Synthesis of the first poly(diaminosulfide)s and an investigation of their applications as drug delivery vehicles

Article abstract of DOI:10.1021/ma2023167

This paper reports the first examples of poly(diaminosulfide)s that were synthesized by the reaction of a sulfur transfer reagent and several secondary diamines. The diaminosulfide group has the general structure of R ₂N-S-NR₂, and although it has been used in the synthesis of small molecules, it has never been utilized in the synthesis of macromolecules until this report. A series of poly(diaminosulfide)s were synthesized at elevated temperatures, and the molecular weights of the polymers were as high as 12-400 g mol^-1 with conversions for the polymerization reaction up to 99%. The rate constants for the transamination reactions that lead to the polymers were measured in several solvents to provide an understanding of the reaction conditions necessary to polymerize the monomers. The degradation of diaminosulfides was studied in D₂O, C₆D₆, CD₃OD, CDCl₃, and DMSO-d ₆/D₂O to demonstrate that they were very stable in organic solvents but degraded within hours under aqueous conditions. These results clearly demonstrated that diaminosulfides are very stable in organic solvents under ambient conditions. Poly(diaminosulfide)s have sufficient stabilities to be useful for many applications. The ability of these polymers to function as drug delivery vehicles was studied by the fabrication of nanoparticles of a water-insoluble poly(diaminosulfide) with a dye. The microparticles were readily absorbed into human embryonic 293 cells and possessed no measurable toxicity toward these same cells.

Full text of DOI:10.1021/ma2023167

Article
pubs.acs.org/Macromolecules
Synthesis of the First Poly(diaminosulfide)s and an Investigation of
Their Applications as Drug Delivery Vehicles
Jun Yoo,† Sheetal R. D’Mello,‡ Tyler Graf,† Aliasger K. Salem,‡ and Ned B. Bowden*,†
†Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States
‡College of Pharmacy, University of Iowa, Iowa City, Iowa 52242, United States
ABSTRACT: This paper reports the first examples of poly-
(diaminosulfide)s that were synthesized by the reaction of a
sulfur transfer reagent and several secondary diamines. The
diaminosulfide group has the general structure of R2N−S−NR2,
and although it has been used in the synthesis of small molecules,
it has never been utilized in the synthesis of macromolecules until
this report. A series of poly(diaminosulfide)s were synthesized at
elevated temperatures, and the molecular weights of the polymers
were as high as 12 400 g mol−1 with conversions for the polymerization reaction up to 99%. The rate constants for the
transamination reactions that lead to the polymers were measured in several solvents to provide an understanding of the reaction
conditions necessary to polymerize the monomers. The degradation of diaminosulfides was studied in D2O, C6D6, CD3OD,
CDCl3, and DMSO-d6/D2O to demonstrate that they were very stable in organic solvents but degraded within hours under
aqueous conditions. These results clearly demonstrated that diaminosulfides are very stable in organic solvents under ambient
conditions. Poly(diaminosulfide)s have sufficient stabilities to be useful for many applications. The ability of these polymers to
function as drug delivery vehicles was studied by the fabrication of nanoparticles of a water-insoluble poly(diaminosulfide) with a
dye. The microparticles were readily absorbed into human embryonic 293 cells and possessed no measurable toxicity toward
these same cells.
INTRODUCTION
■
The integration of new functional groups into polymer
chemistry opens new avenues for research and possible
commercial applications. For instance, the development of
well-defined carbene catalysts based on Mo, W, and Ru in the
1980s and 1990s increased the types and complexities of
polymers that could be synthesized and the problems in
macromolecular science that could be addressed.1−12 These
Figure 1. (a) A polymerization to yield a poly(diaminosulfide). (b)
Sulfur transfer reagents that are commonly used in small molecule
synthesis. (c) A polymer of a benzo[1,2,5]thiadiazole.
catalysts led to the development of living ring-opening
metathesis polymerization (ROMP) and acyclic diene meta-
thesis (ADMET) polymerization, which were significant
this functional group have been synthesized using sulfur
reasons the Nobel Prize was awarded to Schrock, Grubbs,
and Chauvin in 2005.13−22 The use of “click” chemistry is
another example, and its use has increased the complexity of
transfer reagents such as those molecules shown in Figure
1b.28−30 The most prominent applications of small molecules
with diaminosulfides have been in the chemical industry for the
the structure of macromolecules and has found widespread
applications in polymer science.23−25 In a recent example by the
high-temperature vulcanization of rubber and in the con-
struction of polymers with benzo[1,2,5]thiadiazoles along the
Hawker group published in 2010, polymers were synthesized
backbone (Figure 1c).31 Polymers that incorporate benzo-
for the first time with a functional group that was a precursor to
[1,2,5]thiadiazoles have found uses as semiconductors,
ketenes and provided a simple route to synthesize cross-linked
fluorophores, and photoactive components in organic solar
polyethylene to systematically study its materials proper-
cells due to their interesting electro-optical properties.32−40
These polymers link the monomers through carbon−carbon
bonds as shown in Figure 1c rather than through the nitrogen
or sulfur atoms as in poly(diaminosulfide)s. Surprisingly, no
ties.26,27 From these examples and more, it is clear that when
new functional groups are integrated into macromolecules, new
applications are developed that take advantage of their unique
reactivities.
In this article we report the first examples of polymers that
utilize diaminosulfide functional groups along their backbones.
The diaminosulfide functional group has the general structure
of R2N−S−NR2 as shown in Figure 1. Small molecules with
Received: October 15, 2011
Revised: December 9, 2011
Published: January 6, 2012
© 2012 American Chemical Society
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Figure 2. Synthesis of two sets of sulfur transfer reagents. (a) The synthesis of a dithiosuccinimide. (b) The synthesis of a diaminosulfide in two
steps. Molecules C and E were purified by distillation. (c) Molecule F was synthesized using the same procedure as molecule E.
one has used diaminosulfides to bond monomers together as
shown in Figure 1a, and these polymers are the focus of this
report.
