Synthesis of a new triple-responsive biocompatible block copolymer: Self-assembled nanoparticles as potent anticancer drug delivery vehicle

Article abstract of DOI:10.1016/j.reactfunctpolym.2020.104679

There has been a continuous effort towards a synthesis of new stimuli-responsive polymer nanoparticle systems for improved cancer chemotherapy over the last decade. In this context, we have presently developed a temperature, pH, and redox-responsive amphiphilic block-copolymer capable of forming nanoparticles in the aqueous medium, targeted towards drug delivery applications. The copper-catalyzed azide-alkyne cycloaddition reaction was utilized to tie the ends of two copolymers - a thermo-responsive poly(N-isopropylacrylamide) based copolymer with an azide end group and a pH-responsive hydrophobic polymer with redox responsive disulfide bond and an alkyne end group, producing a new triple responsive amphiphilic block copolymer (PHNP) that self-assemble in water to produce nanoparticles. Upon heating above the cloud point of poly(N-isopropylacrylamide), these nanoparticles experienced further aggregation to produce larger sized particles as measured by dynamic light scattering, UV–visible spectroscopy, and scanning electron microscopy techniques. PHNP was found to be capable of encapsulating drugs like doxorubicin (DOX) and also fluorescent probes alike Nile Red. The drug release kinetics divulged that in a period of 24 h more than 90percent of the encapsulated DOX was released in pH 5.4 buffer having 10 mM glutathione (GSH) at 40 °C, an environment prevailing in cancer tissues. In vitro studies including live-dead assay and rhodamine-DAPI staining showed that PHNP was non-cytotoxic. DOX-loaded PHNP was observed to be more effective in prohibiting bone cancer cell (MG63) line in comparison to free DOX, demonstrated by the significant reduction of IC₅₀ values. The uptake studies showed that DOX-encapsulated PHNP was more effective for morphometric distortion of MG63 cells in comparison to bare DOX. Therefore, the present research on the development of a biocompatible thermal, pH, and redox-responsive polymer opens up new opportunities in the area of polymeric carrier systems for drug delivery to cancer cells.

Full text of DOI:10.1016/j.reactfunctpolym.2020.104679

Journal Pre-proof
Synthesis of a new triple-responsive biocompatible block
copolymer: Self-assembled nanoparticles as potent anticancer
drug delivery vehicle
Puja Poddar, Pritiprasanna Maity, Saikat Maiti, Satyagopal Sahoo,
Santanu Dhara, Dibakar Dhara
PII:
S1381-5148(20)30096-1
DOI:
https://doi.org/10.1016/j.reactfunctpolym.2020.104679
REACT 104679
Reference:
To appear in:
Reactive and Functional Polymers
Received date:
Revised date:
Accepted date:
19 January 2020
28 May 2020
15 June 2020
Please cite this article as: P. Poddar, P. Maity, S. Maiti, et al., Synthesis of a new
triple-responsive biocompatible block copolymer: Self-assembled nanoparticles as potent
anticancer drug delivery vehicle, Reactive and Functional Polymers (2019),
https://doi.org/10.1016/j.reactfunctpolym.2020.104679
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Synthesis of a New Triple-Responsive Biocompatible
Block Copolymer: Self-Assembled Nanoparticlesas
Potent Anticancer Drug Delivery Vehicle
Puja Poddar#, Pritiprasanna Maity$, Saikat Maiti#, Satyagopal Sahoo#, Santanu Dhara,$,*
and Dibakar Dhara#,*
#Department of Chemistry
$School of Medical Science and Technology
Indian Institute of Technology Kharagpur
Kharagpur, West Bengal 721302, India

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ABSTRACT
There has been a continuous effort towards a synthesis of new stimuli-responsive polymer
nanoparticle systems for improved cancer chemotherapy over the last decade. In this context,
we have presently developed a temperature, pH, and redox-responsive amphiphilic block-
copolymer capable of forming nanoparticles in the aqueous medium, targeted towards drug
delivery applications. The copper-catalyzed azide-alkyne cycloaddition reaction was utilized
to tie the ends of two copolymers - a thermo-responsive poly(N-isopropylacrylamide) based
copolymer with an azide end group and a pH-responsive hydrophobic polymer with redox
responsive disulfide bond and an alkyne end group, producing a new triple responsive
amphiphilic block copolymer (PHNP) that self-assemble in water to produce nanoparticles.
Upon heating above the cloud point of poly(N-isopropylacrylamide), these nanoparticles
experienced further aggregation to produce larger sized particles as measured by dynamic
light scattering, UV-visible spectroscopy, and scanning electron microscopy techniques.
PHNP was found to be capable of encapsulating drugs like doxorubicin (DOX) and also
fluorescent probes alike Nile Red. The drug release kinetics divulged that in a period of 24 h
more than 90% of the encapsulated DOX was released in pH 5.4 buffer having 10 mM
glutathione (GSH) at 40 °C, an environment prevailing in cancer tissues. In vitro studies
including live-dead assay and rhodamine-DAPI staining showed that PHNP was non-
cytotoxic. DOX-loaded PHNP was observed to be more effective in prohibiting bone cancer
cell (MG63) line in comparison to free DOX, demonstrated by the significant reduction of
IC50 values. The uptake studies showed that DOX-encapsulated PHNP was more effective for
morphometric distortion of MG63 cells in comparison to bare DOX. Therefore, the present
research on the development of a biocompatible thermal, pH, and redox-responsive polymer
opens up new opportunities in the area of polymeric carrier systems for drug delivery to
cancer cells.
KEY WORDS
Nanoparticles, RAFT polymerization, Michael addition, azide-alkyne cycloaddition, live-
dead assay, Cellular uptake, Doxorubicin, Cancer therapy

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INTRODUCTION
In recent times, self-assembled nanoparticles from amphiphilic block copolymers have been
frequently examined as potential carriers for anticancer drugs because of their ability to
encapsulate and deliver the hydrophobic drugs in cancer cells[1][2]. Low critical aggregation
concentrations (CACs) of these self-assembled nanoparticles and high stability of their
micellar[3][4] and vesicular[5][6] forms make them potent drug delivery vehicles. However,
it is also a common knowledge that the clinical success of these nanoparticles is constrained
by ineffectual drug discharge at the pathological sites[7][8]. Hence, there is always a demand
for design and synthesis of new self-assembled polymeric nanoparticles which are adequately
stable under extracellular surroundings but efficiently release its payloads in presence of
intracellular stimuli, like pH[9], temperature[10], redox agent[11] and enzyme[12] on
reaching their target sites.
Recent studies have described that the normal tissues and the cancer tissues have some
different properties, such as pH < 6.0, and temperature ~ 40 to 43 °C for cancer
circumstances (pH 7.4, and temperature ~ 37 °C for normal cells)[13][14] [15]. Also it is
reported that the cytosol and cell nucleus of cancer tissues have glutathione (GSH)
concentration (~10 mM) about 100 times higher compared to blood plasma[16][17].
Considering these issues, it is important to design nanoparticles that remain stable in the
extracellular environment but efficiently release the payloads in a proficient way in malignant
cells rather than in normal cells[18][15][19]. To exploit these differences between cancerous
and healthy cells, several reports on different kinds of stimuli-responsive polymer systems
have been published. Among these, a substantial number of pH-responsive nanocarriers were
proposed with acid-sensitive chemical linkages, like acetal[20][21], imine[22] [9],
hydrazone[23][24], and so on. These acid-sensitive linkages were cleaved in the cancer tissue
surroundings (pH~ 4.0-6.0) in order to transport the drugs at an intracellular level[25].

