Supporting Information
Creating Microenvironments Using Encapsulated Polymers
Muris Kobašlija1, Andrew R. Bogdan1, Sarah L. Poe1, Fernando Escobedo2 & D. Tyler
McQuade3
1
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York
14853, USA, 2School of Chemical Engineering, Cornell University, Ithaca, New York
14853, USA, 3Department of Chemistry and Biochemistry, Florida State University,
Tallahassee, Florida 32306, USA
mcquade@chem.fsu.edu
A. Materials and Methods
1. Materials
Methanol (Mallinckrodt), toluene (Mallinckrodt), chloroform (J. T. Baker),
cyclohexane (Mallinckrodt), poly(ethyleneimine) (Aldrich 10,000 MW), tolylene 2,4diisocyanate (Aldrich, technical grade, 80%), Span 85 (Sigma), 5dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride, Invitrogen/Molecular
Probes), lissamine rhodamine sulfonyl chloride (Acros), acetic anhydride (Mallinckrodt),
hexanes (Mallinckrodt), polystyrene (Polysciences, Inc. 50,000 MW), tetrahydrofuran
(Mallinckrodt), acetone (Mallinckrodt), poly(vinyl alcohol) (Aldrich, 89,000-98,000
MW, 99% hydrolyzed), poly(methylene (polyphenyl) isocyanate) (Polysciences, Inc.),
tetraethylenepentamine (Aldrich), ethanol (Pharmco), and diethyl ether (J. T. Baker) were
used as received.
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2. Instrumentation
Transmitted light images were obtained on a Bio-Rad MRC-600, equipped with an
argon-krypton laser for excitation and attached to a Zeiss Axiovert 10 inverted
microscope at Cornell Nanobiotechnology Center’s (NBTC) Confocal/Multiphoton
Microscope Facility. Images were analyzed using provided Lasersharp and Confocal
Assistant software.
Confocal microscopy was performed on a Leica TCS SP2 Spectral Confocal
Microscope System at Cornell’s Microscopy, Imaging & Fluorimetry Facility. Images
were analyzed using provided Leica software.
Digital images of microcapsules containing dansyl-labeled PEI and of bulk phase
separation of dansyl-PEI/methanol/toluene mixture were obtained with Sony DSC-F717
digital camera and hand-held UV lamp.
Temperature controlled studies were done in a UV/Vis cuvette mounted on the BioRad system described above and attached to VWR Programmable Temperature
Controller, Model 1167P.
Gas chromatographic (GC) analyses were performed using a Varian CP-3800 GC
equipped with a Varian CP-8400 autosampler, a flame ionization detector (FID) and a
Varian CP-Sil 5CB column (length = 15 m, inner diameter = 0.25 mm, and film thickness
= 0.25 μm. The temperature program for GC analysis held the temperature constant at 50
ºC for 1 minute, and then heated samples from 50 to 90 ºC at 17 ºC/min. Inlet and
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detector temperatures were set constant at 220 and 250 ºC, respectively. Mesitylene was
used as an internal standard.
Sigma Plot 8.02 software for Windows by SPSS Inc. was used to plot the points of
the ternary phase diagram.
3. Experimental Details
3.1. Labeling of PEI
With lissamine rhodamine. Polyethyleneimine (PEI, 99%, MW 10,000, 53.0 g)
was stirred with lissamine rhodamine B sulfonyl chloride (0.185 g, 0.3 mmol) in
methanol (400 mL) overnight at room temperature. Methanol was evaporated in vacuo
and the residue dissolved in a minimal amount of water (about 10 mL). The solution was
dialyzed against deionized water for 2 days while contained within a SnakeSkin dialysis
bag (Pierce, 34 mm dry flat width, 3.7 mL/cm, MWCO 3,500) or until no more color
leached out. The remaining residue was lyophilized overnight and acylated as follows.
To the lissamine rhodamine labeled polymer (1 mL) in methanol (10 mL) acetic
anhydride was added (1 mL). The mixture was stirred overnight. Methanol was
evaporated in vacuo and dialysis and lyophilization procedure was repeated as above. A
pink powder (1.07 g) was obtained. The powder was fully soluble in methanol for
subsequent encapsulation.