To illustrate a possible application of poly(diaminosulfides),
we completed initial experiments to investigate the application
of a poly(diaminosulfide)s as a delivery vehicle for drugs. Many
drugs suffer from poor bioavailability, poor water solubility,
short serum circulation lifetimes, and inadequate mechanisms
to enter cells or have serious side effects that limit the amount
of drug that can be administered. To overcome these and more
limitations, drugs are often condensed with synthetic,
biodegradable polymers into nanoparticle delivery vehicles
that are administered to patients.65−73 The polymer protects
the drugs from degradation in the bloodstream and allows their
delivery to tumors by the enhanced permeation and retention
effect where they can be taken into cancer cells. The polymers
used in this field degrade slowly in the bloodstream but have a
rapid rate of degradation when taken into the acidic
compartments of cellsthe endosome and lysosomewhere
they release their cargo.74−77 It is critically important that the
polymer be biodegradable such that it will not accumulate
within the body and cause a toxic response.78,79 In this article,
some of the characteristics of poly(diaminosulfides) as drug
delivery vehicles were investigated, including the stabilities of
diaminosulfides in water under basic, acidic, and neutral
conditions, whether nanoparticles fabricated from these
polymers were internalized by cells, and whether any in vitro
toxicity was observed from the nanoparticles. These studies are
meant to illustrate an interesting application of poly-
(diaminosulfide)s in medicine.
We report the synthesis of a small molecule that is a highly
successful sulfur transfer reagent and how this molecule can be
used to synthesize the first poly(diaminosulfide)s reported in
the literature. Some of the key, initial studies of a
diaminosulfide in numerous solvents are reported to demon-
strate their stabilities and, by extension, the stabilities of
poly(diaminosulfide)s. Finally, one example of a poly-
(diaminosulfide) was fabricated into microparticles and studied
for their ability to be internalized by human embryonic kidney-
293 (HEK-293) cells and whether they showed any toxicity
toward these cells.
An important characteristic of the diaminosulfide group is
that it is based on inorganic atoms (one sulfur and two
nitrogens). Most functional groups that are used to synthesize
polymers are based on organic functional groups such as esters,
amides, anhydrides, acetals, cyclic olefins, vinyl groups,
carbonates, urethanes, and epoxides. Although many monomers
are known to possess inorganic functional groups, it is
uncommon that an inorganic functional group is transformed
in the polymerization reaction and used to link monomers
together as shown in Figure 1a. Most inorganic functional
groups found in monomers or polymers are not transformed
during the polymerization reaction. Three notable examples of
inorganic functional groups that have been polymerized include
the polymerization of thiols into poly(disulfides), the polymer-
ization of cyclic phosphazenes into poly(phosphazenes), and
the polymerization of cyclic siloxanes into poly(siloxanes).41−50
Inorganic functional groups are interesting targets for polymer
synthesis because they can be expected to have new reactivities
that differ from those of organic functional groups and they
have the potential to act as ligands for metals.51−54 The use of
inorganic functional group transformations in the synthesis of
polymers is understudied and represents a potentially rich
source of functional group diversity in macromolecular science.
One part of our motivation to synthesize polymers through
the polymerization of diaminosulfides was based on the
chemical properties of this functional group in small molecule
synthesis. These polymers are structurally related to polythiazyl
(SN)x which was first synthesized in 1953 from S2N2.55−59 This
polymer is electrically conducting at room temperature and
superconducting at low temperatures.55 In prior work by
others, molecules with diaminosulfides were stable and readily
isolated by traditional methods (distillation or chromatog-
raphy).28,29,60−63 In addition, some examples of the synthesis of
molecules containing diaminosulfides proceeded with isolated
yields of 80% or higher. Although promising, these results do
not predict immediate success in a step-growth polymerization.
In these polymerizations, the degree of polymerization, Xn, is
related to the fractional monomer conversion, P, by the
equation Xn = 1/(1 − P).64 Thus, to synthesize poly-
(diaminosulfide)s with modest to high molecular weights via
a step-growth polymerization, the yield of the coupling reaction
must be >95%.
RESULTS AND DISCUSSION
■
Synthesis and Reactions of Sulfur Transfer Reagents.
We hypothesized that poly(diaminosulfide)s could be synthe-
sized by reacting secondary diamines with a sulfur transfer
reagent as shown in Figure 1a. Many secondary diamines were
commercially available or easily synthesized, so the challenge in
the polymerization was to develop a useful sulfur transfer
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Figure 3. Kinetics of transamination reactions. (a) The reaction that was studied in a sealed NMR tube. (b) The conversion of the transamination
reactions as a function of time. The conversion was defined as the sum of the S−N(CH3)Bn bonds divided by the sum of all of the S−N bonds for
molecules E, H, and I. (c) The plot of the initial data points used to find the rate constants for the reaction in each solvent. More data points were
used to find the rate constant for the experiment in C6D6, but they are not shown here.
reagent. Although SCl2 is used in the synthesis of small
molecules with diaminosulfides, its use has several draw-
backs.80−85 This molecule has a low boiling point (59 °C),
must be handled under inert atmospheres, is challenging to
purify, reacts with multiple functional groups such as alcohols
and alkenes, and releases HCl. Because of these limitations, we
have not pursued the synthesis or use of SCl2.
Two different sulfur transfer reagents were studied (Figure
2). Molecule B was initially explored as a sulfur transfer reagent
based on the rapid reactions of thiosuccinimides with
amines.86,87 Although the synthesis of B was straightforward
and did not require any chromatography, its purification was
challenging because of its poor solubility in many solvents.
Molecule B was mostly insoluble in benzene, chloroform,
DMSO, and methylene chloride. Molecule B was cleaned by
washing the crude product with hexanes, and an isolated yield
of 69% was obtained. To increase the purity of molecule B, it
was recrystallized from methanol. Replacement of N-
chlorosuccinimide with N-chlorophthalimide in the second
step yielded a diphthalimide sulfur transfer reagent that also
possessed limited solubility in organic solvents.
Although B was partially soluble in DMSO, it was not used to
synthesize polymers for several reasons. First, the synthesis of B
had poor atom efficiency. The addition of one sulfur (atomic
weight: 32 g mol−1) to yield a diaminosulfide functional group
along the backbone of a polymer would require the use of 2
equiv of tributyltin chloride (MW: 326 g mol−1) and 2 equiv of
N-chlorosuccinimide (MW: 134 g mol−1). Thus, significant
amounts of waste were produced in the synthesis of molecule
B. Second, the poor solubility of molecule B made it
challenging to use in solvents that dissolve many polymers.
Furthermore, it decomposed when heated in CDCl3 and
DMSO-d6.
A second sulfur transfer reagent was synthesized (molecule E
in Figure 2) based on a literature procedure. In the first step, an
excess of ethylmethylamine was reacted with sulfur chloride at
−78 °C. Reactions run at 0 °C had unidentified side products,
but the reaction at −78 °C yielded molecule C in high purity.