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Recently, our and other groups have also worked on polymer nanoparticles containing
disulfide bonds[26][27][28]. A molecule containing disulfide bonds can be reversibly cleaved
to form thiols in presence of GSH[29][30]. Temperature is other stimulant that is capable of
bringing definite structural changes in polymer nanoparticles, which may be exploited to
release an encapsulated drug molecule[31][32]. Among the thermoresponsive polymers,
poly(N-isopropylacrylamide) (PNIPA, LCST in water ~ 32 °C) and PNIPA based copolymers
are largely sought polymers [33]. Several anticancer drugs like doxorubicin (DOX), cisplatin,
paclitaxel, camptothecin were delivered via PNIPA based polymers, some of which were
recently reviewed by Wei et al[34]. For further improvement of chemotherapeutic efficiency,
couple of stimuli-sensitive systems could be connected into one polymer nanoparticle
arrangement to take advantage of the differential physiological environment that exists in
deceased cells[35][36]. Hence, multi stimuli-responsive polymer nanoparticles possess
remarkable prospect in delivering drugs to pathological sites[19].
There are several reports of temperature and pH dual-sensitive polymers constructed from
poly(N-isopropylacrylamide)[37] and poly(acrylic acid)[38] for drug delivery applications.
Recently, an enzyme and temperature dual-sensitive amphiphilic polymeric systems were
reported by the Jayakannan and coworkers[39]. Nonetheless, there are few reports on triple-
responsive polymer systems where three different stimuli were combined in a polymer
nanoparticle system for drug delivery. Zhang et al. have published pH, redox, and photo-
sensitive nanocarrier systems for doxorubicin delivery, but for these systems released drug
percentages were quite less [40]. Yulin Li group has reported a triple responsive nanogel
system for doxorubicin delivery, but they did not reveal the drug-releasing property in detail;
also the drug release percentage was low[41]. In this report, we have synthesized a new triple
responsive polymer scaffold that can disassemble and release drugs, preferably at pH 5.4,
above 40 °C as well as in presence of GSH ( 10 mM). We have synthesized two different

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polymers by RAFT technique - one of them having disulfide linkage with alkyne end group
synthesized from a pH-responsive monomer (HBCEEA) while the other is a copolymer of
NIPA and poly(ethylene glycol methyl ether methacrylate) (PEGMA) with azide end group.
Finally, alkyne-azide cycloaddition reaction was utilized to synthesize a triple-responsive
block copolymer that forms self-assembled nanoparticles that were investigated as a
promising drug delivery system (DDS) for delivering doxorubicin (DOX), an exemplary
chemotherapeutic agent, into malignant cells. The in vitro cytotoxicity, cellular uptake
efficiency, and intracellular DOX release performance of these polymer nanoparticles were
evaluated in cancer cells.
EXPERIMENTAL SECTIONS
Materials: 3,4-Dihydroxybenzaldehyde, n-bromohexane, potassium carbonate (K2CO3),
potassium iodide (KI), sodium borohydride (NaBH4), acryloyl chloride, methacryloyl
chloride,
2-mercaptoethanol,
n-butylamine,
triethylamine (Et3N), dimethyl methyl
phosponate (DMMP), acetonitrile, N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDTA),
n-isopropylacrylamide, poly(ethylene glycol methyl ether methacrylate), copper bromide
were purchased from Sigma Aldrich. Solvents and other compounds were acquired from
local sellers and purification was done following reported procedures. Synthesis of 4-
(hexyloxy)benzaldehyde
was
done
according to
an earlier
report[42].
(4-
(Hexyloxy)phenyl)methanol was prepared by reduction of 4-(hexyloxy) benzaldehyde with
sodium borohydride[43].
Synthesis of 4-((3-azidopropoxy carbonyl)-2-cyanobutan-2-yl ethyl carbonotrithionate)
(CTA1,
Scheme
1):
At
first,
we
have
synthesized
4-cyano-4-
(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid following a reported approach[44].
Then, 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid (200 mg, 0.495
mmol), 3-azido propanol (55.1 mg, 1.1 eq.) were mixed in 5 ml dry dichloromethane (DCM)

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and the mixture was chilled to 0 C. DMAP (9.08 mg, 0.15 eq.) was added to it, and stir for
15 min. Then, DCC (122.49 mg, 1.2 eq.) in 5 ml DCM was added drop by drop and stirred
for 24 h at 27 °C. After completion, the concoction was worked up with 1N HCl and DCM.
The DCM layer was dried by anhydrous MgSO4, and DCM was removed that resulted in a
yellowish liquid product which was separated from impurities by a silica gel column of
1
60−120 mesh using 30% ethylacetate-hexane as eluent (yield 50%). H NMR (600 MHz,
Chloroform-d) (Figure S1) δ 4.21 (dt, 2H), 3.42 (m, 2H), 3.34 (m, 2H), 2.65 (m, 2H), 2.52
(m, 2H), 1.94 (m, 2H), 1.70 (s, 3H)1.57 (m, 3H), 1.27 (m, 18H), 0.89 (t, J = 7.0 Hz, 3H). 13C
NMR (150 MHz, Chloroform-d) (Figure S1, Electronic Supplementary Material, ESM) δ
216.90, 171.37, 118.98, 37.09, 33.83, 31.92, 29.74, 29.62, 29.55, 29.42, 29.34, 29.07, 28.93,
28.05, 27.67, 24.92, 22.69, 14.13.
Scheme 1: Synthetic procedure for CTA1
S
CN
S
1. NaOH, CS2, Et2O
ACVA, reflux
S
S
S
S
COOH
SH
S
S
11
11
11
11
Ethylacetate, 20h
S
2. I2, Et2O, room temp.,
1h
dodecane-1-thiol
4-cyano-4-
bis-(dodecyl-
(dodecylsufanylthiocarbony
l)salfanyl pentanoic acid
sulfanylthiocarbonyl)
disulfide
DCC, DMAP
OH
N3
dry DCM, 0 °C-
3-azidopropan-1-ol
room temp.
S
CN
O
N3
S
S
11
O
CTA1
Synthesis of 2-((2-(2-(2-(prop-2-ynyloxy)ethyl)disulfanyl)ethoxy)carbonyl)ethyl benzyl
carbonotrithionate (CTA2, Scheme 2): 3-(Benzylsulfanyl thiocarbonylsulfanyl) propionic
acid (BTPA)[45] and 2-(2-(2-(prop-2-ynyloxy)ethyl)disulfanyl)ethanol (PYEDE)[46] were
synthesized according to the previously reported procedures. Subsequently, CTA2 was
prepared by DCC/DMAP coupling between BTPA and PYEDE. BTPA (400 mg, 1.468
mmol), PYEDE (338.85 mg, 1.3 eq.) were kept in 5 ml dry DCM and was chilled to 0 C.

