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can endow onto the shell of PUMC. We believe this study pro-
vides a universal strategy for the convenient and facile synthesis
of new multifunctional PUMC, facilitating wider applications
of PUMC in agricultural and biomedical fields.
were washed with methanol repeatedly to remove loosely adsorbed
DAB. Microcapsules were further extracted with methanol for another
4
8 hr and dried to yield PUC+Q. PUC with residue isocyanate groups
−4
completely quenched with hexane solution of ethanol (1.4 × 10 mol
−1
L ) was also treated with tetrahydrofuran solution DAB as described
above to serve as a control sample for PUC+Q.
Quantification of Residue Isocyanate Group on the Shell of PUC:
Isocyanate groups on PUC were quantified using azide-labelling method.
An IR-active compound with primary amine, AEA, was synthesized to
serve as a labelling or reporting agent, which could be covalently grafted
onto the microcapsules through a reaction with residue isocayante
groups on the shell of PUC. After the freshly prepared PUCs were partially
quenched with ethanol/hexane solution for 1 min, they were added into
AEA solution (typically 20 μL, 10%, v/v, in anhydrous tetrahydrofuran)
for labelling. After 2 min of contact with gentle agitation, PUCs were
washed with methanol repeatedly and were subjected to infrared spectra
examination using KBr pellet method. In the case of the control sample,
the system was quenched with pure ethanol for 20 min to ensure
maximum quenching effect. After baseline correction, the characteristic
4
. Experimental Section
Reagents: Polyethylenimine branched (PEI, avg. Mw = 25,000 by
LS, avg. Mn = 10,000 by GPC), 7-diethylamino-4-methylcoumarin
(coumarin-1, a model drug), 2,4-tolylene diisocyanate (TDI) and Span
8
5 (Sorbitane trioleate, surfactant) were purchased from Sigma-Aldrich
(
(
(
Oakville, ON); the PEI solution in anhydrous dimethyformamide
DMF) was dried over molecular sieves (4 Å) overnight before use.
Dimethylamino)pentan-1-ol was purchased from Karl Industry (Aurora,
OH), and 17-alpha,21-dihydroxypregn-4-ene-3,11,20-trione (cortisone,
a model drug) was purchased from Tokyo Chemical Industry (TCI)
America (Portland, OR). All other reagents were purchased from Sigma-
Aldrich (Oakville, ON) and were used as received. MDR ESBL-E. coli
was obtained from the CANWARD (Canadian Ward Surveillance) study
assessing antimicrobial resistance in Canadian hospitals (www.canr.ca).
Synthesis of AEA: AEA was synthesized according to a previous
−
1
band of azide group (2108 cm ) was normalized with Amide I band
−
1 [26]
(1651 cm ).
grafted on PUC.
Data obtained reflected the relative amount of AEA
Quantification of DAB density on the shell of PUC: DAB density, which
is presented as the amount of fluorescein bound to PUC per unit surface
area, was measured using fluorescein titration method. PUCs were
immersed in fluorescein solution (1 mL 1 wt%) for 5 min under gentle
vibration. After removal of fluorescein solution, PUCs were thoroughly
washed with Borex buffer (0.1 M, pH = 9.5). The microcapsules were
then extracted with CTAC solution (1 mL 0.1 wt%, containing 0.1 M Borex
buffer, pH = 9.5) for three times under gentle agitation. The resultant
solution of desorbed fluorescein was measured with UV-vis spectroscopy
at 500 nm.
[25]
report. Briefly, sodium azide (3.6 g) was added to 2-bromoethylamine
−
1
hydrobromide aqueous solution (30 mL, 0.167 g mL ). The mixture was
stirred overnight at 75 °C. After the reaction mixture was cooled to room
temperature, NaOH (976 mg) was added. The solution was further
extracted with CH Cl (3 × 50 mL). The combined organic extracts
were then washed with saturated NaCl solution. The organic phase was
dried over anhydrous Na SO . After filtration, the organic solvent was
2
2
2
4
removed using a rotary evaporator to yield a clear, light-yellow liquid of
-azidoethylamine (1.85 g, 88%) that required no further purification.
2
1
H NMR (400 MHz, CD SOCD ): δ 3.21 (t, J = 6 Hz, 2H; CH N ),
Synthesis and Characterization of PUMC: PUMC was synthesized
3
3
2
3
[
12]
2
2
.67 (t, J = 6 Hz, 2H; CH NH ), 1.44 (s, br, 2H, NH ). The structure of
following the emulsion interfacial polymerization method
with
2
2
2
−
1
-azidoethylamine is shown in Scheme 1.
modifications. Briefly, disperse phase (0.6 mL, typically a 50 mg mL
Synthesis of DAB: To the solution of lauryl bromide (1.48 g,
.94 mmol) in MeCN (15 mL) was added 5-(dimethylamino)pentan-1-ol
0.72 g, 5.48 mmol), and the resulting solution was heated to reflux for
2 h. Excessive solvent and lauryl bromide were removed under vacuum
PEI solution in DMF) was injected into viscous cyclohexane (15 mL,
poly(isobutylene) cyclohexane solution, 7.38 wt%, 158.9 cP, with
typically 0.3 v/v% Span 85) in a 30 mL-beaker. The system was sealed
and agitated with a magnetic flea at 1200 rpm for 10 min to obtain an
emulsion. TDI was added immediately after emulsification leading to
instant formation of microcapsules. The system was agitated at 500 rpm
for another 2 min to allow for further diffusion of TDI into microcapsules.