Molecule C could be carried onto the next step without
purification, or it could be purified by distillation. In the second
step, C reacted with SO2Cl2 to yield D that was not isolated.
Rather, D was slowly added to ethylmethylamine to yield the
sulfur transfer reagent E. This procedure was followed to
synthesize F using dimethylamine in both steps. Both E and F
were readily purified by distillation and yielded clean products
as shown by 1H and 13C NMR spectroscopy and high-
resolution mass spectrometry. Because no chromatography was
necessary for the synthesis of E or F, these reactions could be
scaled up to yield large amounts of product in a short period of
time.
Kinetics of Transamination Reactions. To synthesize
polymers via transamination reactions between molecule E and
secondary diamines, the second-order kinetics of the reaction
between molecule E and benzylmethylamine was studied in
four solvents (Figure 3). Benzylmethylamine was chosen for
these reactions because of the easily identified benzylic CH2
1
group that shifted downfield in the H NMR spectra when
proceeding from benzylmethylamine to H to I.
The reactions between molecule E and 2 mol equiv of
benzylmethylamine were studied, and the rate constants were
measured in CD2Cl2 (7.81 × 10−5 M−1 s−1), DMSO-d6 (4.89 ×
10−5 M−1 s−1), CDCl3 (2.79 × 10−5 M−1 s−1), and C6D6 (5.47 ×
10−6 M−1 s−1). The rate constants were found using the data
points for conversions of less than 10% using the assumption
that the reaction was irreversible. Although the reaction was
reversible, this assumption has been commonly used to find
rate constants for reversible reactions at low conversions.88 It is
important to note that the ethylmethylamine (boiling point =
36 °C) remained in the sealed NMR tube.
Although the reaction was most rapid in CD2Cl2 and reached
equilibrium in 14 h, small amounts of unidentified side
products were visible. The presence of side products made
methylene chloride a poor choice for the polymerization. The
reaction in CDCl3 took 8 days to reach equilibrium, and the
reaction in C6D6 did not reach equilibrium after 8 days. Despite
the slow rates for reactions in these solvents, the reactions were
clean and no side products were observed. The reaction in
DMSO-d6 also did not show any side products after 3 days, but
this reaction reached 37% conversion and did not proceed any
further. The final conversion was less than 50% because
molecule I had limited solubility in DMSO-d6 due to the apolar
structure of molecule I and the polar structure of DMSO-d6.
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1
The H NMR spectra of this reaction in DMSO-d6 showed a
conversions. The polymerization with piperazine (entry 7)
yielded an insoluble polymer in all solvents.
lower than expected concentration of molecule I even after 3
days.
The polymer synthesized in entries 3 and 4 had limited
stability. When this polymer was precipitated into methanol
and water, it rapidly degraded as shown by the presence of
The reaction between molecule E and benzylmethylamine
only reached 51% conversion in 17 h when completed at 40 °C
in an uncapped NMR tube, despite the low boiling point of
ethylmethylamine. Prolonged reaction times resulted in a slow
increase in conversion, but this reaction was judged to be too
slow. Molecule F was synthesized for the polymerization
reactions because of the low boiling point of dimethylamine
(boiling point 7 °C) which would make it simple to remove
from a reaction.
1
numerous, unidentified peaks in the H NMR spectra. To
isolate the polymer with minimal degradation, benzene was
removed under vacuum after the polymerization was complete,
and the polymer was characterized without further purification.
1
The GPC and H NMR spectra were consistent with the
indicated polymer. We believe that the internal, tertiary amine
reacts with the diaminosulfide through an intramolecular
reaction and was the source of the instability of this polymer.
The polymer shown in entry 6 was characterized by
elemental analysis to provide further evidence that it possessed
the indicated composition. The calculated weight composition
of the repeat unit was carbon (64.95%), hydrogen (10.06%),
nitrogen (11.65%), and sulfur (13.34%). The measured weight
composition of the polymer was carbon (64.70%), hydrogen
(9.97%), nitrogen (11.76%), and sulfur (13.44%). The
agreement between the calculated and measured elemental
compositions provided strong evidence that there was only one
sulfur atom bridging between the nitrogens.
Stability of Diaminosulfides in Organic Solvents and
in Water. Although numerous small molecules possessing
diaminosulfide functional groups have been synthesized, no
report on their long-term stabilities in organic solvents or water
have been published. The stability of this functional group was
investigated to estimate the stabilities of poly(diaminosulfide)s
for future work. Molecule E and an internal standard of
diethylene glycol dimethyl ether were added to CDCl3, DMSO-
d6/D2O (10/1 by volume), and C6D6 and allowed to sit at
room temperature in capped NMR tubes (Figure 5). Periodic
1H NMR spectra were collected to determine the percent
decomposition by the mole ratio of molecule E to the ether.
After 32 days the amount of decomposition ranged from no
detectable decomposition in C6D6 to 38% decomposition in
DMSO-d6/D2O. Because molecule E was not soluble in
methanol, the stability of molecule K was studied in CD3OD.
After 32 days, 15% of molecule K degraded.
Reactions between molecule F and benzylmethylamine were
studied in CDCl3, DMSO-d6, and C6D6 in vented reaction
vessels to allow dimethylamine to boil off (Figure 4 and Table
Figure 4. A transamination reaction with dimethylamine as the leaving
group.
Table 1. Transamination Reactions of Molecule F and
Benzylmethylamine
a
entry
solvent
temp (°C)
reaction time (h)
conv (%)
1
2
3
4
5
6
CDCl3
CDCl3
DMSO-d6
C6D6
50
50
50
50
50
85
24
72
73
24
72
24
39
93
84
41
C6D6
84
C6D6
>97
a
The conversion was defined as the sum of the S−N(CH3)Bn bonds
divided by all of the S−N bonds in molecules F, J, and I.
1). Each of the reactions in Table 1 did not show any impurities
1
by H NMR spectroscopy even when heated to 85 °C for
extended periods of time. The conversions for the reactions
were high for each solvent for reactions at 50 °C but went to
quantitative conversions for reactions in C6D6 at 85 °C.
Synthesis of Poly(diaminosulfide)s. Poly-
(diaminosulfide)s were synthesized by reaction of secondary
diamines and molecule F at elevated temperatures (Scheme 1
These results demonstrated that the diaminosulfide func-
tional group was stable in apolar, aprotic solvents but that it
very slowly degraded in polar, protic solvents. The rate of
degradation was slow enough that polymers with diamino-
sulfide functional groups are expected to have reasonable
stabilities in these solvents, and this stability was observed for
the prepared poly(diaminosulfide)s. The polymers were
synthesized in benzene and chloroform at elevated temper-
atures and isolated by precipitation into methanol. Despite
these conditions, the polymers possessed high degrees of
polymerization.