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Scheme 2: Synthetic procedure for CTA2
S
1. NaOH, CS2
2.
SH
HOOC
S
HOOC
Ph
Br
S
Ph
3-mercaptopropanoic
acid
BTPA
Br
S
O
HO
S
S
OH
HO
S
PYEDE
NaH, THF, 0°C
room temp., 10h
2, 2'-dithiodiethanol
DCC, DMAP, dry
DCM, 0 °C -room
temp., overnight
Ph
S
S
O
S
S
S
O
O
CTA2
DMAP (338.85 mg, 0.2 eq.) was mixed to it and then the mixture was stirred for 15 min.
Then, DCC (423.37 mg, 1.4 eq.) in 5 ml DCM was mixed drop by drop. It was stirred for 24
h at 27 °C. The reaction mixture was worked up with 1N HCl solution and DCM. The DCM
layer was dried over anhydrous MgSO4 and the DCM was evacuated to get a yellowish liquid
product which was purified through a silica gel column using 30% ethylacetate-hexane in as
1
mobile phase (yield 55%). Product was characterized by H and 13C NMR spectroscopy
1
(Figures S2, ESM). H NMR (400 MHz, Chloroform-d) δ 7.42 – 7.20 (m, 5H), 4.61 (s, 2H),
4.37 (t, J = 6.5 Hz, 2H), 4.19 (s, 2H), 3.79 (t, J = 6.4 Hz, 2H), 3.63 (t, J = 6.9 Hz, 2H), 3.00 –
2.87 (m, 4H), 2.80 (t, J = 6.9 Hz, 2H), 2.45 (s, 1H). 13C NMR (150 MHz, Chloroform-d) δ
223.00, 171.24, 135.00, 129.3, 128.84, 127.93, 79.52, 74.96, 68.20, 62.99, 58.35, 41.67,
38.84, 37.35, 33.24, 31.45.
Synthesis of 4-(hexyloxy)benzyl acrylate (1c, Scheme 3). (4-(Hexyloxy)phenyl)methanol
(1.40 g, 6.72 mmol) in 20 ml dry DCM was kept in a 100 ml double neck round bottomed
flask. Et3N (1.4 ml, 10.08 mmol) was mixed to it. It was chilled to 0 C. Acryloyl chloride
(705.98 μl, 8.74 mmol) in 5 ml dry DCM was mixed drop by drop to the reaction mixture.

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Scheme 3. Preparation of pH-responsive 2-(2-((4-(hexyloxy)benzyloxy)carbonyl)ethylthio)ethyl
acrylate (HBCEEA, 1e, Scheme 3)
CHO
Br
HO
CHO
K2CO3, DMF, 18 hr.,
reflux, N2 atm.
1a
O
4-hydroxybenzaldehyde
NaBH4, THF
O
CH2OH
O
Cl
O
dry Et3N, dry DCM
O
O
1b
1c
O
n-BuNH2,
HS
OH
acetonitrile
O
Cl
OH
O
S
S
O
O
O
O
dry Et3N, dry DCM
1e
O
O
1d
HBCEEA
Then, it was stirred for overnight at 30 °C. The reaction was quenched by water; the content
was extracted with DCM (100 mL). The DCM part was dried-up by anhydrous MgSO4 and
purified by a silica gel column using 10% ethylacetate-hexane in as eluent to get a yellowish
1
product (l). The formation of 4-(hexyloxy)benzyl acrylate was established by H NMR and
1
13C NMR (Figure S3, ESM). Yield = 1.695 g (96.3 %). H NMR (400 MHz, Chloroform-d) δ
7.31 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 6.43 (dd, J = 17.3, 1.4 Hz, 1H), 6.14 (dd, J
= 17.4, 10.4 Hz, 1H), 5.82 (dd, J = 10.4, 1.5 Hz, 1H), 5.13 (s, 2H), 3.95 (t, J = 6.6 Hz, 2H),
1.78 (dt, J = 14.5, 6.6 Hz, 2H), 1.45 (dt, J = 14.8, 7.7 Hz, 2H), 1.33 (dt, J = 7.1, 3.5 Hz, 4H),
0.94 – 0.87 (m, 3H). 13C NMR (100 MHz, Chloroform-d) δ 166.26, 159.40, 131.01, 130.24,
128.59, 127.84, 114.65, 68.18, 66.37, 31.71, 29.34, 25.85, 22.74, 14.17.
Synthesis of 4-(hexyloxy)benzyl 3-(2-hydroxyethylthio)propanoate (1d, Scheme 3). N-
Butylamine (79.10 μl, 0.8 mmol) and DMMP (28.9 μl, 0.267 mmol) were added to 2-
mercaptoethanol (225.35 μl, 3.2 mmol) in 20 ml acetonitrile. 4-(Hexyloxy)benzyl acrylate

