Aromatic Nitrogen Mustard-Based Autofluorescent Amphiphilic Brush Copolymer as pH-Responsive Drug Delivery Vehicle

Article abstract of DOI:10.1021/acs.biomac.8b01468

Delivery of clinically approved nonfluorescent drugs is facing challenges because it is difficult to monitor the intracellular drug delivery without incorporating any integrated fluorescence moiety into the drug carrier. The present investigation reports the synthesis of a pH-responsive autofluorescent polymeric nanoscaffold for the administration of nonfluorescent aromatic nitrogen mustard chlorambucil (CBL) drug into the cancer cells. Copolymerization of poly(ethylene glycol) (PEG) appended styrene and CBL conjugated N-substituted maleimide monomers enables the formation of well-defined luminescent alternating copolymer. These amphiphilic brush copolymers self-organized in aqueous medium into 25-68 nm nanoparticles, where the CBL drug is enclosed into the core of the self-assembled nanoparticles. In vitro studies revealed ～70% drug was retained under physiological conditions at pH 7.4 and 37 °C. At endolysosomal pH 5.0, 90% of the CBL was released by the pH-induced cleavage of the aliphatic ester linkages connecting CBL to the maleimide unit. Although the nascent nanoparticle (without drug conjugation) is nontoxic, the drug conjugated nanoparticle showed higher toxicity and superior cell killing capability in cervical cancer (HeLa) cells rather than in normal cells. Interestingly, the copolymer without any conventional chromophore exhibited photoluminescence under UV light irradiation due to the presence of "through-space" π- π interaction between the C=O group of maleimide unit and the adjacent benzene ring of the styrenic monomer. This property helped us intracellular tracking of CBL conjugated autofluorescent nanocarriers through fluorescence microscope imaging. Finally, the 4-(4-nitrobenzyl)pyridine (NBP) colorimetric assay was executed to examine the ability of CBL-based polymeric nanomaterials toward alkylation of DNA.

Full text of DOI:10.1021/acs.biomac.8b01468

Subscriber access provided by Stockholm University Library
Article
Aromatic Nitrogen Mustard-Based Autofluorescent Amphiphilic
Brush Copolymer as pH-Responsive Drug Delivery Vehicle
Biswajit Saha, Neha Choudhury, Soma Seal, Bhuban Ruidas, and Priyadarsi De
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01468 • Publication Date (Web): 06 Dec 2018
Downloaded from http://pubs.acs.org on December 7, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
Aromatic Nitrogen Mustard-Based Autofluorescent Amphiphilic Brush
Copolymer as pH-Responsive Drug Delivery Vehicle
a
a
b
c
a,*
Biswajit Saha, Neha Choudhury, Soma Seal, Bhuban Ruidas, and Priyadarsi De
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aPolymer Research Centre and Centre for Advanced Functional Materials, Department of
b
Chemical Sciences, Department of Biological Sciences, Indian Institute of Science Education and
Research Kolkata, Mohanpur - 741246, Nadia, West Bengal, India.
cCentre for Healthcare Science and Technology, Indian Institute of Engineering Science and
Technology, Shibpur - 711103, West Bengal, India.
*
Corresponding Author: E-mail: p_de@iiserkol.ac.in
ABSTRACT: Delivery of clinically approved nonfluorescent drugs is facing challenges
because it is difficult to monitor the intracellular drug delivery without incorporating any
integrated fluorescence moiety into the drug carrier. The present investigation reports the
synthesis of a pH-responsive autofluorescent polymeric nano-scaffold for the administration
of nonfluorescent aromatic nitrogen mustard chlorambucil (CBL) drug into the cancer cells.
Copolymerization of poly(ethylene glycol) (PEG) appended styrene and CBL conjugated N-
substituted maleimide monomers enables the formation of well-defined luminescent
alternating copolymer. These amphiphilic brush copolymers self-organized in aqueous
medium into 25-68 nm nanoparticles, where the CBL drug is enclosed into the core of the
self-assembled nanoparticles. In vitro studies revealed ~70% drug was retained under
o
physiological conditions at pH 7.4 and 37 C. At endolysosomal pH 5.0, 90% of the CBL was
released by the pH-induced cleavage of the aliphatic ester linkages connecting CBL to the
maleimide unit. Although the nascent nanoparticle (without drug conjugation) is nontoxic,
the drug conjugated nanoparticle showed higher toxicity and superior cell killing capability in
1
ACS Paragon Plus Environment

Biomacromolecules
Page 2 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
cervical cancer (HeLa) cells rather than in normal cells. Interestingly, the copolymer without
any conventional chromophore exhibited photoluminescence under UV light irradiation due
to the presence of “through-space” π-π interaction between the C=O group of maleimide unit
and the adjacent benzene ring of the styrenic monomer. This property helped us intracellular
tracking of CBL conjugated autofluorescent nanocarriers through fluorescence microscope
imaging. Finally, the 4-(4-nitrobenzyl)pyridine (NBP) colorimetric assay was executed to
examine the ability of CBL-based polymeric nanomaterials towards alkylation of DNA.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
KEYWORDS: Autofluorescent polymer, chlorambucil, drug delivery, cell imaging, DNA
alkylation
INTRODUCTION
Despite having several potential anticancer agents, the clinical payoff still remains inadequate
because of their severe host toxicity.1,2 The best permissible way to diminish toxicity is to
develop a technology for a controlled targeted drug delivery system (DDS) and release to
eradicate cancer cells while sparing normal cells. Currently, chemotherapy is still considered
as the most common method to treat numerous forms of cancer. Among all the existing
chemotherapeutic agents, alkylating agents were the first compounds identified to be useful
for cancer chemotherapy, because of their deoxyribonucleic acid (DNA) damaging ability by
3
virtue of DNA cross-linking, mispairing of bases etc. In this regard, nitrogen mustards have
emerged as suitable candidates for cancer therapy owing to their ability to form reactive
aziridinium ring by intramolecular displacement of chloride ion by the amine nitrogen.4
Among all types of nitrogen mustards, chlorambucil (CBL) has been extensively utilized for
5
cancer therapy, especially chronic lymphocytic leukemia (CLL). CBL is a clinically
2
ACS Paragon Plus Environment

Page 3 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
approved bifunctional alkylating agent and it is worth to mention that the carboxylic side
6
chain of CBL is pharmacokinetic but not dynamic. Therefore, incorporation of a non-toxic
delivery system to the carboxylic side chain will only affect its absorption and half-life, not
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
8
the effectiveness. The major drawback to the use of CBL is that it is nonfluorescent, thus
prevent the tracking of intracellular drug delivery through fluorescence microscopy, as a
consequence, it will be difficult to have a knowledge regarding cellular drug uptake.
The recent past has witnessed the attachment of a fluorescent tag into the drug delivery
vehicle to aid in the analysis of the intracellular distribution of nonfluorescent drugs. Several
9
10
11
kinds of a fluorescent moiety such as coumarin, spiropyran-coumarin, carbazole have
been reported to assist in the tracking of intracellular drug delivery. However, these current
approaches still suffer from limitations, notably low drug loading capabilities, water
solubility etc. Also, the addition of a fluorophore to a nonfluorescent drug may alter its
intracellular distribution.12 Thus, to eliminate such concerns without affecting the
performance of the drug, new approaches should be developed. Typically, the material with
the ability to conquer these bottlenecks should; (i) have more number of conjugation sites to
have high drug loading capacity, (ii) possess high water solubility and low toxicity, and (iii)
not hamper the cellular uptake ability of the drug. Integration of all these facets is not trivial,
and hence till date, there is no report of any DDS satisfying all the above features in the
context of the delivery of nonfluorescent drugs. These necessitate the development of
different kinds of prodrugs which can provide the above prerequisites.
Existing literature helps us to find out the compound which can meet all the criteria; (i)
to (iii). Stimuli-responsive polymeric prodrugs may be potentially useful in this regard, as
13
they afford great therapeutic efficacy, a high percentage of drug loading capacity, controlled
drug release and selective recognition of the tumor microenvironment to target the cancer
1
4
cells avoiding the undesired side effects. These intelligent carriers release their payload as a
3
ACS Paragon Plus Environment