Scheme 1. Polymerization of Diamines with the Sulfur
Transfer Reagent
and Table 2). These polymerizations were run for 24 to 96 h,
and the resulting polymers were characterized by GPC against
polystyrene standards, H NMR spectroscopy, and 13C NMR
To further explore the stability of the diaminosulfide
functional group, molecule L was synthesized and studied in
water (Figure 6). Molecule L and an internal standard of tert-
butanol were added to D2O with 9 mol equiv of acetic acid
(acidic conditions), 9 mol equiv of KOH (basic conditions), or
no additional acid or base (neutral conditions). The rate
constants for the decomposition of this molecule were 1.29 ×
10−4 s−1 under neutral pH conditions and 9.88 × 10−5 s−1 under
basic conditions. Under acidic conditions, molecule L
1
spectroscopy.
The polymers in entries 1, 2, 5, and 6 had high molecular
weights and degrees of polymerization. The degrees of
polymerization were determined by two methods using the
molecular weight measured by GPC and by end-group analysis
in the H NMR spectra of the polymers. These values for the
degree of polymerization agreed with each other and
demonstrated that these reactions cleanly proceeded to high
1
1
completely degraded by the time the first H NMR spectrum
was obtained so only a lower limit of the rate constant was
calculated (1.70 × 10−2 s−1).
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Table 2. Synthesis of Poly(diaminosulfide)s
a
b
The Mn and PDI were measured using size exclusion chromatography versus polystyrene standards. The degree of polymerization were based on
c
d
1
the values for Mn measured by GPC. The degree of polymerization were based on H NMR spectra. The polymer was insoluble.
1
The only product of degradation determined by H NMR
spectroscopy was the secondary diamine used in the synthesis
of molecule L. From prior work by others, it was known that
diaminosulfides react in water to form sulfur monoxide, which
possessed a half-life of seconds and decomposed to release SO2
and elemental sulfur.89,90
Fabrication of Microparticles from a Poly-
(diaminosulfide) and Their Uptake into Cells. Synthetic
polymers are widely used in drug delivery. In this field a
polymer and drug are fabricated into nano- to micrometer sized
particles and delivered to the body. Most of the polymers used
in this field are based on polyestersalthough other polymers
are under investigationbecause of the need to have the
polymer degrade in vivo before it accumulates in the body and
provokes a toxic response. Polyesters are widely used because
they degrade in the body under neutral or acidic conditions
without the need for enzymes. This observation of the role of
polyesters in drug delivery and the degradation of diamino-
sulfides in water led us to speculate that poly(diaminosulfide)s
may be useful as drug delivery vehicles. The diaminosulfide
functional group degrades several orders of magnitude faster
than ester bonds under acidic conditions, and they possess
reasonable stabilities in water under neutral conditions.91 Some
of the first key experiments to demonstrate the ability of
poly(diaminosulfide)s to function as drug delivery vehicles are
described here, and more results will be published in
subsequent articles.
Figure 5. Stability of two molecules were studied at room temperature
in organic solvents including DMSO-d6/D2O (10/1 v/v).
A polymer with the structure of entry 6 in Table 2 was used
to fabricate microparticles that were studied as potential drug
delivery vehicles (Figure 7). The microparticles were prepared
according to a water/oil/water double emulsion-solvent
evaporation method using poly(vinyl alcohol) as a surfactant.
Briefly, the poly(diaminosulfide) was insoluble in water, and it
was added to dichloromethane with a dye (FITC-dextran). A
surfactant solution of water with 1 wt % poly(vinyl alcohol) was
added to the dicholoromethane and sonicated to produce the
particles. This solution was diluted with more water and
poly(vinyl alcohol) and further sonicated. After removal of the
dichloromethane by evaporation, the microparticles were
Figure 6. Degradation of molecule L was studied in D2O under acidic
(with acetic acid), neutral, and basic (with KOH) conditions. The
amount of the diaminosulfide that degraded as a function of time was
plotted.
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The cell viability of HEK-293 cells was investigated to
determine whether the microparticles derived from poly-
(diaminosulfide)s were toxic. The toxicity of microparticles
fabricated from the polymer with the structure shown in entry 6
of Table 2 was studied via a MTS assay that is widely accepted
as one method to determine cell viability in the presence of
foreign molecules.92,93 Briefly, the MTS assay measures the
mitochondrial activity of the cells and is used as an indication of
the cell growth and viability. In living cells the MTS reagent (a
yellow, water-soluble tetrazolium salt) is cleaved by the
mitochondrial enzyme dehydrogenase (NADH-dependent
reduction of the tetrazolium ring in MTS) to generate a
water-soluble purple product called formazan. The concen-
tration of formazan can be measured, and in this way, the
relationship between the cell number and the amount of
formazan generated is established since the absorbance is
directly proportional to the number of viable cells. Damaged or
dead cells exhibit a reduced or diminished enzyme activity and
therefore less or no formazan production. Here, the incubation
period of 24 h ensured the exposure of the cells to the different
treatments in their exponential growth phase. Figure 9 shows
Figure 7. SEM micrographs of microparticles fabricated from the
polymer shown in entry 6 of Table 2.
filtered and isolated. The particles were spherical in shape and
possessed a smooth, nonporous surface. The z-average particle
size determined by dynamic light scattering was 660 nm and
consistent with the SEM micrograph shown in Figure 7. The
surface charge determined to be −11.6 0.8 mV.
Microparticles were fabricated and loaded with fluorescein
isothiolate-dextran (FITC-dextran) to appear green under
optical microscopy. These microparticles were incubated with
HEK-293 cells at 37 °C for 24 h to study if they were
internalized into the cells. After 24 h, the cells were washed
with PBS buffer twice to remove any microparticles not
internalized into cells. The cells were then fixed with
paraformaldehyde and stained with 4′,6-diamidino-2-phenyl-
indole (DAPI) and phalloidin as described in the Experimental
Section. The results in Figure 8 clearly demonstrated that the
Figure 9. Cell viabilities of HEK-293 cells incubated with increasing
concentrations of poly(diaminosulfide)-based microparticles.
the cell viability as a function of the concentration of
microparticles and demonstrates excellent biocompatibility of
these novel polymeric microparticles in HEK-293 cells.
