Norbornene derived doxorubicin copolymers as drug carriers with pH responsive hydrazone linker

Article abstract of DOI:10.1021/bm201478k

The synthesis and complete characterization of both norbornene-derived doxorubicin (mono 1) and polyethylene glycol (mono 2) monomers are clearly described, and their copolymerization by ring-opening metathesis polymerization (ROMP) to get the block copolymer (COPY-DOX) is vividly elaborated. The careful design of these conjugates exhibits properties like well-shielded drug moieties and well-defined nanostructures; additionally, they show solubility in both water and biological medium and also have the important tendency of rendering acid-triggered drug release. The drug release profile suggests the importance of having the hydrazone linker that helps to release the drug exactly at the mild acidic conditions resembling the pH of the cancerous cells. It is also observed that the drug release from micelles of COPY-DOX is significantly accelerated at a mildly acidic pH of 5.5-6, compared to the physiological pH of 7.4, suggesting the pH-responsive feature of the drug delivery system with hydrazone linkages. Confocal laser scanning microscopy (CLSM) measurements indicate that these COPY-DOX micelles are easily internalized by living cells. MTT assays against HeLa and 4T cancer cells showing COPY-DOX micelles have a high anticancer efficacy. All of these results demonstrate that these polymeric micelles that self-assembled from COPY-DOX block copolymers have great scope in the world of medicine, and they also symbolize promising carriers for the pH-triggered intracellular delivery of hydrophobic anticancer drugs.

Full text of DOI:10.1021/bm201478k

Article
pubs.acs.org/Biomac
Norbornene Derived Doxorubicin Copolymers as Drug Carriers with
pH Responsive Hydrazone Linker
Vijayakameswara Rao N,† Shivshankar R. Mane,† Abhinoy Kishore,‡ Jayasri Das Sarma,*,‡
and Raja Shunmugam*,†
‡
†Polymer Research Centre, Department of Chemical Sciences and Department of Biological Sciences, Indian Institute of Science
Education and Research Kolkata (IISER K), India
S
* Supporting Information
ABSTRACT: The synthesis and complete characterization of
both norbornene-derived doxorubicin (mono 1) and poly-
ethylene glycol (mono 2) monomers are clearly described, and
their copolymerization by ring-opening metathesis polymer-
ization (ROMP) to get the block copolymer (COPY-DOX) is
vividly elaborated. The careful design of these conjugates
exhibits properties like well-shielded drug moieties and well-
defined nanostructures; additionally, they show solubility in
both water and biological medium and also have the important
tendency of rendering acid-triggered drug release. The drug
release profile suggests the importance of having the
hydrazone linker that helps to release the drug exactly at the
mild acidic conditions resembling the pH of the cancerous
cells. It is also observed that the drug release from micelles of COPY-DOX is significantly accelerated at a mildly acidic pH of
5.5−6, compared to the physiological pH of 7.4, suggesting the pH-responsive feature of the drug delivery system with hydrazone
linkages. Confocal laser scanning microscopy (CLSM) measurements indicate that these COPY-DOX micelles are easily
internalized by living cells. MTT assays against HeLa and 4T cancer cells showing COPY-DOX micelles have a high anticancer
efficacy. All of these results demonstrate that these polymeric micelles that self-assembled from COPY-DOX block copolymers
have great scope in the world of medicine, and they also symbolize promising carriers for the pH-triggered intracellular delivery
of hydrophobic anticancer drugs.
have taken the initiative to overcome this factor by opting for a
production technique like ROMP,7a−i the living ring-opening
metathesis polymerization, which is more attractive for the
preparation of such monodisperse polymeric prodrugs due to
the exceptional functional group tolerance of the Grubbs’
catalyst employed in this process.8a−e
Even though there are a lot of familiar methods and
procedures that can be followed to control the release rate of
drugs by covalently attaching them to a hydrolytically labile
bond, such as an imine,9 acetal,10 oxime,11a−c or orthoester,12
we are very specific about hydrazone bonds for their responsive
acuteness in delivery behavior and also for their usually facile
incorporation of hydrazides into delivery materials.13 Typically,
the release rate of the drug with hydrazone linkage is governed
by the pH of the surrounding media, with faster release
observed in acidic (pH 5−6) environments compared with
physiological media (pH 7.4). However, incorporation of a
hydrazone into polymer prodrugs is still synthetically
challenging and not explored much except for very few
INTRODUCTION
■
Over the past two decades, several first-generation therapeutic
molecular products have emerged in cancer therapy to
galvanize the drug delivery system, which is a multidisciplinary
scientific field undergoing explosive development.1a−c Though
they improved the therapeutic benefit of clinically validated
cancer drugs by enhancing drug tolerability and efficacy, it is a
great challenge for researchers in the field to generate an
effective system that has enormous potential in providing
controlled release and molecular targeting properties in these
products.2a−c The role of functionalized polymers has been
widely investigated in this area of research, which has seen
considerable growth during the above-mentioned periods.3a−c
There are several advantages that can be cited in favor of
polymeric drugs over their monomeric precursors, like longer
retention time in the body, lower toxicity, and a greater
specificity of action.4 Recently, efforts have focused on
developing drugs through self-assembly or high-throughput
processes to facilitate the development and screening of them
with these distinct properties.5a,b But inherent characteristics of
a conventional polymer synthesis, such as molecular weight
distribution of a polymer sample, have induced enormous
regulatory difficulties for the chosen synthetic strategy.6 We
Received: October 20, 2011
Revised: November 20, 2011
Published: November 22, 2011
© 2011 American Chemical Society
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1
solvent. H NMR spectra of solutions in CDCl3 were calibrated to
tetramethylsilane as internal standard (δH 0.00).