Biomacromolecules
Page 4 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
counter interaction with intracellular stimuli such as temperature,15 pH,16,17 redox,7,18
enzyme,19 etc. Amongst the several types of stimuli-responsive system, the pH-sensitive
DDSs have earned a significant amount of attention as the pathological cells has more acidic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
14
microenvironment relative to the normal cells. After addressing all the issues, we believe
that the last key point can also be responded by the use of an autofluorescent polymer.
Fortunately, nowadays researchers are keenly interested in developing different kinds of
2
0
21
luminescent polymers namely, poly(amido amine) (PAMAM), poly(amino ester)s (PAE),
poly(ether amide)s (PEA),22 poly[(maleic anhydride)-alt-(vinyl acetate)] (PMV),23
poly(maleic anhydride-alt-styrene),24 which could emit strong fluorescence in proper
conditions without having conjugated polymeric building block and conventional
fluorophores. Henry and co-workers reported pH-responsive and membrane destabilizing
polymeric carriers based on poly(maleic anhydride-alt-styrene) copolymers for intracellular
2
5
drug delivery. Recently, we also reported such kind of unorthodox luminescent alternating
copolymer based on strictly alternating poly(maleimide-alt-styrene) skeleton, which can
exhibit blue fluorescence in varieties of solvents, and pH- and thermo-responsive
hydrophilic-hydrophobic phase transition in an aqueous medium. Generation of fluorescence
from this non-conjugated polymeric structure was attributed to the “through-space” π-π
communication between the C=O group of maleimide unit and adjacent benzene ring of the
styrenic monomer.26,27 This unconventional macromolecular luminogen as fluorescence
probe was used for selective and sensitive detection of picric acid (PA) with an instantaneous
response in 100% aqueous solution.27
The objective of this contribution is to provide a first proof-of-concept to utilize the
autofluorescence property for intracellular tracking of DDS by fluorescence microscope
imaging from the poly(maleimide-alt-styrene) skeleton, connected to nonfluorescent drug
CBL through a pH-sensitive aliphatic ester linkage and biocompatible poly(ethylene glycol)
4
ACS Paragon Plus Environment

Page 5 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
(
PEG) chains. To this end, we have synthesized well-defined amphiphilic alternating brush
copolymers by the sequence-specific copolymerization of two rationally designed monomers,
PEG bearing styrene and CBL conjugated N-substituted maleimide via reversible addition-
fragmentation chain transfer (RAFT) polymerization. We hypothesize that PEG attachment to
the one segment of the copolymer will confer aqueous solubility, protein resistance
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
8
property, and biocompatibility to the alternating copolymer, widely observed for PEGylated
2
9
systems. The polymer self-assembled to a stable (at neutral pH) nanoparticle in aqueous
solution with CBL drug moieties into the core which is surrounded by long PEG chains,
thereby diminishing premature drug leakage and discriminating between cancer and healthy
tissues. Cellular internalization of the CBL conjugated nanocarrier was confirmed by
fluorescence imaging analysis and in vitro studies exhibited that the self-assembled
nanocarriers were found to deliver their payloads upon contact with intracellular stimuli.
Furthermore, the alkylation activity of CBL nitrogen mustard containing copolymer was
30
performed spectroscopically by using a DNA model, 4-(4-nitrobenzyl)pyridine (NBP), a
colorimetric indicator for carcinogenic alkylating agents. To the best of our knowledge, this
is the first report of synthesis of a DDS for nonfluorescent CBL drug, which could meet all
the aforesaid criteria, thus opens up a new opportunity in the engineering of the
autofluorescent amphiphilic copolymer for anticancer drug administration in cancer therapy.
EXPERIMENTAL SECTION
Materials. Furan (98%, Spectrochem, India), ethanolamine (Spectrochem, 99%),
maleic anhydride (Loba Chemicals, 99%), dicyclohexylcabodiimide (DCC, 99%, Aldrich), 4-
(dimethylamino)pyridine (DMAP, 99%, Aldrich), 4-vinylbenzyl chloride (4-VBC, 90%,
Fluka), p-toluenesulfonyl chloride (p-TsCl, 99%, Fluka), polyethylene glycol (PEG2000, 2000
g/mol, 99%, Aldrich), tetrabutylammonium chloride (Bu NCl, 99%, Aldrich), sodium
4
5
ACS Paragon Plus Environment

Biomacromolecules
Page 6 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hydride (NaH, 99%, Aldrich), chlorambucil (CBL, Aldrich), HPLC water (Sisco Research
Laboratories Pvt. Ltd., India), sodium chloride (99%, Merck), sodium hydroxide (NaOH,
99%, Merck), triethylamine (TEA, 99%, Merck), anhydrous sodium sulphate (99%, Merck)
and sodium bicarbonate (99%, Merck) were used as received. 2,2′-Azobis(2-
methylpropionitrile) (AIBN, 98%, Aldrich) was recrystallized from methanol prior to use.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
,4-Dioxane (99%, Aldrich) was passed through a basic alumina column. The 2-
dodecylsulfanylthiocarbonylsulfanyl-2-methyl-propionic acid (DMP) was synthesized
2
6
following previous literature report. Synthesis, purification and characterization of various
intermediates (Figure S1 to Figure S6) and monomers are provided in the Supporting
Information. NMR solvents such as CDCl (99.8% D), DMSO-d (99% D) and D O (99% D)
3
6
2
were purchased from Cambridge Isotope Laboratories, Inc., USA. Dry methanol,
dichloromethane (DCM) and tetrahydrofuran (THF) were prepared by following standard
procedures. Solvents such as toluene, hexanes (mixture of isomers), diethyl ether, DCM,
isopropanol were purchased from Merck and used as received without any further
purification. Cervical cancer cells (HeLa) and human breast cancer cells (MCF-7) were
maintained in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific).
Human embryonic kidney cells 293 (HEK 293) were maintained in Minimum Essential
Medium (MEM, Thermo Fisher Scientific) containing 10% (v/v) fetal bovine serum (FBS,
Gibco, Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific)
o
at 37 C under a 5% CO humidified atmosphere. Cells were washed with PBS, trypsinized
2
using 0.25% trypsin (Thermo Fisher Scientific ) and seeded in 24-well flat-bottomed plastic
plates (Nunc) for all assays. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) and dimethyl sulfoxide (DMSO, 99.9%, cell culture grade) were purchased
from USB corporation and Ameresco, respectively.
6
ACS Paragon Plus Environment