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(700 mg, 2.67 mmol) was added dropwise and purged with nitrogen gas for 15 min. Then the
concoction was stirred at 40 C for 14 h. After completion, acetonitrile was removed and a
yellowish liquid product was obtained through extraction from organic solvent followed by
column chromatography (1:4 ethylacetate-hexane). The formation of compound 1d was
1
1
established by H NMR and 13C NMR (Figure S4, ESM) (Yield 96.86 %). H NMR (400
MHz, Chloroform-d) δ 7.28 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 5.07 (s, 2H), 3.95 (t,
J = 6.6 Hz, 2H), 3.72 (t, J = 5.9 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H), 2.72 (t, J = 5.9 Hz, 2H),
2.64 (t, J = 7.2 Hz, 2H), 1.45 (dt, J = 15.0, 6.5 Hz, 2H), 1.33 (dt, J = 7.0, 3.6 Hz, 4H), 0.90 (t,
J = 6.9 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 171.95, 159.43, 130.31, 127.60,
114.64, 68.17, 66.67, 60.61, 35.50, 35.06, 31.69, 29.31, 26.69, 25.82, 22.72, 14.16.
Synthesis of HBCEEA (1e, Scheme 3): Synthesis of acid-labile 2-(2-((4-
(hexyloxy)benzyloxy)carbonyl)ethylthio)ethyl acrylate (HBCEEA) was carried out as shown
in scheme 3. 4-(hexyloxy)benzyl 3-(2-hydroxyethylthio)propanoate (900mg, 2.64 mmol) in
25 ml dry DCM was kept in a 100 ml two neck round bottomed flask. Et3N (552.32 μl, 3.96
mmol) was mixed to it. The mixture was cooled to 0 C. Acryloyl chloride (277.698 μl, 3.44
mmol) in 5 ml dry DCM was dropwise mixed to the reaction mixture. Then, the mixture was
stirred for 24 h at 30 C. After the end of reaction, water was added for quenching and the
content was extracted with DCM (100 mL). The product was dried over MgSO4, solvent was
removed, purified by column chromatography (60-120 mesh) using 10% ethylacetate-hexane
1
as mobile phase. The formation of compound 1e was established by H NMR and 13C NMR
1
(Figure S5, ESM). Yield = 940 mg (90 %). H NMR (400 MHz, Chloroform-d) δ 7.30-7.26
(m, 2H), 6.87 (d, J = 8.6 Hz, 2H), 6.41 (dd, J = 17.3, 1.3 Hz, 1H), 6.15-6.07 (m, 1H), 5.84
(dd, J = 10.4, 1.4 Hz, 1H), 5.07 (s, 2H), 4.30 (t, J = 6.8 Hz, 2H), 3.95 (t, J = 6.6 Hz, 2H), 2.85
(t, J = 7.3 Hz, 2H), 2.79 (t, J = 6.8 Hz, 2H), 2.64 (t, J = 7.3 Hz, 2H), 1.81-1.73 (m, 2H), 1.49-
1.41 (m, 2H), 1.36-1.30 (m, 4H), 0.93-0.88 (m, 3H). 13C NMR (100 MHz, Chloroform-d) δ

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171.62, 165.88, 159.30, 131.19, 130.15, 128.13, 127.56, 114.53, 68.06, 66.48, 63.60, 34.98,
31.57, 30.56, 29.19, 27.27, 25.71, 22.60, 14.03.
Scheme 4: Synthetic procedure of poly(HBCEEA) homopolymer (PHB)
O
S
O
Hex
O
S
O
O
O
Ph
S
S
O
S
S
O
CTA1
HBCEEA
AIBN, 1,4-dioxane,
70 °C, 24 h
O
O
O
O
S
Hex
O
S 14
Ph
O
O
S
S
O
S
S
PHB
Synthesis of Poly(HBCEEA) Homopolymer with CTA1 (PHB, Scheme 4): HBCEEA (395 mg,
1.00 mmol), CTA1 (29.92 mg, 0.067 mmol) and AIBN (3.28 mg, 0.02 mmol) were taken in
1.5 ml 1,4-dioxane. After purging by nitrogen for 10 minutes, the mixture was stirred in oil-
bath at 70 C for 26 h. Thereafter, the polymerization was quenched, the polymer was
purified by dialysis (MW Cut-off 3000-5000 Da), freeze dried to obtain yellow viscous liquid.
1
The composition and molecular weight of the polymer were determined by H NMR and gel
1
permeation chromatography (GPC) (Figure S6, ESM and Figure 2 respectively). H NMR
(DMSO-d6, 400MHz): δ7.35 (m, Ph ring of CTA1), 7.12 (m, 5H, Ph ring of CTA1), 6.85 (br,
repeating Ph ring of HBCEEA), 4.97 (br, repeating unit of benzylic CH2 of HBCEEA), 4.68
(2H, Ph-CH2- of CTA1), 1.66 (br, polymer backbone), 1.26-1.35 (br, repeating unit of
HBCEEA), 0.84 (br, -CH2CH3 repeating unit of HBCEEA).
Synthesis of poly(NIPA-r-PEGMA) (PNP, Scheme 5): Poly(NIPA-r-PEGMA) (PNP) was
synthesized following an earlier report[47]. Briefly, N-isopropylacrylamide (1.00 g, 8.84
mmol) was taken in 5 ml 1,4-dioxane. CTA2 (45.76 mg, 0.094 mmol) PEGMA (270.75 mg,

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0.56 mmol) and AIBN (0.2 eq, 3.09 mg) were mixed to it. After purging by nitrogen gas for
10 min the mixture was stirred in oil-bath at 70 °C for 24 h. After quenching, the polymer
was recovered by precipitation from ice-cold diethyl ether. It was filtered and dried under
1
vacuum to get a light yellowish product (s). The polymer was characterized by H NMR and
1
GPC (Figure S7, ESM and Figure 2 respectively) H NMR (CDCl3, 400MHz): δ 3.99 (br, -
CH(CH)3 of NIPA), 3.65 (br, -CH2-CH2-O of PEGMA), 3.37 (s, -CH3 repeating unit of
PEGMA), 1.61 (br, CHCH2 polymer backbone), 1.13 (br, CH(CH3)2 of NIPA), 0.87 (t, 3H,
CH3(CH)11 of CTA2).
Scheme 5: Synthesis of poly(NIPA-r-PEGMA) (PNP)
O
H
O
CH3
~8
N
O
S
S
N3
O
11
O
O
CN
CTA2
S
PEGMA
NIPA
AIBN, 1,4-dioxane,
70° C, 24 h
~8
H3C
O
O
O
CN
S
N3
O
r
S
S
11
82
4.6
O
O
NH
PNP
Synthesis of Poly[HBCEEA-b-(NIPA-r-PEGMA)] (PHNP, Scheme 6): PNP (50 mg, 0.004
mmol), PHB (23.88 mg, 0.004 mmol) were taken in 3 ml dry DMF. To this mixture,
PMDETA (0.56 μl, 0.65 eq) and CuBr (0.17 mg, 0.3 eq) were added. The reaction was
carried out at 25 °C for 24 h. Then the reaction mixture was dialyzed (MW Cut-off 10000-
12000 Da) by pH 7.4 phosphate buffer solution and then freeze dried to get final polymer (s).
1
The final polymer was characterized by H NMR spectroscopy (Figure 1) and by GPC for
molecular weight determination (Table 1).

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Scheme 6: Synthesis of poly[HBCEEM-b-(NIPA-r-PEGMA)] (PHNP)
~8
H3C
O
O
O
O
S
O
O
S
O
CN
S
Hex
N3
O
r
Ph
O
O
O
14
S
S
11
82
4.6
S
O
S
S
S
O
O
NH
CuBr, PMDETA
room temp.
PNP
PHB
N
N
N
PNP
PHB
PHNP
Figure 1: 1H NMR spectrum of PHNP in DMSO-d6
Instrumentation and Methods:
NMR Spectroscopy: Bruker Avance II spectrometer was operated at 400 MHz and 25 °C to
1
record H NMR and 13C NMR spectra in CDCl3 or dimethyl sulfoxide (DMSO)-d6 as a
solvent. The residual solvent signal was considered as an internal standard.