pioneering reports on poly(aspartate hydrazone adriamycin)
based polymers.14a A recent report shows the attachment of the
doxorubicin moiety to methoxy poly(ethylene glycol)-b-poly-
(lactide-co-2,2-dihydroxylmethyl-propylene carbonate) by hy-
drazone linkage,14b but it is done by the post-polymer
modification chemistry. The drawback of post-functionalization
here is that the control over attaching the drug to the polymer
backbone could not be demonstrated precisely.
In this article, we report a simple and unique approach to the
design and synthesis of the block copolymer (COPY-DOX)
that has the potential application as a drug carrier. Motivated by
the hydrolytically labile prospects of hydrazone bonds along
with fascinating self-assembly properties of amphiphilic block
copolymers, this work investigates the design and synthesis of
the COPY-DOX, consisting of hydrazone-tethered DOX and
PEG chains in the norbornene backbone. To avoid the
unfavorable peripheral hydrophobicity,15 a well-shielded
environment for the covalently attached DOX is achieved
when it is copolymerized with poly(ethylene glycol) mono-
methyl ether (PEG) as another block. Also, the addition of
PEG block makes the system advantageous, as it shows no
toxicity and can significantly promote water-solubility and
increase the plasma clearance half-life of nanostructures.16
Fourier Transform Infrared (FT-IR). FT-IR spectra were obtained
on FT-IR Perkin-Elmer spectrometer at a nominal resolution of 2
cm−1.
Ultraviolet (UV) Spectroscopy. UV−visible absorption measure-
ments were carried out on U-4100 spectrophotometer HITACHI
UV−vis spectrometer, with a scan rate of 500 nm/min.
Dynamic Light Scattering (DLS). Particle size of QDs were
measured by dynamic light scattering (DLS), using a Malvern
Zetasizer Nano equipped with a 4.0 mW He−Ne laser operating at
λ = 633 nm. All samples were measured in aqueous as well as
methanol at room temperature and a scattering angle of 173°.
Transmission Electron Microscopy (TEM). Low resolution trans-
mission electron microscopy (TEM) was performed on a JEOL 200
CX microscope. TEM grids were purchased from Ted Pella, Inc. and
consisted of 3−4 nm amorphous carbon film supported on a 400-mesh
copper grid.
Confocal Laser Scanning Microscopy (CLSM). Confocal Micro-
scope images were taken in LSM 710 with microscope axio observer
Z.1, Carl Zeiss.
Synthesis of Norbornene-Derived Doxorubicin Hydrazone Linker
(Mono 1). Doxorubicin hydrochloride, 29 mg (0.05 mmol), and
compound 3, 72.6 mg (0.18 mmol), were dissolved in 10 mL of
anhydrous methanol. Trifluoroacetic acid (3 μL) was added to the
reaction mixture. The reaction mixture was stirred at room
temperature for 24 h, while being protected from light. The reaction
mixture was concentrated to a volume of 1 mL and added to
acetonitrile (20 mL) dropwise with stirring. The resulting solution was
allowed to stand at 4 °C. This product was isolated by centrifugation,
washed with fresh methanol/acetonitrile (1:10), and dried under
vacuum to yield the hydrazone linker of doxorubicin (15 mg, 51%
EXPERIMENTAL SECTION
■
Materials: 5-norborene-2-carboxylic acid (mixture of endo and exo
isomers) and exo-oxabicylo-[2.2.1]hept-5-ene-2,3-dicarboxylic anhy-
dride were prepared following the reported procedure. Doxorubicin
hydrochloride, second generation Grubbs’ catalyst, tertiary butyl
carbazate, poly(ethylene glycol) monomethyl ether (PEG, Mn = 700
and 2000 g/mol), 4-aminobenzoic acid, acetic anhydride, sodium
acetate, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlor-
ide, N-hydroxysuccinimide, dicyclohexyl carbodiimide (DCC), 1-
hydroxybenzotriazole, 4-dimethylamino-pyridine (DMAP), pyrene,
CDCl3, DMSO-d6, acetonitrile, dichloromethane, furan, maleic
anhydride, toluene, vinyl ethyl ether, diethyl ether, methanol, dimethyl
formamide (DMF), ethyl acetate, pentane, acetone, trifluoroacetic
acid, and CD3OD were purchased from Sigma Aldrich and used as
received without further purification. Clear polystyrene tissue culture-
treated 96-well and 6-well plates were obtained from Atlanta Drugs.
Dimethyl sulfoxide (DMSO) was used after purification by distillation
under vacuum and dried with calcium hydride (CaH2). Tetrahy-
drofuran (THF) was refluxed over freshly prepared sodium
benzophenone complex (a deep-purple color indicating an oxygen-
and moisture-free solvent) and then distilled to use immediately.