Microparticles in the concentration range of 1−1000 μg/mL
had no adverse effect on cell viability. Even high concentrations
of the microparticles did not reduce cell viability with cell
survival rates greater than 85% for all the concentrations tested.
CONCLUSIONS
■
Figure 8. A laser scanning microscopic image is shown of HEK-293
cells that were exposed to microparticles loaded with FITC-dextran
(green) for 24 h and then washed to remove microparticles that were
not internalized into the cells. The nuclei of the cells were stained blue
by DAPI, and the cytoplasm/cell membranes were stained red by
phalloidin. This image clearly shows that the microparticles were
internalized into the cells.
This paper described the first synthesis of poly-
(diaminosulfide)s from two simple starting materials. The
sulfur transfer reagent used in the synthesis was readily
synthesized in two steps, and because it was purified by
distillation rather than column chromatography, large quantities
could be synthesized in only a few days. These polymers have
many of the right properties to be used as synthetic polymers
for different applications. For instance, we investigated the
stabilities of diaminosulfides in different solvents so that future
applications of poly(diaminosulfide)s could be envisioned. This
functional group was very stable in organic solvents and not
prone to oxidation; in fact, no evidence of oxidation of the
sulfur was observed in any sample. One exciting application of
these polymers as drug delivery vehicles was explored, and the
results were very promising. A poly(diaminosulfide) was readily
fabricated into nanoparticles that were absorbed into cells.
These nanoparticles were also nontoxic toward HEK-293 cells.
These results were promising, but more work is needed to
investigate the advantages poly(diaminosulfide)s may possess
microparticles were internalized into the HEK-293 cells. In this
image, the microparticles were green, the nucleus was blue (due
to the DAPI stain), and the cytoplasm/cell membrane was red
(due to the phalloidin stain). In control experiments with cells
not exposed to the microparticles and not treated with
phalloidin or DAPI, the cells did not fluoresce green. Thus, it
is clear that there was no autofluorescence from the cells and
that the observed green fluorescence within the cells was due to
the uptake of the fluorescently labeled dextran loaded particles.
This result demonstrates that these microparticles have
potential as new drug delivery vehicles.
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Bis(succinimide) Sulfide (Molecule B).94 N-Chlorosuccinimide
(2.22 g, 16.6 mmol) was slowly added to a solution of molecule A
(5.08 g, 8.30 mmol) in CHCl3 (22 mL) at 0 °C and stirred. After 1.7 h,
the ice bath was removed and the reaction was stirred for 8 h. A yellow
solid stuck to the walls of the flask. The yellow organic phase was
decanted. The yellow solid was washed with hexanes and dried under
over polymers used in drug delivery. We propose a general label
of poly(NSN) for any poly(diaminosulfide) to emphasize the
functional group used in their synthesis and found in their
backbones. Poly(NSN) can be used to describe a general family
of polymers in the same way that the terms polystyrenes and
polyacrylates are used.
1
vacuum to give a crude yellow solid (1.30 g, 69% yield). H NMR
(DMSO-d6): δ 2.57 (s, 8H). 13C NMR (DMSO-d6): δ 29.55, 179.47.
N,N′-Dithio(bisethylmethylamine) (Molecule C).95 A solution
of N-ethylmethylamine (10.5 g, 178 mmol) in petroleum ether (400
mL) was cooled to −78 °C for 30 min. To this solution, sulfur
monochloride (6.00 g, 44.4 mmol) was added dropwise for 10 min.
The solution was stirred for 20 min at −78 °C and another 35 min at
room temperature. The mixture was washed with a saturated NaCl
solution in water. The organic layer was dried over anhydrous
magnesium sulfate and evaporated to give a yellow−green oil (7.00 g).
The product was isolated by vacuum distillation at 30−35 °C to yield a
colorless oil (6.20 g, 78% yield). 1H NMR (CDCl3): δ 1.14 (t, 6H, J =
7.2 Hz), 2.64 (s, 6H), 2.69 (q, 4H, J = 7.1 Hz). 13C NMR (CDCl3): δ
13.81, 46.28, 53.54. HRMS calcd for C6H16N2S2: 180.0755. Found:
180.0759.
One significant characteristic of diaminosulfides is that they
are based on an inorganic functional group. Their structures
differentiate them from the numerous organic functional
groups used in the synthesis of most polymers. We believe
that by working with inorganic functional groups with
reactivities that differ from those of organic functional groups,
new opportunities in macromolecular science will be realized.
EXPERIMENTAL SECTION
■
Materials. Sodium sulfide nonahydrate (Na2S·9H2O), tributyltin
chloride, N-chlorosuccinimide, sulfur monochloride, N-ethylmethyl-
amine, N-benzylmethylamine, N,N′-dimethyl-1,6-hexanediamine, N,N′-
bis[3-(methylamino)propyl]methylamine, 4,4′-trimethylenedipieri-
dine, N,N′-di-sec-butyl-p-phenylenediamine, dimethylamine, p-toluene-
sulfonyl chloride, and 3-methoxypropylamine were purchased from
Aldrich or Acros Organics at their highest purity and used as received.
FITC(fluorescein isothiocyanate)-dextran (Mw 20 kDa) and Mowiol
(poly(vinyl alcohol), PVA, av Mw ∼ 67K, 86.7−88.7% hydrolyzed) was
obtained from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO).
Deionized distilled water produced by Barnstead Nanopure Diamond
Water purification Systems (Dubuque, IA) was used throughout. All
other solvents including petroleum ether (39−56 °C) were reagent
grade and purchased from Fisher Scientific. Because dimethylamine is
a gas at room temperature, it was condensed inside a graduate cylinder
in a −78 °C bath before use. Piperazine (99%) was purchased from
Aldrich and was purified by sublimation under vacuum at 130 °C.
Genduran silica gel 60 (230−400 mesh) and Basic Alumina Brockman
Activity I (60−325 mesh) were purchased from Fisher Scientific were
used for all column chromatography.
N-Ethylmethylsulfenyl Chloride (Molecule D).96 A solution of
molecule C (4.81 g, 26.7 mmol) in CH2Cl2 (70 mL) was precooled to
0 °C for 40 min under N2. Sulfuryl chloride (3.96 g, 29.4 mmol) was
added dropwise to the solution for 17 min under N2. The reaction was
stirred for 30 min at 0 °C and another 50 min at room temperature to
give a crude product (6.70 g, 53.4 mmol), which was used in situ for
the preparation of molecule E.
