Journal of Materials Chemistry B
Paper
was stirred and refluxed at 60 1C for 12 h. NMCS–COOH was Encapsulation of the drug using click chemistry
obtained by centrifugation and washing of the product. Then,
Here, 10 mg of NMCS-N3 was first incubated in PBS (10 mM,
20 mL, pH 7.4), and 10 mg of gemcitabine hydrochloride (GEM)
was added and incubated for 24 nh. Then, gemcitabine-loaded
NMCS were recovered using centrifugation and washing with
PBS buffer followed by drying under vacuum. To quantify the
loading ratio of gemcitabine, the supernatant was collected; the
concentration of gemcitabine was determined from a UV-VIS
spectrophotometer at 264 nm. Next, 40 mg of NMCS-GEM
was dispersed in 20 mL of THF under a N2 atmosphere. A
photo-cleavable linker molecule (1-(5-methoxy-2-nitro-4-(4-oxo-
4-(prop-2-yn-1-ylamino)butoxy)phenyl)ethyl 4-oxo-4-(prop-2-yn-
1-ylamino)butanoate) was slowly added to the solution and
stirred for 6 h at room temperature. After the reaction was
over, the product was washed with diethyl ether and collected.
80 mg of NMCS–COOH was dispersed in 15 mL of PBS, followed
by the addition of EDC/NHS (1 mg mLꢀ1) under stirring for
30 minutes at room temperature. After 1 h, an aqueous solution
of 4-azidianiline (20 mg) was slowly added to the mixture and
stirred for 10 h in the dark at RT. The NMCS-N3 particles were
washed with ethanol and dried under a vacuum.
Synthesis of compound A–C
Compounds A–C were synthesized by following the previously
reported protocol.29 The supportive NMR data for each com-
pound are given in the ESI.†
Synthesis of D
Here, 1 g of compound C was mixed in 10 mL of DMF and the
solution temperature was maintained at 0 1C. EDCꢁHCl, fol-
lowed by NHS (1.5 eq. each), was added with continued stirring
in chilled conditions for 1 h under an inert and dark atmo-
sphere; the reaction extended for the next 12 h at RT. After
completion of the reaction, the excess DMF was washed out
through ethylacetate–water phase separation, dried over
MgSO4, followed by reduced pressure drying. To the resulting
–NHS-linked compound (500 mg) in 5 mL dimethylformamide,
Et3N (2 eq., 70 mg, 1.26 mmol) was steadily added with stirring,
then propargylamine (2 eq., 70 mg, 1.26 mmol) was added. The
reaction mixture was magnetically stirred overnight at RT
under inert/dark conditions. On completion of the reaction,
DMF was removed from the ethylacetate phase with excess
water washing, and dried using a rotary evaporator. The solvent
was concentrated to yield a pale white solid. 1H NMR (400 MHz,
DMSO-d6) d 1.36 (d, 3H), 1.91–1.98 (m, 2H), 2.27 (t, J = 8 Hz,
2H), 2.5 (s, 1H), 3.845–3.86 (m, 2H), 3.90 (s, 3H), 4.00 (t, J = 8 Hz,
2H), 5.23–5.28 (m, 1H), 5.52 (br, S, 1H), 7.35 (s, 1H), 7.52 (s, 1H).
13C NMR (100 MHz, DMSO-d6): d 171.77, 153.86, 146.67, 139.32,
138.49, 81.73, 73.33, 68.60, 64.38, 56.51, 31.72, 30.42, 28.26,
25.62, 24.94, 24.48.
PEG–PEI copolymer synthesis and coating on NMCS-N3-GEM
PEG 1500 (0.3 g, 0.2 mmol), 4-formylbenzoic acid (0.075 g,
10 mmol), EDC (0.095 g, 0.5 mmol) and DMAP (0.013 g,
0.1 mmol) were dispersed in 20 mL of anhydrous DCM and
5 mL of THF. After 24 h of reaction under stirring at 25 1C, the
reaction solution was transferred for filtration. The residual
solvent was evaporated. The product, aldehyde-terminated
PEG, was extracted with DCM against the water phase and
collected over MgSO4. The suspension was precipitated in
chilled diethyl ether to collect the product. The obtained
CHO–PEG dissolved in DMSO and 60 mg of PEI25K in solution
were added very slowly. This reaction was continued for 48 h at
25 1C with stirring. The resulting reaction mixture was sub-
jected to dialysis for purification for 3 days and lyophilized
for the ultimate product PEG–PEI. 1H NMR (400 MHz, D2O)
d 7.97 ppm for –NH2, 6.6 ppm corresponds to –OH group, 3.59
for (–OCH2–CH2), 3.35 ppm belong to –OCH3, 2.92 ppm refers
to –NH(–CH2–CH2), 2.59 ppm for (COCH2CH2). Next, 10 mg of
linker-capped NMCS-gem was stirred with 0.5 mL of PEG–PEI
in DMF overnight. The thermo-responsive polymer-coated
particles were centrifuged and washed with Millipore water
followed by drying under vacuum.
Synthesis of E
Propargylamine (198 mg, 3.6 mmol) was slowly added to a
solution of succinic anhydride (300 mg, 3 mmol) in 3 mL of
THF. The mixture was stirred at RT for 24 h and then CH2Cl2
(20 mL) was added. The resulting precipitate was filtered to give
the desired product (4-oxo-4-(prop-2-yn-1-ylamino)butanoic
acid). The above product underwent esterification with com-
pound D (1 : 1) in dry CH2Cl2 in the presence of 1.2 eq. of DCC
and 4 mol% of DMAP for 6 h. The crude product was filtered
over Celite and the solvent was evaporated to obtain our final
photocleavable linker compound E. 1H NMR (400 MHz, CDCl3)
d 1.45 (d, 3H), 1.99 (s, 2H), 2.08–2.15 (m, 2H), 2.19 (t, J = 2.4 Hz,
2H), 2.4 (t, J = 7.2 Hz, 2H), 3.90 (s, 3H), 3.95–3.97 (m, 2H), 4.03
(t, J = 3.2, 2H), 4.05–4.08 (m, 2H), 5.44–5.49 (m, 2H), 6.70 (br, S,
1H), 7.28 (s, 1H), 7.45 (s, 1H). 13C NMR (100 MHz, CDCl3):
d 172.32, 171.36, 162.71, 153.91, 146.53, 139.17, 137.88, 108.89,
NIR laser-induced hyperthermia
The photothermal behaviors of various concentrations (100, 200,
500 mg mLꢀ1) of NMCS-linker-PEG–PEI were observed by irradiat-
ing their PBS dispersions with a diode laser source (980 nm, 0.72,
1.44 W cmꢀ2) for 15 min, and the temperature evolution was
recorded using a digitized thermocouple. After 10 min of expo-
sure, the sample almost reached thermal equilibrium and satu-
rated temperature was obtained in 15 min. The NIR stability of
our material was studied following the laser on–off method for up
to 3 consecutive cycles. The photothermal conversion efficiency30
(Z) was calculated using the following formula:
hAðTmax ꢀ TsurÞ ꢀ Qdis
Z ¼
(1)
Ið1 ꢀ 10ꢀA
Þ
l
79.66, 71.40, 68.37, 65.39, 56.28, 36.59, 32.48, 31.47, 29.63, h is the heat transfer coefficient, A is the area of the container
29.30, 29.04, 24.73, 24.5.
where hA = mCp/ts, m is the mass of the sample, Cp is the heat
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J. Mater. Chem. B, 2021, 9, 3666–3676
This journal is © The Royal Society of Chemistry 2021
Panda, Snigdharani