After the reaction, the system was diluted to 50 mL with ethanol/hexane
solution (10%, v/v) and was centrifuged at 1000 rpm (rcf = 120 g) for
10 min. The microcapsules at the bottom were subjected to repeated
washing with ethanol/hexane solution (25% v/v, 50% v/v, 75% v/v) and
pure ethanol to remove poly(isobutylnene) and TDI residues.
5
(
1
to yield the crude product, which was further purified on column
chromatography eluting with MeOH/CH Cl (1:1) to give DAB as a white
2
2
1
solid. H NMR (D O, 300 MHz, δ) 3.62 (t, J = 6.7 Hz, 2 H; -CH OH),
2
2
+
+
3
4
.37-3.32 (m, 4 H; -CH N CH -), 3.12 (s, 6 H; N (CH ) ), 1.75-1.85 (m,
2 2 3 2
+
H; -CH CH N CH CH -), 1.68-1.59 (m, 2 H; -CH CH OH), 1.31-1.49
2
2
2
2
2
2
(
m, 20 H; CH of lauryl chain), 0.91 (t, J = 6.6 Hz, 3 H; -CH CH CH );
2
2
2
3
1
3
+
C NMR (D O, 75 MHz, δ) 64.5 (-CH CH N CH CH -), 62.9 (-CH OH),
2
2
2
2
2
2
3
2.3 (-CH CH OH), 29.6, 29.5, 27.3, 24.2, 22.7, 20.4, (29.6 to 22.4, CH
2
2
2
in the lauryl and pentyl alcohol chains), 14.2 (-CH CH CH ); HRMS
Size of the microcapsules was measured manually based on a
microscopy image; particle size distribution (PSD) was calculated based
on a method discribed by Merkus.[ Briefly, digital pictures were taken
with a calibrated camera under an optical microscope. The diameters
of over 200 microcapsules (PUMC = 401, MC = 345, MCQ = 226) were
measured. Size distribution was represented by geometric standard
deviation of diameters (σ ): (σ = D /D ); and by Span value (S): S =
2
2
3
+
(
MALDI-TOF) m/z: [M-Br] calculated for C H NOBr, 300.3266; found,
19 42
27]
3
00.3253. Structure of DAB is shown in Scheme 1.
Synthesis of PUC: PUC was synthesized to facilitate the study of
surface chemistry of synthesis and to evaluate modification methods.
−
1
Disperse phase (10 μL, typically a 50 mg mL PEI solution in DMF) was
carefully injected into a microcentrifuge tube (2.0 mL capacity, round
bottom) containing 1 mL of anhydrous cyclohexane. DMF phase formed
a single droplet at the bottom of the tube. Cyclohexane solution of TDI
g
g
84
50
(D - D )/ D , where D represents diameter at n% point of cumulative
90
10
50
n
undersize PSD based on volume weighted distribution. Average diameter
(
10 μL, 25%, v/v) was carefully added to the cyclohexane phase without
was presented as volume weighted mean diameter μ = D(4,3) = ∑ni
g
4
3
disturbing the DMF droplet at the bottom. Polymerization occurred
instantly at the interface of the droplet, forming a microcapsule. The tube
was gently vibrated and let stand for 5 min to allow for further diffusion
of TDI. The resultant microcapsules were ready for further post-grafting
reaction if necessary. Otherwise, the system was quenched with various
amounts of ethanol/hexane solution after reaction.
Preparation of PUC+Q : After the synthesis of PUC, the supernatant
was carefully removed by pipetting, and DAB solution (typically
5
D / ∑ n D .
i i i
Dissolution Test: In vitro drug release of coumarin-1 from MCQ was
tested using USP Apparatus II on a VanKel 600 Dissolution Apparatus
(Palo Alto, CA, USA).[ Briefly, approximately 20 mg MCQ containing
coumarin-1 was accurately weighed and dispersed in 250 mL PBS (0.1 M,
pH = 7.4). The paddle speed was set at 75 rpm and the test temperature
at 37 ± 0.5 °C. At predetermined time intervals (0.25, 0.5, 0.75, 1, 1.5, 2,
2.5, 3, 4, 6, 12, 18, 24, 36, 48, 72, 120, and 176 h), 1.0 mL suspension
was filtered through a 0.2 μm filter and was subjected to HPLC-UV
measurement. 1.0 mL fresh PBS solution was then replenished to
the dissolution vessels. The HPLC-UV analysis was conducted using
28]
−
1
mg mL ) in anhydrous tetrahydrofuran was added immediately.
After the microcentrifuge tube was gently vortexed for 5 min, the
tetrahydrofuran solution of DAB was removed and microcapsules
8
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© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Funct. Mater. 2012,
DOI: 10.1002/adfm.201200656