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Gel Permeation Chromatography (GPC): The molecular weight and dispersity (Đ = Mw/Mn)
of the polymers was determined using a triple detector GPC, Viscotek TDAmax system
furnished with differential pressure viscometry, refractive index and dual-angle light
scattering (λ = 670 nm, 90° and 7°) detectors along with an Agilent 1200 model isocratic
pump. Tetrahydrofuran (THF) was used as the mobile phase with a flow rate of 1 mL min−1
at 25 °C. The absolute molecular weight of the experimental polymers was determined by
putting dn/dc values of the synthesized polymers, which were obtained from the RI values of
four solutions of various concentrations of each polymer from a differential refractometer
-1
(WGE DrBures, Germany). A polystyrene standard (Mn = 105100 g mol , Ɖ = 1.02, dn/dc =
0.185 mL g-1, and [η] = 0.48 dL g-1 at 25 °C in THF, provided by the supplier) was used to
calibrate detector signals.
Size Determination: Hydrodynamic size and size distribution was determined by dynamic
light scattering measurement using Malvern Zetasizer Nano equipment with a detector at
173° angle. A sample chamber was temperature-controlled by a 4.0 mW He−Ne laser
operating at 632.8 nm. Instrumental software was used to process the raw data to determine
the hydrodynamic diameter (Dh) and size distribution. Field emission scanning electron
microscopy (FESEM) measurement was performed by Field Emission Gun-Scanning
Electron Microscope Laboratory (Merlin) for morphological study of polymeric
agglomerates.
Absorbance and Fluorescence Assessments: A Shimadzu (model number, UV-2450)
spectrophotometer was used to measure UV-vis absorbance and fluorescence spectra were
recorded using a Shimadzu RF-6000 spectro-fluorophotometer. These spectroscopic
measurements were carried out by keeping 1 cm path length using a quartz cuvette at 25 °C.
Turbidity Measurement: A Cary 5000 UV-vis-NIR spectrophotometer (Varian Scientific
Instruments) equipped with a digital heat controller was utilized to measure optical

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transmittance at 500 nm. The sample was heated from 28 °C to 50 °C temperature range and
then cooled from 50 °C to 28 °C to record the cloud temperature.
Critical Aggregation Concentration (CAC) Measurement: PHNP block copolymer (5.0 mg)
was dissolved in 500 μl of THF. Phosphate buffer solution pH 7.4 (1.0 mL) was added to this
solution dropwise. It was then transferred to a dialysis bag (MW Cut-off 10000-12000), and
then dialyzed against pH 7.4 buffer (200 mL) for 48 h. The CAC of the nanoparticles was
determined by using a hydrophobic probe (Nile red). Nile red concentration was affixed as
1.26 mM, and the concentration of polymer was assorted from 0.001 to 0.1 mg/ml. Typically,
a fixed quantity of Nile red solution was taken into a 1 ml eppendorf from its concentrated
solution in methanol. The methanol was then completely dried up and polymer solutions of
different concentration were mixed to the eppendrofs having the Nile red. After sonication for
5 min, the solutions were kept overnight to equilibrate. The Nile red solutions were excited at
552 nm to record the emission spectra. The maxima of emission intensity values were plotted
against polymer concentrations and the CAC was measured from the breakpoint.
In Vitro Doxorubicin Encapsulation in Nanoparticles: Encapsulation of the hydrophobic
drugs in the inner crux of PHNP nanoparticle was examined using doxorubicin (DOX).
DOX·HCl (0.5 mg), neutralized by Et3N and the PHNP block copolymer (0.4 mg) were
dissolved in DMSO (1.0 mL). This solution was mixed to phosphate buffer (pH 7.4) (5.0 mL)
and the mixture was stirred at 25 °C for 12 h. This mixture was then taken into a dialysis bag
(MW Cut-off 10000-12000 kDa). It was then extensively dialyzed against 200 mL phosphate
buffer solution (pH 7.4) for 48 h. This DOX encapsulated polymer solution was freeze-dried
in a lyophilizer. Then, this dried drug-encapsulated nanoparticle was dissolved in DMF (1.0
mL) and the drug encapsulated efficiency (DEE) and drug encapsulated content (DEC) were
determined from absorbance measurement. The extent of encapsulated DOX was determined

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from a calibration curve of a standard solution of DOX in DMF. DEE and DEC were
estimated by following the equations:
Weight of encapsulated drug
DEE (%) =
DEC (%) =
X 100%
Weight of DOX in feed
Weight of DOX in nanoparticle
X 100%
Weight of DOX loaded nanoparticle
MG63 and Fibroblast Cell Culture: MG63 cell lines and fibroblast were purchased from
National Centre for Cell Sciences (NCCS), Pune, India and cultured according to our
previous report[48]. Cells were accumulated from tissue culture medium using 0.25% trypsin
in 1 mM EDTA (Himedia) after 70% confluence, and cell density was estimated. The equal
number of cells (1×105) was plated on samples in a 30 mm plate as well as 96 well plates.
Cell Viability and Cytotoxicity Assessment: MTT assay was implemented on various
concentration of free DOX and DOX-encapsulated PHNP (DOX-POLY) nanoparticles on the
proliferation of MG63 and fibroblast cell lines at one day and three days interval to identify
the half-maximal inhibitory concentration (IC50) values. In short, 96-well tissue culture plates
were seeded by cells in the algorithmic aspect (1× 105 cells per well) to cultivate up to 16 h at
CO2 incubator. Accordingly, the cells were treated by different doses of free DOX and DOX-
loaded PHNP for 48 h. By MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide, Sigma Aldrich) reduction assay, Cell viability assessment was done by our earlier
reported procedure[48].
In Vitro Cytocompatibility Study: The in vitro cytocompatibility of triple responsive PHNP
nanoparticle was assessed by Live–Dead and Rhodamine and DAPI staining (Invitrogen,
Pleasanton, CA 94566, USA) on MG63 cells[49]. The assay was performed on PHNP
nanoparticle and TCP as control at one day and three days’ interval. After a certain period,