Page 7 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
Instruments and Characterizations. The detail instrumentation of size exclusion
1
chromatography (SEC), H NMR, UV-Vis and fluorescence spectroscopy can be found in our
earlier report.27 The fluorescence quantum yield (ɸ ) in solution was determined using
F
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
quinine sulfate in 0.05 M sulphuric acid (ɸ = 52%) as a standard. Positive mode electrospray
F
ionization mass spectrometry (ESI-MS) was performed on a Q-Tof Micro YA263 high
resolution
(Waters
Corporation)
mass
spectrometer.
Matrix-assisted
laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was carried out on a
Bruker UltrafleXtremeTM instrument equipped with a smart beam-II laser in the reflector
mode and an acceleration voltage of 22 kV. Dynamic light scattering (DLS) measurements
were carried out at room temperature in a Malvern Zetasizer Nano instrument equipped with
o
a He–Ne laser system operating at 633 nm (scattering angle = 173 ). Polymer solutions (1.0
mg/mL) were filtered through a 0.45 µm syringe filter prior to measurements. Field emission-
scanning electron microscopy (FE-SEM) study was performed on a Carl Zeiss Sigma SEM
instrument. The TEM images were recorded in a JEOL JEM-2100F instrument at 200 kV
operational mode to visualize the morphologies of the nanoparticles. Samples for TEM were
prepared by drop-casting an aqueous solution (1 mg/mL) on a carbon coated Cu-grid and then
dried for few hours under vacuum at room temperature. An epifluorescence Olympus IX81
model equipped with plan fluorite objective with 40X magnification was used to obtain
fluorescence images.
Procedure for the synthesis of alternating brush copolymers, BPDC1-BPDC3.
Typically, M1, M2, DMP, AIBN, and 1,4-dioxane were sealed in a 20 mL reaction vial
equipped with a magnetic stir bar. The vial was sealed tightly, placed on an ice-water bath
followed by purging with dry N for 20 min. Next, the reaction vial was kept in a preheated
2
o
polymerization block at 70 C for 24 h, after which the reaction was quenched by cooling it
on an ice-water bath and exposing to air. Then, the unreacted PEG macromonomer and
7
ACS Paragon Plus Environment

Biomacromolecules
Page 8 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
maleimide monomer were removed by pouring the reaction mixture into a large excess of
diethyl ether, followed by centrifuging the resultant cloudy dispersion at 3500 rpm for 15
min. The upper portion of the diethyl ether layer was carefully decanted. This was repeated
six times with a fresh batch of diethyl ether each time. Then, the purity of the resultant
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
polymer was verified by H NMR spectroscopy (Figure 1C). Polymerization reactions were
carried out at different feed ratios of [M1 + M2] : DMP : AIBN = 50/100/200 : 1 : 0.5.
Molecular weights and dispersity (Ð) values of all the resulting polymers are summarized in
Table S1.
In addition, we have synthesized a copolymer without incorporation of CBL (Scheme
S1). Briefly, 2-hydroxyethyl maleimide (HEMI, 13.3 mg, 0.094 mmol), M2 (200 mg, 0.094
mmol), DMP (0.7 mg, 0.002 mmol), and AIBN (0.164 mg, 0.001 mmol, from stock solution)
were dissolved in 1,4-dioxane in a 20 mL septa sealed reaction vial equipped with a magnetic
stir bar. The vial was purged with N for 20 min, and placed in a preheated reaction block at
2
7
0 °C for 24 h. Purification of the resultant polymer was executed using the same above
mentioned procedure.
Self-assembly study. Self-assembly of the resultant brush copolymers (BPDCx) was
performed by dissolving the polymer in de-ionized (DI) water. Typically, 5 mg of each
polymer was dissolved in 1 mL of DI water, a selective solvent for the PEG segment. The
resulting solution was allowed to stir for overnight. Size distribution and morphology of the
self-assembled polymeric nanoaggregates were determined by DLS measurement, FE-SEM
and TEM study at a concentration of 1.0 and 0.1 mg/mL, respectively.
pH-Triggered degradation of BPDC2 polymeric nanoaggregates. The hydrolysis of
the ester linkage of BPDC2 polymeric aggregates was determined by DLS method via
measuring the hydrodynamic size distribution value. Briefly, the pH values of self-assembled
BPDC2 polymeric nanoaggregates (2.0 mg/mL) were adjusted to 7.4 and 5.0. After being
8
ACS Paragon Plus Environment

Page 9 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
hydrolyzed at room temperature for predetermined time intervals, the hydrodynamic
diameter, D , of hydrolyzed polymer solutions were measured by DLS study. Also, after
h
different incubation time intervals, aliquots (20 µL) were taken out for FE-SEM and TEM
measurements.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In vitro drug release. The performance of drug delivery was measured in both neutral
and acidic environment. For the neutral condition, BPDC2 was dissolved in DI water (1
mg/mL) and sealed in a 2000 Da cut-off dialysis bag. Then, the dialysis bag was immersed in
o
5
0 mL of phosphate buffered saline (PBS) solution at pH 7.4 at 37 C with a magnetic stir
bar. At each predetermined time point, 2 mL of the PBS solution was removed and the
release of chlorambucil was determined by measuring its absorbance at 260 nm by UV-Vis
spectroscopy. For the acidic condition, buffer solutions (pH = 6.4 and 5.0) were used as the
o
release medium at 37 C.
Cell viability assay (MTT assay). The cytotoxicity of free CBL, CBL conjugated
polymeric nanoparticles and polymeric nanoparticle alone was studied in HEK 293 and HeLa
4
cell lines using tetrazolium salt, MTT. In a 24-well plate, 5 × 10 cells were seeded per well
with 1.0 mL DMEM containing 10% FBS and allowed to adhere for 24 h. CBL conjugated
polymeric nanoparticles and polymeric nanoparticle alone are dissolved in PBS to perform
the cytotoxicity assay, whereas free CBL was dissolved in DMSO as it is hydrophobic in
nature. Prior to drug treatment, media from the cells was aspirated, and replaced with fresh
media. Then, various concentration of free CBL, drug conjugated polymeric nanoparticles
and nascent polymeric nanoparticles were added to the wells. Cells were incubated for 24 h at
o
37 C with 5% CO . Control cells received equal volume of vehicle and were incubated for
2
the same time period. All experiments were run in triplicate. After 24 h, 100 µL freshly
prepared solution of MTT (5 mg/mL) was added to each well and incubated for 4 h.
Following incubation media was then aspirated from the cells, and the purple formazan
9
ACS Paragon Plus Environment