Bis(N-ethylmethyl) Sulfide (Molecule E).96 A solution of
molecule D (6.70 g, 53.4 mmol) in CH2Cl2 (40 mL) was slowly
added to a solution of N-ethylmethylamine (7.89 g, 13.3 mmol) in
CH2Cl2 (60 mL) at 0 °C under N2 and stirred for 1 h. The reaction
was washed with a saturated NaCl solution in water. The organic
phase was dried over anhydrous magnesium sulfate and evaporated to
give a yellow−green oil (4.22 g). The product was purified by
distillation under vacuum at room temperature to yield a colorless oil
1
(2.53 g, 32% yield). H NMR (CDCl3): δ 1.14 (t, 6H, J = 7.2 Hz),
2.95 (s, 6H), 3.11 (q, 4H, J = 7.1 Hz). 13C NMR (CDCl3): δ 14.24,
46.29, 54.89. HRMS calcd for C6H16N2S: 148.1034. Found: 148.1033.
N,N′-Dithiobisdimethylamine.95 A solution of dimethylamine
(8.01 g, 178 mmol) in anhydrous ether (400 mL) was cooled to −78
°C for 44 min. Sulfur monochloride (6.00 g, 44.4 mmol) was added
dropwise to the solution for 14 min. The solution was stirred for 30
min at −78 °C and another 30 min at room temperature. The mixture
was washed with a saturated NaCl solution in water. The organic layer
was dried over anhydrous magnesium sulfate and evaporated to give a
colorless oil (6.66 g, 99% yield), which could be used directly for the
preparation of molecule F without further purification. 1H NMR
(CDCl3): δ 2.63 (s, 12H). 13C NMR (CDCl3): δ 48.31. HRMS calcd
for C4H12N2S2: 152.0442. Found: 152.0444.
Dulbecco’s Modified Eagle Medium (DMEM, with high glucose 1X
and 4 mM L-glutamine), Trypsin-EDTA (0.25%, 1X solution), and
Dulbecco’s phosphate buffered saline (PBS) were purchased from
Gibco (Invitrogen, NY). Fetal bovine serum (FBS) was obtained from
Atlanta Biologicals (Lawrenceville, GA). Gentamycin sulfate (50 mg/
mL) was purchased from Mediatech Inc. (Manassas, VA). MTS cell
growth assay reagent (Cell Titer 96 Aqueous One Solution cell
proliferation assay) was purchased from Promega Corp, Madison, WI.
The HEK-293 cell lines were obtained from the American Type
Culture Collection (ATCC, Manassas, VA).
1
Characterization. H and 13C NMR spectra were recorded on a
Bruker DPX 300 at 300 and 75 MHz, respectively. CDCl3 was used as
the NMR solvent with tetramethylsilane (TMS) as an internal
standard. Size-exclusion chromatography (SEC) was performed using
tetrahydrofuran as the mobile phase (1.00 mL min−1) at 25 °C. A
Shimadzu LC-10AT HPLC pump and one Varian column (PLgel 5
μm MIXED-D) were used in series. A Shimadzu RID-10A refractive
index detector and a Shimadzu SCL-10A system controller were used
to measure molecular weights of polymers based on a polystyrene
standard calibration curve.
N-Dimethylsulfenyl Chloride.96 A solution of N,N′-dithiobisdi-
methylamine (6.03 g, 39.6 mmol) in anhydrous Et2O (50 mL) was
cooled to 0 °C for 1 h under N2. Sulfuryl chloride (5.88 g, 43.6 mmol)
was added dropwise to the solution under N2. The reaction was stirred
for 36 min at 0 °C and another 50 min at room temperature to give a
crude product (8.84 g, 79.2 mmol), which was used in situ for the
preparation of molecule F.
Bis(N,N′-dimethyl) Sulfide (Molecule F).96 A solution of N-
dimethylsulfenyl chloride (8.84 g, 79.2 mmol) in anhydrous Et2O (50
mL) was slowly added to a solution of dimethylamine (17.9 g, 39.6
mmol) in anhydrous Et2O (75 mL) at −5 °C under N2 and stirred for
1.2 h. The reaction was washed with saturated aqueous NaCl. The
organic phase was dried over anhydrous magnesium sulfate, and the
solvent was removed after freezing the product at −5 °C to give
yellow-green oil (7.0 g). Further purification was achieved by
distillation under vacuum at 30 °C to yield a colorless oil (4.39 g,
Bis(tributyltin) Sulfide (Molecule A). This molecule was
prepared according to a literature procedure.94 A solution of sodium
sulfide nonahydrate (Na2S·9H2O) (42.0 g, 175 mmol) in deionized
water (34.8 mL) was prepared. This solution was added to a solution
of tributyltin chloride (28.5 g, 87.4 mmol) in THF (174 mL). Extra
deionized water (17.4 mL) was used to transfer the Na2S·9H2O to the
flask. The mixture was reacted at 65 °C for 5 h. After cooling the
reaction, the organic layer was evaporated. The residue was extracted
with Et2O. The extract was dried over anhydrous magnesium sulfate
1
46% yield). H NMR (CDCl3): δ 3.02 (s). 13C NMR (CDCl3): δ
1
and evaporated to give a colorless oil (21.8 g, 81% yield). H NMR
49.69. HRMS calcd for C4H12N2S: 120.0721. Found: 120.0719.
Bis(N,N′-(3-methoxypropyl(triethylene glycol monomethyl
ether) sulfide)) (Molecule L). In a flask was added N-(3-
(CDCl3): δ 0.91 (t, 18H, J = 7.2 Hz), 1.08 (m, 12H), 1.34 (m, 12H),
1.55 (m, 12H). 13C NMR (CDCl3): δ 13.66, 15.85, 27.17, 28.68.
694
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Article
methoxypropyl(triethylene glycol monomethyl ether)) (2.27 g, 9.64
mmol) and 3.6 mL of benzene. Next, molecule F (0.503 g, 4.18 mmol)
was added, and the flask was connected to a reflux condenser and
heated to 85 °C for 48 h. The benzene was removed under vacuum.
The product was cleaned by chromatography on basic alumina oxide
using ethyl acetate. The product was a clear oil (1.54 g, 73% yield). 1H
NMR (CDCl3): δ 1.81 (p, 2H, J = 6 Hz), 3.12 (t, 2H, J = 7.2 Hz) 3.2−
3.4 (m, 10H), 3.5−3.7 (m, 10H). 13C NMR (CDCl3): δ 29.06, 54.83,
57.53, 58.56, 59.02, 70.13, 70.28, 70.45, 70.56, 70.64, 71.95. HRMS
calcd for C22H48N2O8S: 500.3131. Found: 500.3125.