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rhodamine-phalloidin (Life Technologies, Invitrogen) and DAPI (4′,6-diamidino-2-
phenylindole, Life Technologies, Invitrogen) staining was done to assess cell morphology.
Details of Rhodamine and DAPI staining procedure was done by previously reported
method[49]. For Live-Dead assay, a solution consisting of 2 µM calcein aceto-methoxy
(Calcein AM) and 4 µM ethidium homodimer (ETD) was used to incubate (30 min) the cell-
gel assembly at ambient temperature. A fluorescent microscope (Carl Zeiss, Germany) with
excitation filters at 450–490 nm (green, Calcein AM) and 510–560 nm (red, ETD) using ZEN
software was used to monitor the cell-gel construct after incubation.
Uptake Studies: Cellular uptake of bare DOX along with DOX-POLY nanoparticle from
aqueous solution was observed at various time intervals (15 min to 24 h) under the
fluorescence microscope. Subsequently, the cellular uptake of DOX from DOX-POLY
nanoparticles at different temperatures was also evaluated by incubating DOX-POLY loaded
MG63 cells at 25 ºC, 37 ºC, and 40 ºC. The endocytosis of DOX-encapsulated nanoparticles
was studied under a fluorescence microscope (Carl Zeiss, Germany) with excitation filters of
580 nm (red channel) using ZEN software.
RESULTS AND DISCUSSION
Synthesis of pH-Responsive Monomer, HBCEEA: A new pH-responsive monomer was
prepared following steps presented in Scheme 3. First, 4-(hexyloxy)benzaldehyde was
synthesized from 4-hydroxy benzaldehyde and n-hexyl bromide in the presence of potassium
carbonate. 4-(Hexyloxy)benzaldehyde was then reduced to 4-(hexyloxy)phenyl methanol in
the presence of sodium borohydride. Then, 4-(hexyloxy)benzyl acrylate was prepared by
acrylation of 4-(hexyloxy)phenyl methanol which was further treated with 2-mercaptoethanol
and acryloyl chloride in successive steps to form a new pH-responsive 2-(2-((4-
bis(hexyloxy)benzyloxy)carbonyl)ethylthio)ethyl acrylate (HBCEEA). The formation of

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1
HBCEEA was proved by NMR spectroscopy. In H NMR spectrum (Figure S5, ESM), the
peaks at 7.30-7.26 ppm and 6.87 ppm are due to Ar proton, and the peak at 5.07 ppm is due
to benzylic CH2. The peaks at 6.41, 6.15-6.07, and 5.84 ppm for vinylic proton confirmed the
presence of acrylate moiety in HBCEEA. The peaks at 2.85, 2.79, 2.64 ppm are due to CH2
groups adjacent to the Sulphur group. The peaks between 0.88 to 3.95 ppm correspond to an
alkyl group of the O-hexyl group. The formation of the desired monomer was also
established from the 13C NMR spectrum (Figure S5 (bottom)).
Synthesis of Triple-Responsive Block Copolymer (PHNP): We have used RAFT (reversible
addition-fragmentation chain transfer) for polymerization of the pH-responsive monomer
HBCEEA in the presence of CTA1 with alkyne end (Scheme 4). Comparison between the
number of protons in the end group with the number of protons in the repeating units from 1H
NMR spectra provided a number average molecular weight (Mn) of poly(HBCEEA)
homopolymer (PHB) as 5950 g/mol (Table 1). As seen from Figure S6 (ESM), no peak was
found at δ 6.39, 6.15, and 5.85 ppm, which suggests that no monomer was left. At the same
time, poly(NIPA-r-PEGMA) (PNP) was synthesized by RAFT polymerization of a mixture
1
of NIPA and PEGMA in the presence of CTA2 having azide end (Scheme 5). The H NMR
of the synthesized PNP is displayed in Figure S7 (ESM). Peaks at 3.99, 3.37, and 0.87 ppm
are due to protons of isopropyl group of NIPA, terminal CH3 group of PEGMA, and terminal
CH3 group of CTA2, respectively. In this case, the absence of proton peaks between 5.5 to
6.5 ppm confirmed the successful polymerization of the monomers. Mn value for PNP was
1
calculated from H NMR spectra as 12000 g/mol. Finally, a new triple responsive block
copolymer (PHNP) was prepared by an alkyne-azide cycloaddition reaction between alkyne
terminated PHB and azide terminated PNP. All characteristic peaks of the individual
1
precursor polymers are present in the H NMR spectrum of PHNP (Figure 1). Peaks between
7.22-6.84 ppm are from PHB, while peaks between 3.86-3.24 ppm are from PNP. GPC

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analysis revealed a monomodal distribution with narrow dispersity (Đ) for all of the starting
polymers as well as the final block copolymer, confirming good control over polymerization
(Figure 2). Mn value of PHNP was nearly equal to the sum of the Mn of PHB and PNP, which
confirmed tieing of the precursor polymers, PHB and PNP, by azide-alkyne cycloaddition
reaction. Molecular weight data of the polymers are summarized in Table 1.
Table 1: Molecular weight for the polymers in this work
a
b
Polymer Composition
Abbreviation
Mn
Mn
Ɖb
(g/mol)
(g/mol)
Poly(HBCCEA)
PHB
PNP
5950
5850
1.13
1.15
1.14
Poly(NIPA-r-PEGMA)
12000
13300
Poly[(HBCCEA)-b-(NIPA-r-PEGMA)]
PHNP
17950
19500
Mn = Number average molecular weight. Ɖ = dispersity (molecular weight distribution). aMeasured from 1H NMR
spectroscopy. bAquired fromGPC.
Figure 2: GPC Chromatogram of PHB, PNP, PHNP.
Self-Assembly of the Block Copolymer: It is common knowledge that the amphiphilic
polymers form stable nanostructures in aqueous system by self-assembly above a specific
concentration, which is often called critical aggregation concentration (CAC). The value of
hydrodynamic diameter (Dh) of PHNP at 25 C was found to be ~125 nm, whereas the Dh
value of PNP at 25 C was 8.7 nm (polymer concentration of 0.1 mg/ml). This suggests that
there is the formation of some self-assembled nanoparticles by PHNP polymer chains. A
fluorescent probe, Nile red was used to determine the CAC of PHNP in phosphate buffer

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medium (pH 7.4). Nile Red gives a considerably faint emission maximum at 660 nm (λex
=
550 nm) in water. No significant difference in Nile red emission spectra was noticed at low
polymer concentration range, indicates that the surrounding of Nile Red molecules
experiences a comparable situation as in aqueous medium. After a specific concentration of
the PHNP, the emission intensity of Nile Red increased drastically in a non-linear fashion,
which was accompanied by a blue shift of the emission maxima. These results indicate that
the Nile Red must be encapsulated in the hydrophobic domain formed by the assembly of the
PHNP block copolymers. From the point of inflection of Nile red emission intensity versus
polymer concentration plot, the CAC of the PHNP block copolymer (~ 0.02 mg/mL, Figure
3b) was calculated.
Figure 3: (a) Florescence emission spectrum of Nile red in solutions of different PHNP concentration
(mg/ml) in PBS buffer (pH 7.4) at 25 C. (b) Plot of Nile Red emission intensity at 620 nm against
PHNP concentration.
The PNP block in PHNP is also thermo-responsive due to the presence of PNIPA. Solutions
of PNP random copolymer were found to be transparent below 40.5 C, above which the
transparency decreased significantly (Figure S8, ESM). The cloud point was considered to be
the temperature at which a 50 % drop in transmittance was observed. The hydrodynamic size
of PNP was found to increase significantly above the cloud point (Figure S9, ESM). Lower
critical solution temperature (LCST) of PNIPA is known to be 32 C in water[50], but, in this
case, due to the random presence of highly hydrophilic PEGMA, the LCST of PNP was