Biomacromolecules
Page 10 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
crystals that formed as a result of the reduction of MTT by mitochondrial dehydrogenase
enzyme from the cells were dissolved in 500 μL of DMSO (added per well). After 10 min,
the absorbance from formazan crystals was measured at 570 nm. Similar experiments were
also performed in HEK 293 cell line with free CBL, CBL conjugated nanoparticle and
nanoparticle alone.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cell imaging study. HeLa and MCF-7 cells were cultured on the coverslip for 24 h at
o
3
7 C with 5% CO . Then, the cells were treated with BPDC2 (drug concentration = 14
2
o
μg/mL) and incubated for 24 h at 37 C. The imaging process was performed after cells were
washed with PBS buffer and mounted on a glass slide. Fluorescence images were acquired
through a high-resolution epifluorescence microscope with 40X objective lens (Nikon Eclipse
Ti-S, inverted microscopes) under blue, green and red channels.
NBP alkylation assay. Alkylation activity of aromatic nitrogen mustard of
chlorambucil in BPDC2 has been investigated with 4-(4-nitrobenzyl)pyridine (NBP) by
3
1
following the established procedure with some modifications. BPDC2 was dissolved in
Milli-Q water (1.0 mg/mL) in a septa sealed vial. The vial was placed in an ice-water bath at
0 oC. A 1.0 mL aliquot of 0.2 M NaOAc buffer pH 5.0 was added, followed by addition of 40
o
µL 0.5 M NBP in acetone. Then, the resulting solution was heated to 100 C for 20 min.
After cooling the reaction mixture to room temperature, 2.0 mL of triethylamine/acetone
(1:1) was added. The solution was diluted to 10 mL with acetone and shaken by a hand mixer
for 1 min. Afterwards, the absorbance of the violet color solution was measured at 560-570
nm by UV-Vis spectroscopy within an hour. The rate constant of the reaction was calculated
by the following equation (1):
퐴 - 퐴0
∞
푙푛
= 푘푡
(1)
퐴 - 퐴푡
∞
1
0
ACS Paragon Plus Environment

Page 11 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
Where 퐴 is the initial absorbance, 퐴 denotes the absorbance of the adduct when all NBP
0
∞
has been consumed, 퐴 is the absorbance at different time intervals and 푘 (rate constant) is the
푡
slope of the ln{(A - A )/(A - A )} versus time (t) plot.

0

t
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
RESULTS AND DISCUSSION
Synthesis and characterization of nitrogen mustard-based amphiphilic alternating
copolymer. Since polymerization of an equimolar mixture of styrene and maleimide gives
rise to strictly alternating poly(maleimide-alt-styrene) skeleton,32 in the present study
amphiphilic alternating brush copolymer was synthesized by the copolymerization of
chlorambucil functionalized maleimide derivative (MI-CBL, M1) and PEG conjugated
styrene macromonomer (St-PEG, M2) as shown in Scheme 1. The M1 and M2 monomers
were synthesized through multistep reactions as shown in Scheme 1. Maleic anhydride was
converted into 4,10-dioxatricyclo[5,2,1,02,6]dec-8-ene-3,5-dione (1, Figure S1) via Diels-
Alder reaction with furan, and it was subjected to react with ethanolamine to give compound
2 (Figure S2). Compound 2 was then coupled with CBL via dicyclohexylcabodiimide (DCC)/
4
-(dimethylamino)pyridine (DMAP) esterification reaction to produce the compound 3
(Figure S3). The final monomer M1 was synthesized through retro Diels-Alder reaction of
o
1
compound 3 in toluene at 110 C. The formation of M1 was characterized using H NMR
spectroscopy (Figure 1A) and mass analysis (Figure S4). To synthesize the second monomer
(M2), at first 4-vinylbenzyl chloride (4-VBC) was converted into its alcohol derivative (4,
o
33
Figure S5) using a phase transfer catalyst in water at 125 C. Polyethylene glycol
monomethyl ether (molecular weight = 2000 g/mol) was treated with tosyl chloride to yield
PEG2000 tosylate (5, Figure S6). Compounds 4 and 5 were then reacted together in presence of
sodium hydride (NaH) in dry tetrahydrofuran (THF) to produce PEG functionalized styrenic
1
macromonomer M2. The successful synthesis of M2 was confirmed by both H NMR
1
1
ACS Paragon Plus Environment

Biomacromolecules
Page 12 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
spectroscopy (Figure 1B) and matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) mass spectrometry (Figure 1D). MALDI-TOF mass spectrum of M2 showed
a series of peaks, where two major signals are separated by exactly 44 m/z value, which is
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
identical with the molar mass of ethylene glycol (-O-CH -CH -) repeating unit. Furthermore,
2
2
we performed size exclusion chromatography (SEC) analysis of M2 (Figure S7), where we
observed monomodal molecular weight distribution. These results suggest the formation PEG
appended styrenic macromonomer M2.
Scheme 1. Synthetic routes employed for the preparation of maleimide conjugated
chlorambucil monomer (M1), styrenic macromonomer (M2) and pH-responsive amphiphilic
alternating copolymer, BPDCx, via RAFT polymerization.
Subsequently, copolymerization reactions of M1 and M2 were carried out using AIBN
o
as an initiator and DMP as chain transfer agent (CTA) in 1,4-dioxane at 70 C via RAFT
polymerization (Scheme 1). The use of RAFT polymerization can offer unprecedented
latitude in the synthesis of water-soluble amphiphilic architectures with precise molecular
weight and appropriate functionality for the encapsulation or attachment of therapeutic
agents.34,35 The copolymers were named as BPDCx, where x represents the different feed
1
2
ACS Paragon Plus Environment

Page 13 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
ratios of M1 and M2 (BPDC1-BPDC3, Table S1). The CTA to initiator ratio [CTA]/[AIBN]
=
1:0.5 was kept constant during all the polymerization reactions. After purification,
1
1
copolymers were characterized by H NMR spectroscopy and a typical H NMR spectrum of
BPDC2 is shown in Figure 1C, where the absence of vinyl and maleimide proton peaks
ensures the purity of the resulting polymer. Formation of the copolymer was also confirmed
by MALDI analysis. Increase in the molecular mass distribution of M2 after RAFT
polymerization with M1 approves the attachment of both the monomers into the polymer
chain (Figure 1D). The number average molecular weight (Mn,SEC) and molecular weight
distribution (dispersity, Ð) values of the copolymers were evaluated by SEC in THF and
results are summarised in Table S1. SEC plots (Figure S7) of BPDCx polymers showed
unimodal molecular weight distributions with a small trace of unreacted non-functionalized
PEG (Figure S7). The elution volume of the polymers decreases with the increase of
[monomer (M)]/[CTA] ratio in the feed. However, the measured Mn,SEC values are much
lower compared to the theoretical number average molecular weight (Mn,theo) values (Table
S1). This is most probably due to the different hydrodynamic volume of synthesized
polymers than that of poly(methyl methacrylate) (PMMA) standards, which we have used to
construct the conventional calibration for the calculations of Mn,SEC values from SEC analysis.
Several literature reports,36,37 and also our earlier work26 revealed the formation of
perfectly alternating sequences of monomers throughout the polymer chain upon
copolymerization of maleimide and styrene or their derivatives. The integration ratio of the
aromatic peak of CBL moiety at 6.60 ppm and methoxy proton of PEG entity at 3.36 ppm is
almost equal to one (Figure 1C), suggesting a strong propensity of alternating
copolymerization of these monomers. The alternate sequence of the monomers was further
ascertained by MALDI-TOF analysis (Figure S8). Figure S8 revealed that the BPDC2 is a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
ACS Paragon Plus Environment