Anhydrous acetonitrile and dichloromethane were refluxed with CaH2
and then distilled prior to use. All other reagents and solvents of
analytical grade were used as received unless otherwise mentioned.
Cell Studies. Dulbecco’s modified Eagle’s medium (DMEM),
minimal essential medium (MEM), penicillin, streptomycin, and fetal
bovine serum (FBS) were purchased from Invitrogen. 3-(4,5-
Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
was purchased from USB (Cleveland, OH). Vectashield mounting
medium with DAPI was obtained from Vector Laboratories.
Characterization. Gel Permeation Chromatography (GPC).
Molecular weights and PDIs were measured by Waters gel permeation
chromatography in THF relative to PMMA standards on systems
equipped with Waters Model 515 HPLC pump and Waters Model
2414 Refractive Index Detector at 35 °C with a flow rate of 1 mL/min.
HRMS analyses were performed with Q-TOF YA263 high resolution
(Waters Corporation) instruments by +ve mode electrospray
ionization.
1
yield). H NMR (DMSO-d6, 400 MHz): δ 11.8 (s, 1H), 7.69−7.95
(m, 7H), 7.79−7.80 (m, 2H), 6.62 (s, 2H), 5.43 (m, 2H), 5.26 (m,
2H), 5.30 (s, 2H), 4.95−4.97 (m, 1H), 4.85−4.88 (t, 1H), 4.56−4.57
(m, 1H), 4.17−4.18 (m, 1H), 4.21 (m, 1H), 4.02 (s, 3H), 3.55−3.56
(m, 1H), 3.11 (s, 2H), 2.95−3.02 (m, 2H), 2.13−2.15 (m, 2H), 1.88−
1.89 (m, 1H), 1.6−1.69 (m, 1H), 1.16−1.19 (m, 3H). 13C NMR
(CD3OD, 400 MHz): δ 215.0, 187.62, 188.82, 177.20, 162.77, 158.39,
156.46, 137.83, 129.04, 127.98, 122.16, 120.49, 112.12, 100.98, 82.81,
76.86, 71.78, 67.88, 61.0, 59.0, 39, 32.0, 33.81, 29.42, 16.98. IR (KBr,
cm−1): δ 3474, 2992, 2419, 1718, 1799, 1674, 1604, 1567, 1525, 1416,
1299, 114, 1258, 1154, 1026, 1006, 960, 900, 891,788, 733. MS (ESI)
calcd for C8H10O2Na [M + H]+, 824.25; observed, 824.9.
Synthesis of Norbornene Grafted Poly(ethyleneglycol) (Mono
2). Monomethoxy PEG, 100 mg (2.5 mmol), and 20 mg (2.9 mmol)
of compound 4 were dissolved in 5 mL of dry dichlomethane.
Dicyclohexyl carbodiimide, 597 mg (2.9 mmol), and a catalytic
amount, 1.84 mg (10 mol %), of dimethyl amino pyridine were added
to the mixture at room temperature. The reaction mixture was allowed
to stir for 15 h. The resulting solid was removed by filtration and the
filtrate was concentrated to a pasty mass. Dichloromethane/hexane
(1:2) was charged to the pasty mass and kept overnight to obtain a
1
solid (80 mg, 80% yield). H NMR (CDCl3, 400 MHz,): δ 6.0−6.1
(m, 2H), 3.80−4.2 (m, 4H), 3.3 (s, 2H), 3.0 (s, 1H), 2.9 (m, 1H),
2.19−2.28 (m, 2H), 1.2−1.6 (m. 2H). 13C NMR (CDCl3, 400 MHz):
δ 176.0, 138.40, 135.63, 63.45, 61.58, 59.29, 46.19, 42.93, 41.48, 30.26,
26.39.
Homopolymerization of Mono 1. A total of 30 mg (0.0138 mmol)
of mono 1 and second generation Grubbs’ catalyst, 0.81 mg (15 mol
%), were dissolved in a 3 mL solution of dry CDCl3 and CD3OD (9:1
v/v %) under nitrogen while being protected with light. The mixture
was stirred at room temperature for 4 h. The resulting polymer was
isolated by precipitating in cold ether and concentrated under vacuum.
Gel permeation chromatography was done in tetrahydrofuran (flow
rate = 1 mL/min). The molecular weight of the polymer was obtained
using polystyrene standards (Mn = 17400; PDI = 1.17; 10 mg, 33%
yield). 1H NMR (CD3OD, 400 MHz): δ 7.94−7.9549 (m, 2H), 7.79−
7.80 (m, 2H), 7.77−7.78 (m, 1H), 7.36−7.39 (m, 1H), 5.43 (m, 1H),
5.26 (m, 2H), 5.13−4.4.6 (m, 1H) 5.30 (s, 2H), 4.59 (d, 1H), 4.21 (m,
Fluorometry. Fluorescence emission spectra were recorded on a
fluorescence spectrometer (Horiba Jobin Yvon, Fluoromax-3, Xe-150
W, 250−900 nm).