With dansyl. Polyethyleneimine (PEI, 99%, MW = 10,000, 2.0 g) was stirred with
dansyl chloride (0.020 g, 0.07 mmol) in methanol (10 mL) overnight at room
temperature. Methanol was evaporated in vacuo and the residue dissolved in a minimal
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amount of water (about 2 mL). The solution was dialyzed against deionized water for 2
days while contained within a SnakeSkin dialysis bag (Pierce, 34 mm dry flat width, 3.7
mL/cm, MWCO 3,500). The remaining residue was lyophilized overnight and acylated
as follows. To the dansyl labeled polymer (1.19 g) in methanol (10 mL) acetic anhydride
was added (7 mL). The mixture was stirred overnight. Methanol was evaporated in
vacuo and dialysis and lyophilization procedure was repeated as above. A yellow powder
(1.94 g) was obtained. The powder was fully soluble in methanol for subsequent
encapsulation.
3.2. Microscopy
Transmitted light microscopy of PEI containing microcapsules: closed system.
To a 3 mL fluorometer cuvette (in temperature controlled studies 1 mL UV/Vis cuvette
was attached to a heated/refrigerated circulator unit), PEI containing microcapsules were
added (30 mg). The cuvette was fitted with a rubber septum and placed onto a
microscope stage of an inverted confocal microscope (BioRad/Olympus microscope). A
heavy lead ring was placed on top of the cuvette to prevent accidental movement.
Syringes containing methanol and toluene and a vent needle were inserted through the
septum. The images were taken as each of the solvents was added to the microcapsules
with a 10x dry objective.
Confocal laser scanning microscopy of PEI containing microcapsules: open
system. Microcapsules were placed on a microscope slide and a solvent was added. A
cover slip was placed on top of the microcapsules and the edges of the cover slip were
sealed with nail polish to prevent evaporation. The confocal images of the microcapsules
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were taken immediately on a Leica laser scanning confocal microscope with a 40x oil
objective.
Transmitted light microscopy of polystyrene containing microcapsules: open
system. Microcapsules were placed on a microscope slide and a solvent was added. A
cover slip was placed on top of the slip and the specimen was inverted and placed on an
inverted microscope stage (BioRad/Olympus microscope). The cover slip was left
hanging so that a solvent droplet could be added at any time. The images were taken
shortly after each successive solvent addition with a 100x oil objective.
3.3. Ternary Phase Diagram Construction
An arbitrary spot on the phase diagram was picked and the sample prepared as
follows. An exact amount of PEI was weighed (target mass was 0.1g). Based on that
mass and the coordinates of the picked spot, masses of methanol and toluene were
calculated. The calculated mass of methanol was added and the resulting mixture was
stirred in a closed vial until all of the PEI was dissolved. To the solution the prescribed
amount of toluene was added and the mixture was observed for phase separation. The
observation was plotted onto the phase diagram as 1-phase or 2-phase point using Sigma
Plot 8.0. The experiment was repeated for other points in the same fashion until
appropriate number of points was obtained. The results are plotted in Figure S1.
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Figure S1. Ternary phase diagram for PEI/methanol/toluene mixture (black dots indicate one-phase region
and red dots indicate two-phase region).
3.4. Tie-lines determination
After determining the 1-phase and 2-phase regions of the phase diagram, an
arbitrary point in the 2-phase region was picked. Sample was prepared as follows. An
exact amount of PEI was weighed (target mass was 1.0 g). Based on that mass and the
coordinates of the picked spot, masses of methanol and toluene were calculated. The
calculated mass of methanol was added and the resulting mixture was stirred in a closed
vial until all of the PEI was dissolved. To the solution the prescribed amount of toluene
was added and the mixture was transferred to a separatory funnel and the phases were
separated. To each of the phases (1 mL, the amount used for the calibration curves),
mesitylene (20 μL) was added as an internal standard. These samples were then analyzed
by gas chromatography. The weight composition of each phase was calculated based on
the calibration curves previously built for the system (Figures S2A and S2B). The
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determined tie-lines were then plotted in the ternary phase diagram. The phase boundary
was drawn to guide the eye by including all the end points of the tie-lines as well as by
separating two-phase from one-phase region (Figure S3).
Figure S2. (A) calibration curve for amount of methanol present in the sample, and (B) calibration curve
for amount of toluene present in the sample.