Biomacromolecules
Page 14 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
perfectly alternating copolymer, as there is no single molecular mass distribution which is
equal to either the mass of M1 or M2.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Figure 1. H NMR spectra of (A) M1, (B) M2, and (C) BPDC2 in CDCl . (D) MALDI-TOF
3
mass spectra of M2 before (black trace) and after (red trace) RAFT polymerization with M1.
Photophysical properties. Alternating copolymers based on poly(maleimide-alt-
styrene) skeleton without having any conventional fluorophore moiety can exhibit
fluorescence behaviour.26,38 Thus, the photophysical property of BPDC2 was investigated in
different solvents. The absorption spectra of BPDC2 is represented in Figure S9, which
displayed two absorption bands in the UV region at 258 and 304 nm, collectively attributed to
2
7
the π-π* and n-π* electronic transitions of maleimide carbonyl and chlorambucil moiety.
Next, to probe the emission behaviour of BPDC2, the polymer solution was irradiated with
UV (λex = 339 nm) in different solvents (Figure 2A). A broad emission spectrum was
observed with emission maxima at around 422 and 384 nm in water and various organic
solvents, respectively. The fluorescence quantum yield (ɸ ) of BPDC2 in THF was estimated
F
1
4
ACS Paragon Plus Environment

Page 15 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
to be 0.98%. The appearance of this kind of unanticipated fluorescence is due to the “through
space” π-π communication between the benzene ring and the neighbouring C=O group of
2
6
maleimide unit. In the next step, luminescence characteristics of the BPDC2 polymer were
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
studied by virtue of fluorescence microscopy. The BPDC2 polymer was dissolved in water
(0.5 mg/mL), drop cast on a glass slide, dried overnight at ambient condition, followed by
vacuum drying. The fluorescence microscope images (Figure 2B and Figure S10) show that
the BPDC2 polymer can exhibit blue, green and red emission on illumination with UV light
although no conventional fluorophore moiety was employed during the synthesis of
3
9
polymers. Interestingly, the observed emission behaviour of the BPDC2 polymer is in well
accordance with the PL spectra (Figure 2A).
Figure 2. (A) Fluorescence spectra (λ = 339 nm) of BPDC2 in different solvents, and (B)
ex
fluorescence microscopy images of the BPDC2. The images were taken from blue (420-460
nm), green (510-550 nm) and red (575-625 nm) channels, respectively. Scale bar is 100 µm.
Self-assembly of brush copolymers in aqueous medium. It is well-documented that
amphiphilic brush copolymers with densely grafted macromolecular architectures in the
selective solvent can possess a variety of morphologies such as spherical micelles,40
41
42
cylindrical nanostructures, multi-molecular micelle, etc. The generation of these higher-
order molecular assemblies is vastly dependent on the hydrophobicity/hydrophilicity ratios of
the copolymer compositions. Due to the presence of long PEG side chains, the as-synthesized
BPDCx polymers can quickly be dissolved in aqueous media without any external assistance
1
5
ACS Paragon Plus Environment

Biomacromolecules
Page 16 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and endure self-assembly (Figure 3A). First, the self-assembly behaviour of BPDC1-BPDC3
was investigated by dynamic light scattering (DLS) (Figure 3B-3D). The number-average
hydrodynamic diameter (D ) values were found to be 25±5.0 (PDI = 0.265), 50±2.0 (PDI =
h
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
.230) and 68±3.0 (PDI = 0.167) nm for BPDC1, BPDC2, and BPDC3, respectively, with a
monomodal size distribution. The significant increase in D from 25 to 68 nm as a function of
h
[M]/[CTA] ratio can be described by the fact that with the increase of monomer feed into the
copolymer composition, large number of hydrophobic (M1) and hydrophilic (M2) segment
will accumulate into the core and corona of the nanoparticle, respectively, on self-assembly
in water.
Figure 3. Schematic representation of self-assembly of BPDCx in an aqueous medium (A).
DLS curves, FE-SEM and TEM images of (B) BPDC1, (C) BPDC2, and (D) BPDC3.
To gain more information on the formation and morphology of the BPDCx
nanostructures in aqueous solutions, FE-SEM and TEM images were recorded. As shown in
Figure 3B-3D, BPDC1-BPDC3 brush copolymers self-assembled into square shaped,
spherical and a mixture of spherical and non-spherical nanoparticles, respectively with an
average size of 30 to 40 nm. Furthermore, the TEM images have confirmed the existence of
different morphologies having a diameter in the range of 30 to 40 nm. Note that the apparent
hydrodynamic diameters in aqueous medium obtained from DLS are somewhat larger in size
1
6
ACS Paragon Plus Environment

Page 17 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
in comparison to the size measured from FE-SEM and TEM, because electron microscopy
study was done with dried samples, whereas in DLS measurements polymer chains are
hydrated with water molecules.43,44 Nevertheless, the abrupt change in morphologies by
varying chain length could be ascribed to the differences in hydrophobic MI-CBL chain
length and higher PEG grafting density. This observation demonstrates how using RAFT
polymerization to prepare brush copolymer by “grafting through” approach provides a handle
through which the morphology of the self-assembled nanoparticle can be manipulated. To
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
elucidate the mechanism of self-assembly behaviour of as-synthesized amphiphilic
1
copolymers, H NMR spectroscopy was performed in two different solvents, CDCl , and D O
3
2
(Figure S11). The CDCl is a good solvent for both the segments M1 and M2, whereas D O
3
2
1
is a bad solvent for the M1 units present in the polymer chain. The H NMR spectrum of
BPDC2 showed all the characteristics peaks from both the co-monomers in CDCl (Figure
3
S11A), while the resonance signals corresponding to the CBL unit of M1 is completely
vanished in D O (Figure S11B). These results describe the formation of higher-order
2
structures in water through the precise arrangement of hydrophobic (M1) and hydrophilic
(M2) components, where hydrophobic aggregation of the polymer skeleton including M1
unit and styrenic part of M2 is surrounded by the long hydrophilic PEG chains.45
Nevertheless, BPDC2 spherical nanoparticle could be a better drug carrier to exploit the
enhanced permeability and retention (EPR) effect as it possesses the nanoscopic size of 50
nm, which is in between 10-100 nm.46
Acid-triggered activation of BPDC2 polymeric nanoparticles. With the aliphatic
ester linkages connecting CBL moieties to the polymeric backbone, BPDC2 is expected to
undergo hydrolysis under acidic conditions. Therefore, the sensibility of the polymer
nanoparticles containing the acid-labile ester groups was tested by DLS in an acidic buffer
o
(
pH 5.0) at 37 C. Upon incubating BPDC2 in the acidic buffer for 0 to 48 h, the D values
h
1
7
ACS Paragon Plus Environment