Nuclear Magnetic Resonance (NMR). The 1H NMR spectroscopy
was carried out on a Bruker 500 MHz spectrometer using CDCl3 as a
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Scheme 1. Synthetic Methodology of COPY-DOX Block Copolymers
1H), 4.02 (s, 3H), 3.11 (s, 2H), 1.6−1.9 (m, 2H), 2.46 (m, 2H), 1.16−
1.19 (m, 3H), 1.60−1.90 (m, 2H). IR (KBr, cm−1): 3944, 3757, 3690,
3600, 3054, 2987, 2856, 2085, 2521, 2411, 2305, 2155, 2126, 2054,
1715, 1605, 1551, 1422, 1263, 1157, 1101, 1058, 1017, 896.
thaw cycles. Monomer 2 (30 mg) was transferred to the flask
containing the catalyst via a cannula. The reaction was allowed to stir
at room temperature until the polymerization was complete (4 h),
after which the second monomer 1 (12 mg) was added to the flask via
a cannula. The polymerization was allowed to continue for another 24
h before it was quenched with ethyl vinyl ether (0.2 mL). An aliquot
was taken for GPC analysis, and the remaining product was
precipitated from pentane, dissolved again in THF, passed through
neutral alumina to remove the catalyst, and precipitated again from
pentane to get a pure COPY-DOX. Gel permeation chromatography
was done in tetrahydrofuran (flow rate = 1 mL/min). The molecular
weight of polymer using was measured using polystyrene standards.
Mn = 38000, PDI = 1.05. 1H NMR (CD3OD, 400 MHz,): δ 7.65−8.03
(s, q, d, d′, e, e′), 5.17−5.47 (a, a′, x, f, j, y), 4.20 (g′), 4.05 (n), 3.66 (z),
3.58−3.63 (k, w), 3.30 (n′), 3.0−3.03 (c, p′, b), 2.10−2.19 (g), 1.60−
1.90 (g′, t, p, w, r), 1.29−1.32 (h). IR (KBr, cm−1): 3944, 3757, 3690,
3601, 3054, 2987, 2930, 2685, 2521, 2411, 2305, 2155, 2127, 2055,
1968, 1737, 1605, 1550, 1422, 1265, 1155, 1107, 1017, 991, 896, 849.
Cell Culture. Hela cells, a human uterine cervical cancer cell line,
were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
with penicillin (100 U/mL) and streptomycin (100 μg/mL) plus 10%
fetal bovine serum (FBS). Whereas, HEK-293, a human embryonic
kidney cell line, was maintained in MEM containing 10% FBS (fetal
bovine serum) with penicillin (100 U/mL) and streptomycin (100 μg/
Homopolymerization of Mono 2. Mono 2, 100 mg (0.0408
mmol), and second generation Grubbs’ catalyst, 0.346 mg (0.0004
mmol), were dissolved in dry dichloromethane (3 mL) under nitrogen,
and the mixture was stirred at room temperature for 4 h. The resulting
polymer was isolated by precipitating in cold ether and concentrated
under vacuum. Gel permeation chromatography was done in
tetrahydrofuran (flow rate = 1 mL/min). The molecular weight of
the polymer was obtained using polystyrene standards. Mn = 7000,
PDI = 1.04 (80 mg, 80% yield).1H NMR (CDCl3, 400 MHz,): δ 5.0−
5.3 (m, 2H new peaks were observed), 3.80−4.2 (PEG-H, 190H), 3.3
(s, 3H), 1.5−1.7 (m, 39H). IR (KBr, cm−1): 3944, 3690, 3054, 2987,
2685, 2305, 1422, 1265, 1102, 896, 738, 706.
Synthesis of Block Copolymer (COPY-DOX). Known amounts of
monomers 1 and 2 were weighed into two separate Schlenk flasks,
placed under an atmosphere of nitrogen, and dissolved in anhydrous
dichloromethane and methanol (9:1 v/v %). Into another Schlenk
flask, a desired amount of third generation Grubbs’ catalyst 0.63 mg
(15 mol %) was added, flushed with nitrogen, and dissolved in a
minimum amount of anhydrous dichloromethane and methanol (9:1
v/v %). All three flasks were degassed three times by freeze−pump−
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mL) and 4T, a mouse mammary gland cancer cell line was maintained
in RPMI containing 10% FBS (fetal bovine serum) with penicillin
(100 U/mL) and streptomycin (100 μg/mL). All the cell lines were
cultured for a month with 10 passages and maintained at 37 °C with
5% CO2 in their respective medium.
while the signal at δ 3.37 (3H) was responsible for methyl
group of poly(ethylene glycol) methyl ether (Figure 1a). The
Cellular Uptake Study. Confluent monolayers of HEK-293, Hela,
and 4T cells were seeded on 12 mm coverslips plated on 24-well tissue
culture dishes in their respective medium. Confluent monolayer cells
were treated with free Dox of COPY-DOX at different concentrations
(1−20 μg) for 24 h. After 24 h post-treatment, cells were briefly
washed in PBS with Ca2+ and Mg2+, fixed in a methanol/acetone (1:1)
mixture for 2−5 min, successively washed with PBS without Ca2+ and
Mg2+, and mounted in Vectashield with DAPI. The cellular uptake
behavior and the intracellular distribution of free Dox and COPY-
DOX were analyzed using confocal laser scanning microscope (Zeiss,
LSM 710).