Figure S3. Ternary phase diagram for PEI/methanol/toluene mixture with tie-lines and phase boundary
drawn in.
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3.5. Mathematical treatment of PEI/methanol/toluene system using the FloryHuggins theory
We used the framework of the classical Flory-Huggins theory[1] for three
components comprising two solvents and one polymer[2], generalized for the case where
the molar volume of the solvents is unequal. Let 1 and 2 denote the two solvent species
and 3 be the polymer. It can be shown that the “activity” a of each component is given
by:
ln a1 
ln a2 
ln a3 
1  10
kT
2  20
kT
3  30
kT
 ln 1  1  1 
V1
V
V
2  1 3  ( 122  133 )(2  3 )  1  2323
V2
V3
V2
(1)
 ln 2  1  2 
V2
V
V
V
1  2 3  ( 2 121   233 )(1  3 )  2 1313
V1
V3
V1
V1
(2)
V3
V
V
V
V
1  3 2  ( 3 131  3  232 )(1  2 )  3 1212
V1
V1
V1
V2
V1
(3)
 ln 3  1  3 
Where i is the chemical potential of component i in the solution, i0 is the
chemical potential of the pure component i (at identical temperature and pressure as the
solution), i and Vi are the volume fraction and the molar volume of component i in the
solution, and ij is Flory’s interaction parameter between components i and j. If two
phases “prime” and “double prime” are at thermodynamic coexistence, then:
ln a1'  ln a1"
(4)
ln a2'  ln a2"
(5)
ln a3'  ln a3"
(6)
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Once we write the right hand sides of Eqs. (1), (2), and (3) into both sides of Eqs.
(4), (5), and (6), respectively (using volume fractions for the appropriate phase), the latter
will form a set of 3 non-linear equations in the main compositional unknowns 1' ,  2' , 3' ,
1" ,  2" , and 3" (assuming that all the  and the V values are known and are the same in
both phases). Using the constraints that 1'   2'  3'  1 and 1"   2"  3"  1 , it is then
clear that one only needs to specify one of the compositions (say  2' ) to be able to solve
Equations (4)-(6) for the other unknown volume fractions. In practice, we used a
multidimensional Newton-Raphson numerical method (with globally convergent search
method[3]) coupled with a continuation method to find the solutions over a range of
conditions. We then transformed the resulting volume fractions i into weight fractions wi
to be able to make a comparisons with experiment. The lines connecting the calculated
weight fractions w1' , w2' , w3' from one phase with the corresponding fractions w1" , w2" ,
and w3" of the other phase, form the tie lines (which go across the two-phase region). For
the system under investigation, using toluene=1, methanol=2, and PEI=3, we set V2/V1 =
0.45 and V3/V1=94 (the latter assumes that PEI has a number average molecular weight of
10,000 and a density of 1 g/ml). In the absence of reported or experimentally measured
values of the  parameters[4], we estimated them as follows. While methanol and toluene
are fully soluble at room conditions, they form a highly non-ideal solution; their vaporliquid behavior[5] can be approximately fitted by a one-parameter Margules equation – an
activity coefficient model that is essentially equivalent to the Flory-Huggins model –
providing an approximate value of 12 2. Since methanol is a very good solvent for PEI,
we simply assume that 23 0. This leaves as the only fitting parameter the toluene-PEI
13. We clearly must have that 13>23 and based on typical “poor-solvent” polymer pair
13 should be order 1 (4). We thus chose 13 such that it gives a reasonable agreement
with the extrapolated ~55-60 weight % of PEI in the polymer rich phase for the toluene-
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PEI binary system (i.e., 0% methanol; see Figure S3). This yielded 13 =0.82. The
calculated triangular phase diagram was shown in Figure 4B.
References:
[1] P. J. Flory, Principles of Polymer Chemistry (Cornell University Press, Ithaca, 1953).
[2] A. R. Shultz, P. J. Flory, J. Chem. Phys. 17, 268 (1949).
[3] W.H. Press, et al., Numerical Recipes in Fortran 77 (Cambridge University Press,
New York, 2001)
[4] P. C. Painter, M. M.Coleman, Fundamentals of Polymer Science (CRC Press, New
York, 1997).
[5] H. Hori, I. Tanaka, J. Occup. Health 40, 132 (1997).
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