Biomacromolecules
Page 18 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
increased from 25 to 82 nm with unimodal monodisperse size distribution (Figure 4A). After
7
2 h, a remarkable increase in size distribution (D = 295 nm) was observed, presumably due
h
to the formation of hydrophilic alcohol derivative on complete hydrolysis of the ester
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
linkages. In contrast, no significant change was observed in the DLS study when the BPDC2
o
was incubated at physiological pH 7.4 at 37 C (Figure S12). This study confirms the pH-
responsiveness of the aliphatic ester linkages connecting CBL moieties to the polymer
skeleton, which can be readily cleaved by intracellular pH to deliver the drug.47
Figure 4. (A) Analysis of number-average hydrodynamic diameter (D ), (B) FE-SEM and,
h
(C) TEM images of BPDC2 polymeric nanoparticles before and after hydrolysis at pH 5.0.
Additionally, pH-triggered degradation of BPDC2 nanoparticles was further
corroborated by FE-SEM and TEM observations. Figure 4B and 4C reveal that with the
increase of incubation time from 0 to 72 h at pH 5.0, the spherical nanoparticles undergo
fusion with complete disruption of spherical morphologies, resulting in aggregated
nanostructures of higher sizes. This result is consistent with the appearance of higher size
distribution in DLS. The increase in particle size and size distribution, as shown by both
DLS, FE-SEM, and TEM are strong evidence of core-shell nanoparticle aggregation over
48
time. We hypothesize that on hydrolysis of ester linkages, the CBL moieties released from
1
8
ACS Paragon Plus Environment

Page 19 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
the nanoparticles, leading to an imbalance in hydrophobicity/hydrophilicity ratio and result in
particle aggregation.
pH-responsive in vitro release studies. To evaluate the potential of the present DDS,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
49
in vitro drug release kinetics was studied via dialysis method. For this purpose, BPDC2 was
dissolved in de-ionized water (1 mg/mL) and it was sealed in the dialysis membrane (MWCO
=
2000 g/mol). Subsequently, the dialysis bag was immersed in a beaker containing 50 mL of
o
PBS buffer at pH 7.4 and 37 C. The cumulative release of the cargo from the core of the
nanoparticles was determined using UV-Vis spectroscopy by measuring the absorbance at a
wavelength of 260 nm for 10 days. In PBS, BPDC2 nanoparticles are displaying slow
rupturing process as a small amount of CBL (~32%) leached out within 10 days of
incubation. This observation assists us to claim that the synthesized polymeric nanocarrier are
quite stable under physiological conditions and able to stabilize the drug molecule during
prolonged circulation in blood. According to the chemical structure of the BPDCx polymer,
CBL was conjugated to the copolymer backbone through an aliphatic ester bond, which is
susceptible to cleave at endosomal pH (4.5-6.5) resulting in disruption of nanoscaffolds
leading to the delivery of the cargoes (CBL) to the intracellular environment (Figure 5A).47
To demonstrate this effect, the in vitro drug release studies were performed by incubating
BPDC2 in phosphate buffered saline at pH 6.4 and 5.0. The corresponding drug release
profiles are provided in Figure 5B. At pH 6.4, the percentage of drug release was increased
up to ~57% because of the ester hydrolysis at slightly acidic pH. The releasing phenomena
could be further accelerated up to ~90% by lowering the pH to 5.0. This fact can be attributed
to the facile cleavage of the ester bond from the side-chain of the copolymer at
endolysosomal pH. However, a significant difference in CBL releasing behaviour was seized
after 48 h of incubation which is mainly due to the substantial destabilization of the
nanocarrier as shown in DLS measurement (Figure 4A). Interestingly, the slow pH-
1
9
ACS Paragon Plus Environment

Biomacromolecules
Page 20 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
responsive feature of the newly engineered drug delivery vehicle makes them suitable for
once-a-week administration as it releases the drug for more than 7 days in a sustained
5
0
manner. Thus, the present system could be of interest as a potential DDS where a long-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
lasting drug depot is desirable.
Figure 5. (A) Schematic representation of pH-triggered release of CBL from BPDC2
nanocarrier. (B) Cumulative release profile of CBL from interior of the nanocarrier at varying
pH.
Cytotoxicity studies. Having been demonstrated the pH-triggered release of payloads,
in the following step, the intracellular drug delivery capability of BPDC2 and the control
alternating polymer without drug attachment (see BPC in Scheme S1) and free CBL were
investigated by assessing the cytotoxicity in human embryonic kidney cells 293 (HEK 293)
5
1
and cervical cancer (HeLa) cell lines using MTT assay. Both the normal and cancer cell
lines were exposed to various drug concentrations present in the nanoparticles ranging from
0
-28 µg/mL (polymer concentration: 250 to 1000 µg/mL, 7 µg/mL of CBL in 250 µg/mL
o
polymeric scaffold, the drug loading content is 9.29%) for 24 h at 37 C. The cytotoxicity
results are shown in Figure 6A, where more than 75% cell viability is observed with BPC in
both HEK 293 and HeLa cell lines even at a very high dose of 1000 µg/mL, indicating the
biocompatibility of nascent polymeric scaffold for drug delivery applications. Similarly, the
2
0
ACS Paragon Plus Environment

Page 21 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
BPDC2 showed more than 70% cell viability in normal HEK 293 cell line, suggesting
nontoxic nature of the BPDC2 towards the normal cells (Figure 6B), as a proof of which, the
cytotoxic assay of free CBL was carried out in HEK 293 cell lines where only 50% cell
viability was observed which is more toxic than the BPDC2 nanoparticle (Figure 6C). The
drug concentration was maintained as equivalent to the drug conjugated nanoparticle.
Interestingly, in the HeLa cell line, the BPDC2 nanoparticle exhibited superior cell killing
upto 60% and became highly effective to impart toxicity to the cancer cells (Figure 6B).This
could be due to the acidic environment of cancer cells compared to the normal cells, allowing
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
2
the better release of CBL through acid catalyzed ester hydrolysis. It should be noted that the
CBL conjugated polymeric nanoparticles exhibited about 40% killing of HeLa cells at a
concentration of 14 µg/mL, whereas there were almost no cell killing (~4%) phenomena
observed with free CBL (Figure 6C) which shows that our polymeric nanocarrier are more
effective to penetrate the cell membrane and induce their toxicity. However, a similar cell
viability was observed with free CBL in both HEK 293 (~60%) and HeLa (~51%) cell lines
at a concentration of 28 µg/mL. Nevertheless, the macromolecular drug carriers can also
show better uptake capacity into the cancer tissues via EPR effect compared to free drug.53,54
Thus, the present in vitro investigation indicates that the BPDC2 nanoparticles are capable to
accomplish better cell growth inhibition in cancer cells rather than normal cells. The main
hurdle in the use of free CBL as a chemotherapeutic agent is represented by its very poor
water solubility though it has clinical importance in the treatment of Non-Hodgkin
55
Lymphoma. Hence, the higher toxicity of the above mentioned pH-sensitive water-soluble
BPDC2 nanoparticles compared to the BPC towards the malignant cells provides a direct
evidence that our newly engineered alternating brush copolymer can behave as an efficient
drug delivery vehicle for the hydrophobic drugs, such as chlorambucil.
2
1
ACS Paragon Plus Environment