Cytotoxicity Assay of COPY-DOX. Cytotoxicity of COPY-DOX in
HEK, Hela, and 4T cell lines was quantitatively determined using
MTT enzymatic and colorimetric assay. All cell lines were seeded at 1
× 104 cells/well in 96-well plates and maintained in culture for 24 h at
37 °C in their respective medium. After 24 h, the medium was
removed, cells were washed with PBS, and medium containing
different concentrations of COPY-DOX (25−200 μg) was added to
the designated wells. The whole experimental plate was incubated for
72 h. Fresh MTT (20 μL) from 5 mg/mL stock solution was added to
each well, followed by incubation for 4 h at 37 °C. After 4 h, medium
from the wells was removed and 100 μL of DMSO was added to each
well and incubated for 15 min to completely solubilize the cells. The
absorbance of the resulting solution was measured at 515 nm, and cell
survivals were determined by comparison of optical density with
untreated respective control cell cultures.
Cell-Growth Inhibition Assay by Trypan Blue Exclusion Method.
Cells were seeded at 1.25 × 104 cells in 24-well tissue culture plates
and the inhibition activities of cell growth and division of Doxy
polymer were quantitatively determined by visual cell counting using a
hemocytometer chamber. After 24 h of cell plating, different
concentrations of COPY-DOX (25−50 μg) was added to the
respective wells. Designated wells for control were maintained with
respective medium without adding any COPY-DOX. The whole plate
was incubated for an additional 72 h. Cell counting was performed by
the trypan blue exclusion method at 24, 48, and 72 h. The extent of
cell inhibition was determined by viable cell population counts and
compared with untreated control cell culture for each time point.
RESULTS AND DISCUSSION
■
Monomer Synthesis (Scheme 1). Exo-5-norbornene-2-
carboxylic acid was separated from the commercially available
mixture of endo- and exo-5-norbornene-2-carboxylic acid by
the idolactization methods of Ver Nooy and Rondestvedt (see
Supporting Information (SI)). From the 1H NMR, the
carboxylic acid signal was observed at δ 12 ppm (br, 1H;
Figure S7). The signals at δ 6.02−6.06 ppm (m, 2H)
correspond to norbornene olefenic protons, while the signals
at δ 1.97−2.05 ppm (dt, J = 12.7 Hz, 1H) and δ 1.66−1.71 (m,
1H) belong to norbornene-bridged hydrogens. It was further
confirmed by FT-IR (Figure S12) where the stretching mode
due to carboxylic acid of exo-norbonene carboxylic acid was
observed at 1700 cm−1. Norbornene-grafted poly-
(ethyleneglycol) was prepared by coupling reaction between
carboxylic acid and alcohol derivative of monomethoxy PEG
using dicyclohexyl carbodiimide (DCC) and 4-dimethylamino-
pyridine (DMAP). Formation of the monomer was confirmed
1
1
Figure 1. (a) H NMR spectrum of mono 2; (b) H NMR spectrum
of mono 1; (c) mass spectrum of mono 1.
signals at δ 1.5 ppm (d, 1H) and δ 1.75 ppm (d, 1H) were for
norbornene-bridged protons. The signals at δ 6.0−6.4 ppm (m,
2H) correspond to norbornene olefinic protons.
Exo-oxabicylo[2.2.1]hept-5-ene-2,3-dicarboxylic anhydride
was prepared following the reported process.13 3-(4-Carbox-
yphenylcarbamoyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-carboxylic
acid was prepared by using 1 equiv of 4-aminobenzoic acid and
5.1 equiv of acetic anhydride in the presence of 0.29 equiv of
sodium acetate in dimethyl formamide solvent. Product was
1
confirmed by H NMR and FT-IR spectroscopy. The signal at
1
1
δ 13.1 (bs, 1H) ppm was responsible for carboxylic acid group
(Figure S1) while signals at δ 8.0−8.2 (m, 2H) and 7.4−7.5 (m,
2H) ppm were responsible for aromatic protons. The specific
signals at δ 6.6 (s, 2H), 5.34 (s, 2H), and 3.1 (s, 2H) ppm peaks
were for oxo-norbornene protons. Broad stretching frequency
by H NMR and FT-IR spectroscopes. In the product, H
NMR carboxylic acid signals at 12 ppm were absent, which was
evident that exo-norbornene carboxylic acid was completely
reacted with alcohol derivative of monomethoxy PEG. The
signals at δ 3.65−4.3 ppm were due to the PEG (H-PEG),
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Figure 2. FT-IR spectra of (a) free doxorubicin and (b) mono 1.
from FT-IR spectroscopy at 3244 cm−1 was attributed to free
carboxylic acid as shown in (Figure S11a).
methanol as solvent. Observed mass (m/z) and calculated mass
for all monomers were in good agreement, which confirmed the
formation of product.
