Enzyme responsive acetaminophen hydrogels
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Citation
Vemula, Praveen Kumar et al. “Enzyme Responsive
Acetaminophen Hydrogels.” IEEE, 2009. 1–2. © 2009 IEEE
As Published
http://dx.doi.org/10.1109/NEBC.2009.4967713
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Institute of Electrical and Electronics Engineers (IEEE)
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Final published version
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Wed May 25 18:08:19 EDT 2016
Citable Link
http://hdl.handle.net/1721.1/74120
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Detailed Terms
Enzyme Responsive Acetaminophen Hydrogels
Praveen Kumar Vemulaa, Gregory A. Cruikshankb, George Johnb*, Jeffrey M. Karpa*
aHarvard-MIT Division of Heath Sciences and Technology, Department of Medicine
Brigham and Women's Hospital, Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA
bDepartment of Chemistry, The City College of New York, Convent Avenue at 138th Street, New York, NY t0031
Abstract- Utilization of enzyme catalysis as a tool to disassemble
self-assembled hydrogels to control the release encapsulated drug
provides an opportunity to design a wide range of enzymespecific low-molecular-weight hydrogelators (LMWGs). Herein,
we report a novel approach for controlled delivery of multiple
drugs by an enzyme triggered hydrogel degradation mechanism.
In this proof-of-concept work, we report the synthesis of
LMWGs (amphiphiles) from well-known drug acetaminophen
(which is known as Tylenol®), and their ability to self-assemble
into nanoscale structures in aqueous solutions to form hydrogels
that subsequently encapsulate a second drug such as curcumin
which is a known chemopreventive hydrophobic drug. Upon
enzyme triggered degradation, hydrogels showed single and
double drug delivery at physiological conditions in vitro. After
treating with prodrug amp hip hiles, mesenchymal stem cells
(MSCs) retain their stem cell properties such as maintaining
their adhesive and proliferation capacities with high viability.
This new platform approach will have prospective effect on
hydrogel based drug delivery research through developing drug
delivery vehicles from a wide range of prod rug-based gelators.
I. INTRODUCTION
biocompatible fatty acid would surmount existing drawbacks.
Self-assembly proved to be a powerful strategy to develop
molecularly defined and functional material [I]. Hydrogels are
one among them, and particularly they have been widely
applied as intelligent delivery systems in controlled drug
delivery [2]. A study of the literature reveals that there are
only limited self-assembly based systems (low-molecularweight gelators (LMWGs) are available as opposed to
polymeric hydrogels [3]. One of the major rationales for this
scenario is undesired toxicity of unknown metabolites that are
generated from degradation of LMWGs hydrogelators. As a
consequence, here we propose a novel conceptual route to
surpass possible toxicity of in situ generated fragments of
LMWGs (Fig. la). The key step in this approach is selecting
either clinically practiced drugs or appropriate drugs that have
safe metabolic pathway as precursor molecule to generate a
library of amphiphiles which can undergo self-assembly to
form hydrogels. Though covalent conjugation of known-drugs
to the polymer backbone is well documented, however,
polymer degradation can lead to polymer fragments with
heterogeneous chain lengths that may generate potential
toxicity or unwanted side effects. Hence, our approach,
synthesizing prodrug-based amphiphiles which can degrade in
presence of an enzyme to generate parent drug and another
Figure 1. a) Schematic representation of single and multiple drug delivery
a)
b)
..
drug
I
l
synthesis
)t-o-
1
~
H, C
A
OH
a,ro-dircarboxVlic acid
~
DeC, DMAP. dry THF
24 h, rt
amphiphiles
o
.Jl. ~
self·assembly
H, C
Prodrug·based hydrogels
~~
~
~~
~~
~~
~
acetaminophen (Apn )
-0-o· 1"1.Jl
0
~
h
Jl,
0
' OH
n = 6 Apn·7·COOH
n = 12 Apn·13·COOH
~
(with second drug
encapsu lated hydrogels)
1
,yr- , .