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increased to 40.5 C. Thermally-induced self-aggregation of PHNP was examined by
following optical transmittance at 500 nm with increasing solution temperature. PHNP block
copolymer solutions were translucent below 39 °C, but above this temperature the solutions
became cloudy.
Figure 4: Plot of (a) optical transmittance (%) (b) hydrodynamic diameter (Dh) of PHNP copolymer
as a function of temperature in pH 7.4 medium.
In the presence of the hydrophobic PHB block, the cloud point of PHNP in water was
lowered by a couple of degrees compared to the PNP block (Figure 4a). Dynamic light
scattering (DLS) was efficiently utilized to examine this self-aggregation of PHNP block
copolymer. Figure 4b demonstrates the hydrodynamic size distribution of PHNP in phosphate
buffer solution (pH 7.4) as a function of temperature. Below a particular temperature, the size
remained the same, about 125 nm. As temperature reached close to the CAC of the PHNP (39
C), there was a drastic increase in the size. At higher temperatures, the PNP block became
hydrophobic by nature and consequently, the PHNP block copolymers started undergoing
self-aggregation through PNP chains forming large particles. However, these aggregated
particles did not precipitate from solution as indicated by a constant value of hydrodynamic
size and optical transparency over a long time.
Field emission scanning electron microscopy (FESEM) of the PHNP block copolymer
solution was also performed at two different temperatures, one below the cloud point (at 25

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C) and other above the cloud point (at 40 C) for probing of the nature of the self-aggregated
nanostructures. From FESEM, round shaped assemblies of size ~110 ± 10 nm (at 25 C,
Figure 5a) and ~700 ± 50 nm (at 40 C, Figure 5b) were observed at temperatures below and
above the cloud point respectively. Particle size obtained from atomic force microscopy
(AFM) (Figure 5c and Figure 5d for 25 C and 40 C, respectively) matched well with
FESEM data. The particle size obtained from FESEM and AFM was slightly lower than that
acquired from the DLS measurements, which can be interpreted as the dehydration of
nanoparticles during the drying process onto the aluminium foil.
Figure 5. Images of PHNP nanoparticles at pH 7.4: FESEM at (a) 25 °C (b) 40 °C; AFM at (c) 25 °C
(d) 40 °C.
pH and GSH Responsive Solution Behavior. In mildly acidic condition, β-thiopropionate
group undergoes hydrolysis at a comparatively slower rate than many other acid-labile
components[51][52][53]. However, it can be easily synthesized by thiol acrylate Michael
Addition reaction [52]. Due to inductive effect of the sulfur, a fractional positive charge is
created on the adjacent carbonyl carbon, which facilitates the hydrolytic cleavage of the β-
thiopropionate moiety[48]. As
a
result, for PHNP, at low pH, hydrophobic
bis(hexyloxy)phenyl methanol was freed from the polymer chain that resulted in deformation
and swelling of the nanoparticles. DLS study (Figure 6a) shows that at 25 C and in the
absence of GSH, the diameter of the nanoparticles decreased with time after the pH of the
medium was lowered to pH 5.4. To examine the effect of GSH, the nanoparticles were placed
in buffer containing GSH (~10 mM) at pH 7.4 and 25 °C. Figure 6b demonstrates that the
size of the nanoparticles decreased with time. This might have occurred due to breaking of

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the -S-S- bonds present in the polymer resulting in degradation of the self-assembled
structure. It may be noted that as both -S-S- bonds and β-thiopropionate moieties are present
in the hydrophobic domain of the nanoparticles, the access to these labile functionalities in an
aqueous medium is somewhat restricted, and as a result, the degradation of the nanoparticles
happened slowly.
Figure 6: Plot of hydrodynamic size distribution of PHNP as function of time (a) at pH 5.4, 25 °C,
without GSH (no GSH); (b) at pH 7.4, 25 °C, 10 mM GSH.
Doxorubicin encapsulation and in vitro release study: Doxorubicin (DOX) was used as
model drug in order to demonstrate the capability of the thermoresponsive PHNP
nanoparticles to load hydrophobic anti-cancer drug in the hydrophobic domain. The drug
encapsulation content (DEC) of DOX was determined using absorption spectroscopy as ~9%
and drug encapsulation efficiency (DEE) was found to be 80% at 25 C. The effect of DOX
on the self-assembly behavior of thermoresponsive PHNP was further investigated by
evaluating the optical transmittance of the DOX-loaded PHNP nanoparticles as a function of
temperature and the result is presented in Figure S10 (ESM). The aggregation temperature of
the drug-loaded PHNP nanoparticles was determined to be as ~39 °C similar to the PHNP
nanoparticles.
The release of DOX from the PHNP nanoparticles in presence of different stimuli was
determined and the data are presented in Figure 7. Under physiological conditions (phosphate

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buffer solution, pH 7.4, without GSH) only 16% of the loaded DOX was released in 24 h.
Therefore, it can be said that the drug-encapsulated scaffold was stable enough under normal
body environment. Next, we studied the release behavior in the presence of individual
stimulus before applying all three stimuli together. The cumulative release behaviour of
DOX-POLY at various media are shown in Figure 7. Addition of 10 mM GSH to DOX-
POLY at 25 °C and pH 7.4, resulted in release of ~ 40 % of the encapsulated drug. The drug
release was further enhanced to ~ 45 % by lowering the pH to 5.4 of the medium with no
GSH at 25 °C. To find out the effect of temperature on the drug release efficiency,
experiments were carried out at 40 °C, pH 7.4 and without GSH. In this case, ~70 % of the
loaded drug was released. Thus, the release of DOX from the PHNP nanoparticles was
improved by either lowering the pH or presence of GSH or increasing the temperature of the
medium. However, of the three stimuli, temperature had the most significant effect. Finally,
DOX release efficiency was determined in presence of three external stimuli - at 40 °C, pH
5.4, and 10 mM GSH. In this case, ~93% of the encapsulated drug was released within 24 h
that confirmed the effectiveness of the present PHNP block copolymer nanoparticles as a
promising candidate for the delivery of anticancer drugs. After establishing the drug release
capability of PHNP nanoparticles in vitro (PBS buffer), the system was tested for its
capability in vitro (biological samples).