Tertiary butyl hydrazone nadic carboxylate was prepared by
coupling of nadic acid and tertiary butyl carbazate using 1.2
equiv of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hy-
drochloride (EDC) coupling reagent in the presence of 1.2
equiv of 1-hydroxybenzotriazole (HOBT) in dimethyl for-
mamide solvent. Formation of the product was confirmed by
1H NMR and FT-IR spectroscopy. The proton signal at δ 1.26
(s, 9H) ppm was responsible for the tertiary butyl group, while
signals at δ 10.30 (bs, 1H) and δ 8.96 (s, 1H) were for
hydrazone protons. The signals at δ 7.8−7.9 (m, 2H) and δ
7.2−7.26 (m, 2H) were for the benzene ring protons. The
signals at δ 6.60 (s, 2H), 5.34 (s, 2H), and δ 3.11 (s, 2H) ppm
were observed for oxo-norbornene protons (Figure S3). The
stretching frequency at 3244 cm−1 for free carboxylic acid was
shifted to 3277 cm−1, which indicated the formation of amide
group as shown (Figure S11b). In the next step, tert-butoxy
carbonyl group was cleaved using trifluoroacetic to yield the
hydrazide. This product was confirmed by 1H NMR and FT-IR
spectroscopy. The signal at δ 1.26 (s, 9H) ppm for tertiary
butyl group was absent in the product, which indicated the
complete deprotection of tertiary butyl group (Figure S5).
Shifting of stretching frequency from 3277 to 3473 cm−1
indicated the formation of the free amine group (Figure S11c).
Doxorubicin with hydrazone linker (mono 1) was prepared
by addition of doxorubicin hydrochloride and norborne
hydrazide in methanol in presence of trifluoroacetic acid and
the whole reaction set up was protected from light for 24 h.
Polymerization Reactions. Homopolymerization.
Homopolymerization of mono 1 was carried out by using
second generation Grubbs’catalyst at room temperature in dry
CDCl3 and CD3OD (9:1 v/v %) solvent system and monitored
1
by H NMR. New signals were observed at δ 5−5.3 ppm, and
norbornene olefinic protons disappeared at δ 6.6 ppm,
indicating the formation of the product. Molecular weight of
the polymer, poly 1, was determined by the gel permeation
chromotography, as shown in Figure S13 (Mn = 17400 with
PDI 1.17). The stretching frequency at 1567 cm−1 for
norbornene olifinic bond (CC) was absent, which confirmed
the formation of homopolymer (Figure S14). The poly 2 was
prepared by using second generation Grubbs’ catalyst (M/I =
15) at room temperature in DCM. Within 1 h, new peaks were
observed at δ 5.0−5.3 ppm, and norbornene olefinic protons
were disappeared at δ 6.0−6.1 ppm, as shown in Figure S10.
The stretching frequency at 1567 cm−1 for the norbornene
olefinic bond (CC) was absent, which confirmed the
formation of poly 2 (Figure S15).
Block Copolymerization. Known amounts of monomers
(mono 1 and 2) were weighed into two separate Schlenk flasks,
placed under an atmosphere of nitrogen, and dissolved in
anhydrous dichloromethane and methanol (9:1 v/v %). Into
another Schlenk flask, a desired amount of second generation
Grubbs’ catalyst was added, flushed with nitrogen, and
dissolved in minimum amount of anhydrous dichloromethane
and methanol (9:1 v/v %). All three flasks were degassed three
times by freeze−pump−thaw cycles. The reaction mixture was
stirred at room temperature for 4 h. Then monomer 1 was
added to the reaction mixture and stirred at room temperature
1
The product was confirmed by H NMR and IR spectroscopy.
The signals at δ 7.94−7.95 (m, 2H), 7.79−7.80 (m, 2H), 7.77−
7.78 (m, 1H), and 7.36−7.39 (m, 1H) ppm were responsible
for doxorubicin aromatic group protons. The signal at δ 6.62 (s,
2H) was observed for the norbornene olefinic protons (Figure
1b). In mono 1, the band at 1730 cm−1, which was responsible
for carbonyl stretching frequency of free doxorubicin was
absent and that indicated the formation of hydrazone linker
between ketone and hydrazine (Figure 2a,b). All the monomers
were characterized by micromass spectrometer (Q-TOF), using
1
for 24 h. The whole reaction was monitored by H NMR for
every 1 h. New peaks were observed at 5.0−5.3 ppm, and
norbornene olefinic protons disappeared at 6.6 ppm, indicating
the formation of the product. The stretching frequency at 1567
cm−1 for the norbornene olefinic (CC) bond was absent in
COPY-DOX (Figure S16), which confirmed the polymer
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formation. The reaction mixture was quenched with ethyl vinyl
ether (0.2 mL). An aliquot was taken for GPC analysis, and the
remaining product was precipitated from pentane, dissolved
again in THF, passed through neutral alumina to remove the
catalyst, and precipitated again from pentane to get a pure
COPY-DOX. Molecular weight of the polymer was charac-
terized by THF-GPC, which showed Mn = 38000 with PDI
1.05 value (Figure 3b). It was observed from the GPC that all
was also observed from AFM and TEM that the observed
micelles from COPY-DOX were spherical in shape and
assembled in controlled manner.
In Vitro Doxorubicin (DOX) Release. Dialysis Study.