Entry 1, Table 2. Molecule F (0.942 g, 7.83 mmol) was reacted
with N,N′-dimethyl-1,6-hexanediamine (1.13 g, 7.83 mmol) in
refluxing benzene (11 mL) at 85 °C for 24 h. After evaporating the
solvent, the polymer was precipitated into methanol (10 mL). The
polymer was dried under vacuum to yield a brown oil (1.02 g, 75%
yield). 1H NMR (CDCl3): δ 1.29 (m, 4H), 1.54 (m, 4H), 2.94 (s, 6H),
3.07 (t, 4H, J = 7.2 Hz). 13C NMR (CDCl3): δ 26.88, 28.86, 46.90.
61.05.
of CDCl3. After connecting a condenser to the flask, the mixture was
reacted at 50 °C, and the reaction was monitored by 1H NMR
spectroscopy every 24 h showing 9% conversion to J and 88%
conversion to I after 72 h.
The same procedure was followed for the reaction of molecule F
(88.3 mg, 735 μmol) and N-benzylmethylamine (178 mg, 1.47 mmol)
in 1.47 mL of DMSO-d6 showing 13% conversion to J and 77%
conversion to I after 72 h. The reaction of molecule F (82.3 mg, 685
μmol) and N-benzylmethylamine (166 mg, 1.37 mmol) in 1.37 mL of
C6D6 showed 17% conversion to J and 75% conversion to I after 72 h.
Molecule F (89.9 mg, 748 μmol) and N-benzylmethylamine (181
mg, 1.50 mmol) were reacted in 1.5 mL of benzene at 85 °C showing
3% conversion to J and 97% conversion to I after 24 h.
Stability of Molecule E in Organic Solvents. The stability of
molecule E was studied in CDCl3, DMSO-d6/D2O (10/1 v/v), and
C6D6 following the same procedure. Molecule E (34.4 mg, 2.32 × 10−4
mol) was added to an NMR tube with 1 mL of solvent. Next,
diethylene glycol dimethyl ether (31.2 mg, 2.32 × 10−4 mol) was
added. The NMR tube was capped, and 1H NMR spectra were
periodically collected. The amount of decomposition was determined
by the difference in ratio of the peaks due to molecule E and the ether
measured on days 1 and 32.
Entry 3, Table 2. Molecule F (0.186 g, 1.55 mmol) was reacted
with N,N′-bis[3-(methylamino)propyl]methylamine (0.268 g, 1.55
mmol) in refluxing benzene (1.4 mL) at 85 °C for 72 h. The benzene
was removed under vacuum. When the polymer was redissolved in 4
mL of CH3OH and precipitated into 9 mL of water, the polymer
decomposed to unknown products, and the 1H NMR spectrum
became too complicated to assign the peaks. Therefore, after the
polymerization was complete the polymer was dried under vacuum to
yield a brown oil (0.310 g, 97% yield) that was used without further
Stability of Molecule L in D2O. Molecule L (31.4 mg, 6.27 × 10−5
mol) was added to an NMR tube. A 1 mL solution in D2O of tert-
butanol (5.96 mg, 6.27 × 10−5 mol) and acetic acid (30.3 mg, 5.02 ×
10−4 mol) was added to the NMR tube, and it was vigorously shaken.
The first 1H NMR spectrum after 271 s showed no evidence of
molecule L and showed the secondary amine as the only degradation
product.
1
purification. H NMR (CDCl3): δ 1.71(m, 4H), 2.21 (s, 3H), 2.32 (t,
4H, J = 7.5 Hz), 2.95 (s, 6H), 3.12 (t, 4H J = 7.1 Hz). 13C NMR
(CDCl3): δ 25.59, 42.43, 46.90, 55.38, 59.01.
The same procedure was followed except that no acetic acid was
added (the neutral conditions). The decomposition of molecule L was
followed by 1H NMR spectroscopy. The same procedure was followed
except that no acetic acid was added and KOH (9 mol equiv) was
added (the basic conditions). The decomposition of molecule L was
Entry 5, Table 2. Molecule F (0.186 g, 1.55 mmol) was reacted
with 4,4′-trimethylenedipiperidine (0.326 g, 1.55 mmol) in CHCl3 (1.6
mL) at 60 °C for 72 h. After evaporating the solvent and redissolving it
in CH2Cl2 (4 mL), the polymer was precipitated into methanol (8
1
1
followed by H NMR spectroscopy.
mL) to give a white-yellow powder (0.330 g, 88% yield). H NMR
Formulation of Microparticles. Microparticles were fabricated
from the polymer in entry 6 of Table 2 using a double emulsion-
solvent evaporation method that is widely used for the encapsulation
of hydrophilic drugs. The surfactant solution (1 wt % PVA in water,
internal water phase or W1) was added to the polymer solution (in
dichloromethane, oil phase or O) under microtip probe sonication for
30 s (10 W energy output, Fisher Scientific sonic dismembrator Model
100) to form the first emulsion (W1/O). This was then immediately
added to the second PVA solution (in water, external water phase or
W2) and further sonicated at the same speed for another 30 s to form
the second emulsion (W1/O/W2). These processes were carried out
under an ice bath. The final emulsion was then added to aqueous PVA
solution under magnetic agitation and stirred at room temperature and
under atmospheric pressure until complete evaporation of dichloro-
methane. The microparticles were collected by centrifugation at 8500
rpm for 10 min (Fischer Scientific Accuspin 400), washed twice with
water, and freeze-dried for 48 h (FreezeZone 4.5, Labconco). The
FITC-dextran loaded microparticles were prepared in the same
manner by dissolving FITC-dextran in the internal water phase used in
making the primary emulsion.
Determination of Particle Size (Hydrodynamic Diameter)
and Size Distribution. Particle size and particle size distribution of
microparticles were analyzed at a concentration of approximately 1 mg
particles/1 mL of deionized water. Appropriate dilution of the particle
suspension is necessary in order to avoid multiscattering events. The
measurements were carried out on microparticle suspensions using a
Zetasizer Nano-ZS (Malvern Instruments). The particle size and size
distribution by intensity were measured by dynamic light scattering
(He−Ne laser with a fixed wavelength of 633 nm, 173° backscatter at
25 °C) in 10 mm diameter cells.
(CDCl3): δ 1.22(m, 12H), 1.59 (m, 4H), 3.08 (t, 4H, J = 11.0 Hz),
3.44 (m, 4H). 13C NMR (CDCl3): δ 23.68, 34.02, 34.96, 36.72, 58.57.