Biomacromolecules
Page 22 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. MTT cytotoxicity assay of nascent BPC nanoparticle (A), BPDC2 nanoparticle
(B), free CBL (C) in both HEK 293 and HeLa cell lines.
Cellular internalization of BPDC2. Encouraged by the autofluorescent behavior, we
examined the cellular internalization of BPDC2 towards HeLa and MCF-7 cells by
fluorescence microscopy. For this purpose, HeLa and MCF-7 cells were incubated with
o
BPDC2 for 24 h at 37 C. The CBL concentration used for the treatment was 14 µg/mL with
respect to 500 µg/mL nano-scaffold. We observed blue, green and red emissions in both the
cell membrane on excitation in blue, green and red channels, respectively (Figure 7). The
fluorescence microscope images captured in both the cells strongly suggest the efficient
internalization and accumulation of CBL conjugated nanocarrier in the peri-nuclear region,
where the intracellular release of CBL takes place upon degradation of ester linkages by
means of intracellular stimuli in the cancerous cells, confirmed by the above pH-responsive
in vitro release studies and MTT assay. Furthermore, the fluorescence images of HeLa cells
incubated with BPDC2 for 10 h were recorded (Figure S13). The comparison of these
fluorescence images revealed that the cellular internalization of BPDC2 at 10 h is similar to
those of 24 h. This result implies that the autofluorescent polymeric nanoparticle was capable
of undergoing cleavage into the cells within 10 h of incubation.
2
2
ACS Paragon Plus Environment

Page 23 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Epifluorescence images of HeLa (A) and MCF-7 (B) cells incubated with BPDC2
for 24 h, in blue, green and red channels. Scale bar is 10 µm.
Alkylating activity assessment of BPDC2. The NBP assay is a suitable technique to
evaluate the activity of alkylating agents.30,56 The NBP can serve as a DNA model, because
of the similar reactivity of NBP and N7 position of guanine in DNA.57,58 This method is based
on the formation of a new chromophore, resulting from the reaction between the alkylating
agent (CBL) and the nucleophile, NBP (Figure 8A). Moreover, the nucleophilicity constant
of NBP and N7 position of guanine is almost identical (3.5-3.6), as determined by Swain-
Scott nucleophilicity relationship.30 Since CBL is known to alkylate DNA through the
reaction at the N7 position of guanine, we used NBP assay to analyse the alkylating activity
of CBL conjugated copolymer BPDC2. UV-Vis spectroscopic measurements were carried
out for the determination of activity of BPDC2 towards alkylation in water as a sole solvent
during the course of the reaction, which potentially increases the similarity of the assay
condition with the biological systems. We have prepared various concentrations of BPDC2 in
water and a particular amount of NBP (in acetone) was added followed by addition of 1.0 mL
o
NaOAc buffer to each reaction mixture. Then the solutions are allowed to heat at 100 C for
20 min. After cooling at room temperature, a violet colour product was formed upon
2
3
ACS Paragon Plus Environment

Biomacromolecules
Page 24 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
basification with triethylamine (Figure 8A). It is observed that the colour intensity and the
absorbance of the solutions increased upon increasing the polymer concentration as shown in
Figure S14 and Figure 8B, respectively, which shows a linear increase of absorbance as a
function of concentration (see inset in Figure 8B). Thus, we inferred that the coloured adduct
obeys the Lambert-Beer’s law. The colour of the alkylated NBP-CBL adduct was stable up to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
h at room temperature. However, it should be noted that polymer without drug (CBL)
conjugation (BPC) does not form any colour on reaction with NBP (data not shown),
signifying the utility of CBL moiety in BPDC2 as an alkylating agent.
Figure 8. (A) Schematic representation illustrating the S 2 reaction between NBP and CBL
N
conjugated BPDC2 polymer. (B) Evolvement of absorption spectra with the increase of
polymer concentration. (C) Pseudo-first-order kinetic plot of NBP alkylation.
To understand the alkylation mechanism by NBP, we performed a kinetic study of the
reaction. Using a time-dependent analysis at a particular concentration of BPDC2 (1.0
mg/mL), we found that initially the absorbance increases with time and it becomes saturated
indicating no further alkylation after 25 min (Figure S15). In order to determine the rate
constant of the reaction, the absorbance values are plotted versus time. The rate constant of
2
4
ACS Paragon Plus Environment

Page 25 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
59
the reaction was calculated by using the equation (1). As illustrated in Figure 8C, the NBP
alkylation reaction follows pseudo-first-order kinetics. The rate constant is estimated to be
-1
60
0.186 min , which is much higher as compared to only CBL and other derivatives of CBL.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
This is presumably due to the existence of a higher number of pendent CBL entity in the
copolymer chain. Thus, the newly synthesized CBL conjugated luminescent copolymer has
better alkylation power compared to the free CBL.
CONCLUSIONS
In summary, the present strategy demonstrates the synthesis of an autofluorescent brush
copolymer, where anticancer aromatic nitrogen mustard drug CBL is conjugated to the
polymer side-chain through pH sensitive bonds that are stable at physiological pH 7.4 but are
cleavable in the mildly acidic pH (corresponding to the endolysosomal pH) and releases
CBL. The strictly alternating placement of M1 and M2 along the polymer main chain leads to
photoluminescence under UV light irradiation via “through-space” π-π interaction between
the C=O group of maleimide unit and adjacent benzene ring of the styrenic monomer.26,27
Thus, the brush copolymer is capable of showing blue, green and red emission under UV
light irradiation albeit it does not consist of any traditional fluorophore moiety.
The correct amphiphilicity of the copolymer chains assists them to self-assemble into
nanoparticles in aqueous media and the morphology of self-assembled nanoparticles could be
tuned by changing the polymer chain length.61 The nascent nanoparticle (without drug
conjugation) is nontoxic to both normal (HEK 293) and cancer (HeLa) cells, may be due to
the presence of densely grafted biocompatible PEG chains. Interestingly, the BPDC2
nanoparticle exhibited superior cell killing and high affectivity to impart toxicity into the
cancer cells rather than into the normal cells, probably due to acid catalyzed ester hydrolysis
2
5
ACS Paragon Plus Environment

Biomacromolecules
Page 26 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and release of CBL in the acidic environment of cancer cells. The NBP colorimetric assay in
the aqueous medium provided a clear evidence on the proficiency of the CBL alkylating
agent functionalized BPDC2 copolymer for DNA alkylation. Because of the
autofluorescence in the copolymer, fluorescence microscope imaging could be successfully
performed for intracellular tracking of BPDC2. Thus, the present study offers delivery and
monitoring of intracellular drug delivery of clinically approved nonfluorescent aromatic
nitrogen mustard drug CBL, although no fluorescence moiety is integrated into the present
DDS. This feature differentiates them from traditional DDS where the systems mainly deal
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
2
with the fluorescent drug, doxorubicin (DOX). We believe that this approach may open a
new avenue to accomplish intracellular drug delivery of clinically approved nonfluorescent
drugs by using autofluorescent polymeric materials.
ASSOCIATED CONTENT
Supporting information
1
Detailed synthetic methods of monomers including detailed synthetic methods and H NMR
spectra of all the intermediates for the synthesis of M1 and M2, ESI-MS spectra of M1, SEC
traces of brush copolymers, MALDI analysis of BPDC2, UV-Vis spectra of BPDC2
polymer, fluorescence microscope images of BPDC2, self-assembly study of copolymer by
1H NMR, degradation study of self-assembled nanoparticles at pH 7.4, fluorescence images
of HeLa cells incubated with BPDC2 for 10 h, digital photographs of alkylated NBP-CBL
adduct, time-dependent NBP alkylation assay. This material is available free of charge via the
Internet at http://pubs.acs.org.
2
6
ACS Paragon Plus Environment

