition, PBS (pH 6.0) and acetate buffer (pH short time. The conjugation of a NIR dye to the polymer makes
5.4) was used as the release media instead.
it a theranotic agent for the imaging-guided drug delivery, and
this kind of novel polymer is potential for the future cancer
treatment.
Size Transformation of the Micelles in Acidic Condition
20 mL TP micellces solution was kept at 37oC in a 100 mL
beaker. 0.1 M HCl aqueous solution was used to adjust the pH
to several predetermined point. After balance, 0.1 mL of the
solution was taken out to measure the size and size
distribution by DLS.
Acknowledgements
This work is supported by the National Natural Science
Foundation of China (No. 51373162), and the Natural Science
Foundation of Anhui Province (No. 1408085MKL03).
Cytotoxity
The cytotoxity of the polymers was measured with a methyl
tetrazolium (MTT) viability assay against MCF7 and HepG2
cells. Experiments in both the cells were carried out in a similar
way. In short, cells were seeded in 100 μL DMEM in 96-well
plates at the density of 3000 cells per well, followed by
incubation for 24 h. After replacing DMEM, different
concentrations of free Dox or the three kinds of polymers we
synthesized were added. Following cultivation for 24 h, 20 μL
MTT with the concentration of 5 mg/mL was added to each
well and incubated for another 4 h. Then the plates were
shaken for 10 min with the addition of 150 μL DMSO. The
absorption of the wells was measured at 570nm.
Notes and references
‡ Footnotes relating to the main text should appear here. These
might include comments relevant to but not central to the
matter under discussion, limited experimental and spectral data,
and crystallographic data.
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