For the drug release profile of COPY-DOX, pH 7.4 as well as
acidic conditions were studied, as the normal pH of blood for
sustaining human life is about 7.4, while the endosomes and
lysosomes of cells have a more acidic environment.11c
Therefore, the drug release study of COPY-DOX micelles
was carried out in pH 7.4 in phosphate buffer solution and pH
5.5 and 6 in acetate buffer solutions, respectively.18 A total of 1
mg of block copolymer (COPY-DOX) was dissolved in 1 mL
of distilled water and loaded in a dialysis tube (3500; Dalton
cutoff) and dialyzed against 70 mL of a buffer solution whose
pH was maintained at 5.5. An aliquot of the sample was
removed and its absorbance at 480 nm was measured as an
indication of the release of doxorubicin. Fluorescence also
recorded by exciting the solution at 510 nm. Emissions from
the free DOX released from COPY-DOX were observed at 560
and 590 nm. The sample was then added back to the solution
to maintain the volume of the solution. This procedure was
repeated for every 1 h and results were observed for 48 h. It was
observed that, after 24 h, there was no significance increase in
the intensity of fluorescence and UV intensity (Figure 5a). The
similar procedure was repeated to monitor the drug release at
pH 6.0 (Figure 5b), as well as pH 7.4, and the results are shown
in Figure 5c. The DOX release from COPY-DOX at pH 7.4
was very minimal (less than 5%), which was very interesting to
observe, as it clearly demonstrated the COPY-DOX micelle’s
stability in the physiological condition. It was also very worthy
to note that the maximum as well as fast release was observed at
pH 5.5 compared to pH 6 or 7.4, which suggested the
importance of having the acid-labile hydrazone linker in the
COPY-DOX.
To Investigate the Ability of the COPY-DOX To Affect
the In Vitro Efficacy. Slow release of Dox, through acid-labile
linkages, inside acidic cancerous cells and endosomes should
give our COPY-DOX with pH linker additional selective edge
over the free drug. To study the in vitro efficacy of the release
of COPY-DOX with pH linker three different cell lines were
chosen such as human embryonic kidney cell line (HEK-293)
and two cancerous cell lines: a human uterine cervical cancer
cell line (Hela cells) and a mouse mammary gland cancer cell
line (4T). Three different cell lines were grown in culture with
similar kinetics and the pH of the cell medium.
Uptake of COPY-DOX in all the cells was through
endocytosis and occurred relatively slowly inside the cell
compared to free DOX. The uptake of free DOX occurred in
about 6−8 h in all the cell lines, which could be clearly visible
by fluorescent microscope after 12 h of post treatment (data
not shown). Interestingly, the uptake of COPY-DOX appeared
to be different. Post treatment of COPY-DOX at 24 h did not
show any intracellular accumulation in the HEK-293 cells, but a
diffused intracellular accumulation was observed in Hela cells
and 4T cells. There was a clear endosomal/lysosomal COPY-
DOX penetration that was observed (Figure 6).
Figure 3. (a) Table comprising molecular weight and PDI of poly 1
and 2 and COPY-DOX; (b) gel permeation chromatogram of COPY-
DOX confirms the formation of block copolymer. It was calculated
that 1 mg of COPY-DOX (Mn = 38000) contains 2.631 × 10−6 mol %
of doxorubicin.
the macroinitiator was completely converted to the block
copolymer (Figure 3b) with very narrow PDI. All the
polymerization showed the controlled behavior and produced
monodisperse polymers (Figure 3a).
Determination of Critical Micelle Concentration of
COPY-DOX. Critical micelle concentration (CMC) was
measured by using pyrene as an extrinsic probe.17 Pyrene (4
μg) was dissolved in methanol. Several samples were prepared
with different concentration of COPY-DOX. The COPY-DOX
was dissolved in 1000 μL of methanol and 5 mL of water was
added to the samples. The solution was stirred at room
temperature to evaporate the organic solvent. The fluorescence
intensities of prepared solutions were measured by spectro-
fluorometer. The concentration of pyrene was maintained at
0.2 μM and final concentration of COPY-DOX was changed
from 0.01 to 1 μg/mL, with the excitation wavelength set at 339
nm; the emission intensities were determined at 371, 382, and
396 nm. The relative emission fluorescence intensities of 396/
371 nm was varied as a function of COPY-DOX concentration
(Figure 4a). The CMC was determined by taking copolymer
concentration value at which the relative fluorescence intensity
ratio began to change. The observed CMC was 0.923 μg/mL.
Morphology of COPY-DOX Micelles. A total of 1 mg of
block copolymer (COPY-DOX) was dissolved in 10 mL of
methanol and stirred for 1 h to ensure the complete polymer
solubilization. From this solution, 2.5 mL of sample was taken
in a vial to which 18% of water was added under constant
stirring. Dynamic light scattering (DLS) analysis was performed
on the solution and the particle size was measured (Figure 4b).