Page 27 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
AUTHOR INFORMATION
Corresponding Author
*
E-mail: p_de@iiserkol.ac.in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACKNOWLEDGMENTS
The authors thank Dr. Sankar Maiti (Department of Biological Sciences, Indian Institute of
Science Education and Research Kolkata) for providing cell culture facility and fruitful
suggestions during the manuscript preparation. B.S. acknowledges Council of Scientific and
Industrial Research (CSIR), Government of India, India, for his senior research fellowship
(SRF). S.S. acknowledges Science and Engineering Research Board (SERB) for her National
Post-doctoral Fellow (NPDF) fellowship.
REFERENCES
(1) Danhier, F.; Feron, O.; Préat, V. To Exploit the Tumor Microenvironment: Passive and
Active Tumor Targeting of Nanocarriers for Anti-Cancer Drug Delivery. J. Controlled
Release 2010, 148, 135-146.
(2) Liu, J.; Pang, Y.; Zhu, Z.; Wang, D.; Li, C.; Huang, W.; Zhu, X.; Yan, D. Therapeutic
Nanocarriers with Hydrogen Peroxide-Triggered Drug Release for Cancer Treatment.
Biomacromolecules 2013, 14, 1627-1636.
(
3) Kondo, N.; Takahashi, A.; Ono, K.; Ohnishi, T. DNA Damage Induced by Alkylating
Agents and Repair Pathways. J. Nucleic Acids 2010, 2010, 543531-543537.
4) Polavarapu, A.; Stillabower, J. A.; Stubblefield, S. G. W.; Taylor, W. M.; Baik, M.-H.
(
The Mechanism of Guanine Alkylation by Nitrogen Mustards: A Computational Study. J.
Org. Chem. 2012, 77, 5914-5921.
2
7
ACS Paragon Plus Environment

Biomacromolecules
Page 28 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
5) Robak, T.; Kasznicki, M. Alkylating Agents and Nucleoside Analogues in the Treatment
of B Cell Chronic Lymphocytic Leukemia. Leukemia 2002, 16, 1015-1027.
(6) Omoomi, F. D.; Siadat, S. D.; Nourmohammadi, Z.; Tabasi, M. A.; Pourhoseini, S.;
Babael, R. A.; Saffari, M.; Ardestani, M. S. Molecular Chlorambucil-Methionine Conjugate:
Novel Anti-Cancer Agent Against Breast MCF-7 Cell Model. J. Cancer Sci. Ther. 2013, 5,
075-084.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(7) Saha, B.; Haldar, U.; De, P. Polymer-Chlorambucil Drug Conjugates: A Dynamic
Platform of Anticancer Drug Delivery. Macromol. Rapid Commun. 2016, 37, 1015-1020.
(8) Jana, A.; Nguyen, K. T.; Li, X.; Zhu, P.; Tan, N. S.; Ågren, H.; Zhao, Y. Perylene-
Derived Single-Component Organic Nanoparticles with Tunable Emission: Efficient
Anticancer Drug Carriers with Real-Time Monitoring of Drug Release. ACS Nano 2014,
8
, 5939-5952.
(9) Barman, S.; Mukhopadhyay, S. K.; Gangopadhyay, M.; Biswas, S.; Dey, S.; Singh, N. D.
P. Coumarin–Benzothiazole–Chlorambucil (Cou–Benz–Cbl) Conjugate: An ESIPT Based pH
Sensitive Photoresponsive Drug Delivery System. J. Mater. Chem. B 2015, 3, 3490-3497.
(10) Barman, S.; Das, J.; Biswas, S.; Maiti, T. K.; Singh, N. D. P. A Spiropyran–Coumarin
Platform: An Environment Sensitive Photoresponsive Drug Delivery System for Efficient
Cancer Therapy. J. Mater. Chem. B 2017, 5, 3940-3944.
(11) Venkatesh, Y.; Karthik, S.; Rajesh, Y.; Mandal, M.; Jana, A.; Singh, N. D. P. Three-
Arm, Biotin-Tagged Carbazole–Dicyanovinyl–Chlorambucil Conjugate: Simultaneous
Tumor Targeting, Sensing, and Photoresponsive Anticancer Drug Delivery. Chem. Asian J.
2
016, 11, 3482-3486.
(12) Cunningham, C. W.; Mukhopadhyay, A.; Lushington, G. H.; Blagg, B. S. J.; Prisinzano,
T. E.; Krise, J. P. Uptake, Distribution and Diffusivity of Reactive Fluorophores in Cells:
Implications Toward Target Identification. Mol. Pharmaceutics. 2010, 7, 1301-1310.
2
8
ACS Paragon Plus Environment

Page 29 of 35
Biomacromolecules
1
2
3
4
5
6
7
8
9
(
13) Ulbrich, K.; Holá, K.; Šubr, V.; Bakandritsos, A.; Tuček, J.; Zbořil, R. Targeted Drug
Delivery with Polymers and Magnetic Nanoparticles: Covalent and Noncovalent Approaches,
Release Control, and Clinical Studies. Chem. Rev. 2016, 116, 5338-5431.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(14) Kanamala, M.; Wilson, W. R.; Yang, M.; Palmer, B. D.; Wu, Z. Mechanisms and
Biomaterials in pH-Responsive Tumour Targeted Drug Delivery: A Review. Biomaterials
2016, 85, 152-167.
(15) Johnson, R. P.; Jeong, Y. –Il.; John, J. V.; Chung, C.-W.; Kang, D. H.; Selvaraj, M.;
Suh, H.; Kim, I. Dual Stimuli-Responsive Poly(N-isopropylacrylamide)-b-Poly(L-histidine)
Chimeric Materials for the Controlled Delivery of Doxorubicin into Liver Carcinoma.
Biomacromolecules 2013, 14, 1434-1443.
(16) Wang, L.; Liu, G.; Wang, X.; Hu, J.; Zhang, G.; Liu, S. Acid-Disintegratable
Polymersomes of pH-Responsive Amphiphilic Diblock Copolymers for Intracellular Drug
Delivery. Macromolecules 2015, 48, 7262-7272.
(
17) Mukherjee, S.; Sarma, J. D.; Shunmugam, R. pH-Sensitive Nanoaggregates for Site-
Specific Drug-Delivery as well as Cancer Cell Imaging. ACS Omega 2016, 1, 755-764.
18) Li, D.; Bu, Y.; Zhang, L.; Wang, X.; Yang, Y.; Zhuang, Y.; Yang, F.; Shen, H.; Wu, D.
(
Facile Construction of pH- and Redox-Responsive Micelles from a Biodegradable Poly(β-
hydroxyl amine) for Drug Delivery. Biomacromolecules 2016, 17, 291-300.
(19) Kashyap, S.; Singh, N.; Surnar, B.; Jayakannan, M. Enzyme and Thermal Dual
Responsive Amphiphilic Polymer Core-Shell Nanoparticle for Doxorubicin Delivery to
Cancer Cells. Biomacromolecules 2016, 17, 384-398.
(20) Wang, D.; Imae, T. Fluorescence Emission from Dendrimers and its pH Dependence. J.
Am. Chem. Soc. 2004, 126, 13204-13205.
2
9
ACS Paragon Plus Environment