Reactions of Molecule E and N-Benzylmethylamine (Figure
3). Molecule E (46.3 mg, 312 μmol) was dissolved in 1.35 mL of
CD2Cl2, and 1 mL (34.4 mg, 232 μmol) of the solution was
transferred to a NMR tube. After the addition of N-benzylmethyl-
amine (56.3 mg, 464 μmol) and sealing the NMR tube with a rubber
1
septum, H NMR spectra were continually recorded for 3 days. The
reaction was monitored by conversion of the benzyl hydrogens in N-
benzylmethylamine at 3.71 ppm to the benzyl hydrogens in molecule
H at 4.31 ppm and in molecule I at 4.38 ppm.
The same procedure was also followed for the kinetics in CDCl3.
The conversion from molecule E to molecules H and I was monitored
by comparing the benzyl peak (3.74 ppm) of N-benzylmethylamine
with the benzyl peak (4.32 ppm) of molecule H and the benzyl peak
(4.38 ppm) of molecule I for 10 days.
For the kinetics in DMSO-d6, molecule E (51.3 mg, 345 μmol) was
dissolved in 1.49 mL of DMSO-d6, from which 1 mL (34.4 mg, 232
μmol) was added to an NMR tube. After adding N-benzylmethylamine
(56.3 mg, 464 μmol) and sealing the NMR tube with a rubber septum,
the reaction was monitored by 1H NMR spectroscopy for 5 days. The
conversion was observed by comparing the benzyl hydrogens in N-
benzylmethylamine at 3.63 ppm with the benzyl hydrogens in
molecule H at 4.29 ppm and in molecule I at 4.36 ppm.
For the kinetics in C6D6, molecule E (49.4 mg, 333 μmol) was
dissolved in 1.44 mL of C6D6 and 1 mL (34.4 mg, 232 μmol) was
added to an NMR tube, followed by the addition of N-benzylmethyl-
amine (56.3 mg, 464 μmol) and sealing the NMR tube with a rubber
septum. The conversion from molecule E to molecules H and I was
monitored by comparing the benzyl hydrogens in N-benzylmethyl-
amine at 3.62 ppm with the benzyl hydrogens in molecule H at 4.34
ppm and in molecule I at 4.36 ppm.
Measurement of Surface Charge. The zeta potential of
microparticles was analyzed by dispersing the microparticles in
deionized distilled water at a concentration of 1 mg/mL using folded
capillary cells. Sample dilution is often necessary in order to eliminate
particle interactions. Zeta potential is an indicator of the charge on the
Transamination Reaction of Molecule F and N-Benzylme-
thylamine (Table 1). N-Benzylmethylamine (153 mg, 1.26 mmol)
was added to a solution of molecule F (75.8 mg, 631 μmol) in 1.26 mL
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surface of the microparticles. The surface charge measurements of the
blank microparticles were performed using the electrophoretic laser
scattering method (Laser Doppler Microelectrophoresis, He−Ne laser
633 nm at 25 °C).
Scanning Electron Microscopy (SEM). The shape and the
surface morphology of the microparticles were studied using a
scanning electron microscope. The particles were mounted on silicon
wafers which were placed on aluminum specimen stubs using adhesive
carbon tape. The mount was then coated by ion sputtering (K550
Emitech sputter coater, set at 10 mA for 2.5 min) with conductive gold
and examined using a Hitachi Model S-4800 SEM, operated at 4 kV
accelerating voltage.
cell viability(%) = [absorbance intensity of cells treated with
MPs]/[absorbance intensity of cells
without any treatment (control)] × 100
The cytotoxic effect of different treatments was calculated as a
percentage of cell growth with respect to the control. Values are
expressed as mean SEM for each microparticle concentration (n =
6).
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: ned-bowden@uiowa.edu; Tel: (319) 335-1198.
Cell Culture. The cells were maintained in DMEM supplemented
with 10 vol % FBS and gentamycin at a concentration 50 μg/mL in a
humidified incubator (Sanyo Scientific Autoflow, IR direct heat CO2
incubator) at 37 °C containing 95% air and 5% CO2. The cells were
plated and grown as a monolayer in 75 cm2 polystyrene cell culture
flasks (Corning Inc., Corning, NY) and subcultured (subcultivation
ratio of 1:4) after 80−90% confluence was achieved. Cell lines were
started from frozen stocks, and the medium was changed every 2−3
days. The passages used for the experiment were between 4 and 15.
Investigation of FITC-Dextran Loaded Microparticle Uptake
by HEK-293 Cells Using Confocal Microscopy. To determine the
qualitative in vitro intracellular uptake of microparticles, cells were
plated at a density of 50 000 cells/well in a clear, flat-bottom, 8-
chambered glass slide with cover (Lab-Tek, Nunc, NY) that were
previously coated with 0.1 wt % poly(L-lysine). The cells were allowed
to attach overnight, and the next day the cell culture medium was
removed and the cells were treated with an aliquot of a suspension of
FITC-dextran loaded microparticles in medium and further incubated
at 37 °C for 24 h. The experiment was terminated by removing the
particulate suspension and washing the cell monolayer two times with
PBS in order to remove particles not internalized by the cells. The cells
were then fixed with 4 vol % paraformaldehyde, followed by
permeabilization of cells with 0.2 wt % Triton X-100 (Sigma, Sigma-
Aldrich, St. Louis, MO). The cells were later treated with phalloidin
and finally mounted with Vectashield, Hardset mounted medium with
DAPI (H-1500, Vector Laboratories, Inc., Burlingame, CA). The cells
were washed with PBS during every step in the process. Cellular
uptake of FITC-dextran loaded microparticles and their intracellular
distribution was visualized by confocal microscopy (Carl Zeiss LSM
710, 60× oil objective lens) by using DAPI, FITC, and phalloidin
filters equipped with Zen 2009 imaging software.
ACKNOWLEDGMENTS
■
N.B.B. gratefully thanks the NSF for generous funding (CHE-
0848162). A.K.S. gratefully acknowledges support from the
American Cancer Society (RSG-09-015-01-CDD), the National
Cancer Institute at the National Institutes of Health
(1R21CA13345-01/1R21CA128414-01A2/UI Mayo Clinic
Lymphoma SPORE), and the Pharmaceutical Research and
Manufacturers of America (PhRMA) Foundation. A.K.S thanks
the Core Microscopy Research Facility for use of imaging
equipment.
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dx.doi.org/10.1021/ma2023167 | Macromolecules 2012, 45, 688−697