The size of the micelles was about 105 nm with 0.25 PDI. The
morphology of the micelles was determined by AFM (Figure
4c) as well as TEM (Figure 4d). From TEM and AFM analysis,
the particle size was also measured to be about 100 nm, which
was in good agreement with DLS measurements (Figure 4b). It
The pH of the medium was relatively low in 4T cells in
culture compared to HEK-293 and Hela, consecutively the
release of DOX from COPY-DOX was more pronounced in 4T
cells compare to Hela and HEK-293. The known acidic
environment formed during endosomal uptake process, would
induce DOX release from the COPY-DOX in 4T and Hela
cells compared to HEK-293. Increasing the incubation time did
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Figure 4. (a) Plot of concentration of COPY-DOX vs intensity ratio of emissions at 375 and 396 nm from pyrene. The observed CMC was 0.923
μg/mL. (b) DLS measurement of COPY-DOX micelles in aqueous solution. The size of the micelles was about 105 nm with 0.25 PDI. (c)
Representative AFM image of COPY-DOX micelles spin-coated on a silicon surface. (d) TEM images of COPY-DOX micelles dip-casted on
carbon-coated copper grids. The inset shows the proposed self-assembled structure of COPY-DOX block copolymers in an aqueous environment.
not change the rate or amount of internalization (data not
shown). Mean intensity of fluorescence (released DOX) was
plotted from 10 frames for each concentration (Figure 6) and it
was noted that the amount of release of Dox from the COPY-
DOX was highest in 4T compare to that followed by Hela
compared to HEK-293. It was a very interesting observation to
note as it emphasized the importance of our design of COPY-
DOX with the hydrazone linker that released more Dox in 4T
because 4T cells are more acidic than Hela cells.
to 200 μg) for 72 h, at which time the viability of the cells was
determined by MTT assay. HEK-293 cells showed significant
cytotoxic effect at relatively higher concentration (200 μg),
whereas Hela cells showed significant cytotoxic effect from the
concentration of 100 μg, but 4T cells were more susceptible for
COPY-DOX, and the significant effect of cytotoxicity was
observed as low as 25 μg/mL concentration. Interestingly, at
higher concentration (100−200 μg), all three cells lines were
showing cytotoxicity, which suggested that there was an
imperfect uptake of nanoparticles into the cells above the
threshold, as shown in Figure 7. The observed cytoxicity could
The effect of COPY-DOX on the cell viability was evaluated
by incubating COPY-DOX with increasing concentration (25
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Figure 6. (a) Uptake of Doxy block copolymer in HEK-293, Hela, and
4T cells. Cells were plated on coverslips inserted into the wells of 24-
well tissue culture plates. Confluent monolayer cells were loaded with
Dox and COPY-DOX at different concentrations (1−20 μg) for 24 h.
Uptake studies were performed at 24 h post loading of Doxy and were
analyzed using confocal laser scanning microscope (LSM 710; Carl
Zeiss). Mean intensity fluorescence as an index of release of Dox from
COPY-DOX after 24 h of incubation at different concentrations were
plotted. Ten different frames were acquired from each concentration
of treatment in a LSM 710 confocal microscope. Area of each frame
was 45176.68 μm2. Mean intensities were calculated for each frame
and the mean of mean intensity was plotted against the concentration.
Ten frames were taken from the each concentration at 40× in a
LSM710 Zeiss confocal microscope.
lines. Significant cell growth inhibition was observed in both
Hela cells and 4T, whereas no significant cell growth inhibition
was observed in HEK-293 cells at a concentration range of 25−
50 μg (Figure 7).
CONCLUSION
■
This paper describes a novel approach by which a polymeric
micelle exhibits well-shielded drug moieties, significant water, as
well as biological medium, solubility, well-defined nanostruc-
tures, and acid-triggered drug release to elevate the efficacy and
safety of the loaded drugs. To prepare such micelles,
amphiphilic COPY-DOX block copolymer that has shell-
forming PEG block along with core-forming DOX functionality
conjugated through acid-sensitive hydrazone bonds to the side
chains of the norbornene block are synthesized. To our
knowledge, this report represents the simplest route to produce
block copolymers containing a narrow polydispersity and a high
density of drugs that are delivered efficiently in the acidic
environment (pH 5−6). This newly designed block copolymer
self-assembled into a spherical nanostructure in aqueous
solutions that is confirmed by DLS, AFM, and TEM analysis.
Figure 5. Drug release studies of COPY-DOX polymer. Fluorescence
response was measured with varying time intervals at (a) pH 5.5; (b)
pH 6; (c) DOX release profile from COPY-DOX micelles at 37 °C in
comparison with pH 5.5, 6, and 7.4; (d) A cartoon representation of
breaking of the hydrazone linkage at acidic pH and releasing the drug.
be attributed to the damage to cytoplasmic, endosomal, and
lysosomal membranes, which could lead to cell death.
Internalization and release of DOX-induced cytotoxicity could
have a profound effect on cell growth and cell division.
The effect of COPY-DOX on cell growth and cell division
was observed by incubating the nanoparticle in all three cell
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new modalities for the creation of cancer therapy with
macromolecular drug carrier to interact with living organisms.
Future work in this area by our group will concentrate on
improving the targeted delivery by incorporating folate
functionality to COPY-DOX micelles not only to selectively
control intracellular environment-sensitive drug release, but
also to significantly enhance drug delivery efficiency by affecting
drug efficacy with short exposure times.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional analytical data, synthetic scheme, procedures, and
references. This material is available free of charge via the
Internet at http://pubs.acs.org.
ACKNOWLEDGMENTS
■
R.N.V., S.R.M., and A.K. thank IISER-Kolkata for the research
fellowship and R.S. thanks DST, New Delhi, for Ramanujan
Fellowship. R.S. and J.D.S. thank IISER-Kolkata for the
infrastructure and start up funding. Authors also thank Mr.
Ritabrata Ghosh for his assistance with confocal measurement.
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