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Figure 7: Cumulative release of DOXfrom DOX-POLY nanoparticle at different condition.
Biological Studies of DOX-Encapsulated PHNP nanoparticles in MG63 bone cancer Cells.
Assessment of IC50 of DOX and DOX-POLY Nanoparticles: To assess the cell inhibitory
potency of DOX-POLY nanoparticle, MTT assay was performed on normal cell lines
(fibroblast) as well as on bone cancer cells (MG63) to identify the half-maximal inhibitory
concentration (IC50) by treating different concentration of bare DOX and DOX-POLY
nanoparticles (0.1-1.0 µM) (Figure S11, ESM). In the fibroblast cell line, the IC50 value of
bare DOX (0.879 µM) was increased to 0.931 µM for DOX-POLY nanoparticles (Figure 8a).
The IC50 value for both bare DOX and DOX-POLY nanoparticles was lower for MG63 cell
lines than fibroblast cell line, which confirmed that the effectiveness of DOX towards cancer
cells both in free as well as in encapsulated form. However, the IC50 value of DOX-loaded
PHNP nanoparticle in MG63 cell lines was significantly lower (0.257 µM) than free DOX
(0.563 µM) (Figure 8b) which showed the improved efficiency of DOX-POLY towards
cancer cells. One probable reason for this may be due to directed targeting of DOX-loaded
nanoparticles to the specific endocytic compartment through selective endocytic
compartmentalization and concurrent release of drug and localization thereof within the cell

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nucleus, possibly due to the enhanced permeability and retention effect (EPR effect) as
compared to free DOX.
Figure 8: Absorbance value during MTT assay for (a) IC50 calculation for bare DOX (DOX) and
DOX-POLY in MG63 (MG) cell line and fibroblast (FB) cell line (b) bare DOX (DM) and DOX-
POLY on MG63 cell (DPM) at different concentration mentioned at the top.
The % of cell viability assessment at a various concentration (0.1 µM, 0.2 µM, 0.4 µM, 0.6
µM, 1 µM) of bare DOX and DOX-POLY showed that DOX-POLY was more effectively
internalized to the MG63 cells and inhibit its proliferation with increasing concentration.
With increasing the drug concentrations, the DOX-POLY displayed a considerable
improvement in efficiency in contrast to that of bare DOX. For example, the bare DOX
exhibited 38 % cell killing, while the DOX-POLY nanocarrier achieved approximately 60 %
cell killing at 0.4 µM concentration (Table 2 and Figure S12, ESM). Thus, DOX-loaded
PHNP nanoparticles were found to be less toxic to normal cells, while was able to effectively
inhibit the growth of cancer cells Therefore, DOX-POLY nanoparticles could be
advantageous for drug administration purposes and could be efficient nanocarriers for
anticancer theranostics.

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Table 2 Cell viability % of bare DOXand DOX-POLY on MG63 cells at various concentrations.
Cell viability %
DOXconc.
(µM)
Free DOX
DOX-POLY
84.15
0.1
0.2
0.4
0.6
1.0
96.95
79.61
61.50
50.67
39.70
68.24
42.13
27.74
15.78
Assessment of cytotoxicity of PHNP nanoparticles: Cytotoxicity assessment of PHNP
nanoparticles on the chondrocyte cell (normal bone cartilage cell) (Figure S13, ESM) and the
MG63 cell line (Figure 9) show increases in the number of cells after 72 h, which indicates
the biocompatibility of PHNP nanoparticles. To assess the potency of PHNP nanoparticles as
drug delivery vehicles, Live-Dead, Rhodamin-DAPI staining were performed to analyze the
adhesion and morphological characteristics of chondrocyte cell as well as MG63 cells in the
presence of PHNP nanoparticles. After three days of culture, the PHNP nanoparticle was
established to be non-toxic, exhibited good cell adhesion potential and a higher rate of
proliferation without extensive alteration of cell morphology as assessed through Rhodamine-
DAPI and Live-Dead staining. Figure 9 showed relatively higher cell adhesiveness and
proliferation after three days of culture as compared to one-day culture demonstrating the
cytocompatible character of the PHNP nanoparticles. Nonexistence of a significant number of
dead cells, as indicated by the absence of red stain after live-dead staining, that also indicates
its non-cytotoxic character (Figure 9a, 9b). Subsequently, one day along with three days of
culture, Rhodamine-DAPI staining affirmed identical morphology of chondrocyte cells
(Figure S13, ESM) and MG 63 cells (triangle-like) (Figure 9c, 9d) in the presence of PHNP
nanoparticle.

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Figure 9: Estimation of cell proliferation and cytotoxicity of PHNP nanoparticles on MG63 cell.
Adhesiveness, growth and morphological characteristics of MG63 seeded with PHNP nanoparticles
displayed infinitesimal dead cells with invariable cellular morphology - (a, b) Live-Dead analysis and
(c, d) Rhodamine–DAPI staining (Pseudo Color).
Time-dependent cellular uptake study: To further establish the comparative cellular uptake,
MG63 cells were incubated with both DOX-POLY nanoparticle and bare DOX. Endocytosis
of drug (after 15 min, 30 min, 1 h, 3 h, 24 h, respectively) was noticed under a fluorescence
microscope with excitation filters of 560 nm (red channel) using ZEN software. Figure 10
showed that the endocytosis of the drug was initiated at 15 min in the cytoplasm and
subsequently increased with time. From Figure 10, it is shown that the cell morphology was
changed as an outcome of the penetration of DOX into the nucleus. As DOX is a nucleus
targeting drug, bare DOX is expected to permeate inside the nucleus directly, but in the case
of DOX-POLY nanoparticle, the drug was released by a slow and sustained manner through
the cytoplasm to the nucleus and after 24 h distinctive cell distorted morphology was
observed.

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Figure 10: Uptake of bare DOX (DOX) and DOX-POLY (DOX-Poly) by MG63 cells at different
time evidenced by fluorescence microscope.
The temperature dependent drug release behavior was monitored by the microscopic
technique. DOX release from DOX-POLY nanoparticle in MG 63 cells (Figure 11) showed

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that drug release at 25 ºC was very low whereas at 40 ºC incubation (incubation time ~30min)
depicted higher release in comparison to 37 ºC.
Figure 11: Temperature dependent discharge of DOX from DOX-POLY in MG63 cell. BF: Image in
bright field mode, FL: image in fluorescence mode. Scale bar: 50 µm.
CONCLUSION
A triple-responsive amphiphilic block-copolymer (PHNP) was designed and synthesized by
tieing the ends of two copolymers - a thermo-responsive poly(N-isopropylacrylamide) based
copolymer with an azide end and a pH-responsive hydrophobic polymer with redox
responsive disulfide bond and an alkyne end. The self-assembled nanoparticles formed by
this amphiphilic block copolymer in aqueous medium was capable of encapsulating
doxorubicin (DOX), an anticancer drug, and also releasing higher than 90% of the
encapsulated DOX in a period of 24 h in a pH 5.4 buffer having 10 mM glutathione at 40 °C,
an environment prevailing in cancer tissues. PHNP was established to be non-cytotoxic to
both fibroblast, and bone cancer cells (MG63). In vitro studies including determination of
IC50 values, Live-Dead assay, and rhodamine-DAPI staining showed that DOX-POLY was