1
,yr- ,
enzyme catalysis
(hyd rogels degradation)
~. ~
, ~
" single drug" delivery
" multiple drug" delivery
from prodrug·based hydrogels in response to enzyme catalysis. b) Synthesis
of acetaminophen based amphiphiles. Scanning and transmission electron
microscope images of curcumin encapsulated hydrogels (c and d,
respectively).
Despite of having various stimuli such as temperature, pH
and ionic strength etc to trigger drug delivery, an enzyme
triggered drug delivery considered to be most safe method. In
addition, often it allows the release of encapsulated
therapeutics in desired locations. In this present approach we
have overcome existing hurdles to develop biocompatible
amphiphilic prodrugs as LMWGs to encapsulate hydrophobic
drugs and subsequently release single and multiple drugs upon
enzyme mediated gel degradation (Fig. la) in this
investigation. To demonstrate the proof-of-concept, we
N-(4choose
the
well-known
drug
Tylenol,
hydroxyphenyl)acetamide, which is also known as
acetaminophen (ApD), its drug activity is well studied [4,5].
APD is a common analgesic and antipyretic drug that is used
for the relief of fever, headaches, and other minor aches and
pains. It is remarkably safe in recommended doses and
importantly its route of metabolism in humans has extensively
been investigated and found to be safe. A set of amphiphiles
were synthesized in a single step from APD as shown in Fig.
lb.
II. MATERIALS AND METHODS
Prodrug-based amphiphiles (ApD-7-COOH and APD-13COOH) were synthesized using a single step esterification
reaction from acetaminophen (Fig. Ib). Typically, in a round
bottom flask, a ,ro-dicarboxylic acid (12 mmol) was dissolved
in dry THF, to that 1.2 equivalent of DCC and catalytic
amounts of DMAP was added at room temperature. After
stirring for 1 hr, 4-Hydroxyacetanilide (Apn, 1.36 g, 10
mmol) was added in one lot and stirring continued for 24 hrs.
After completion of the reaction, mixture was filtered to
remove DCU, washed with THF. After removal of THF,
slightly acidic water was added, and thrice extracted with
chloroform; organic layer was dried over anhydrous Na2S04.
After evaporating the solvent, the obtained crude products
were purified by silica gel column chromatography using
methanol:chloroform (1:9) as eluent, afforded pure products
as white solid. Yields of the reactions were - 50%.
III. RESULTS AND DISCUSSION
Gelation abilities of Apn based amphiphilic prodrugs have
been investigated thoroughly in a wide range of solvents. The
amphiphile Apn-7-COOH forms excellent gels in water and
other aqueous buffer solutions even in the presence of salts. In
addition, Apn-7-COOH is soluble in basic solutions (PH
greater than 8), on contrary it forms hydrogel in acidic and
neutral pH solutions. This clearly shows that terminal
carboxylic acid groups play a vital role in self-assembly
through formation of a hydrogen bonding network. However,
due to the longer hydrophobic chain Apn-13-COOH was
sparingly soluble in water and thus additional co-solvent (5-15
v/v% of alcohol such as methanol, ethanol or isopropanol) is
necessary to make the hydrogel. Hydrogels of Apn-7-COOH
or Apn-13-COOH were found to encapsulate high
concentrations of curcumin (up to 0.5 mmol); this observed
enhanced concentration of curcumin could be due to
localization of hydrophobic curcumin within the hydrophobic
pockets of the hydrogen's three-dimensional network.
Morphology of curcurnin encapsulated hydrogels were
examined under electron microscope; SEM and TEM images
(Fig. 1 c and d, respectively) showed that prodrug gels form
branched or entangled fibrous/sheet-like gel networks with
fiber thickness of 50-400 nm, and fiber lengths of several
microns.
To examine multiple (two) drug delivery (Fig. 2a), lipase
was added to the curcurnin encapsulated hydrogel of Apn-7COOH (2 wt%) and kept it at 37 °C. Absorbance spectra were
recorded on aliquots which were collected (after the addition
of enzyme to the hydrogel) at regular intervals. Interestingly,
initial aliquots did not show any absorbance peak, but aliquots
collected after 4 hrs showed absorption maxima at 425 nm,
which corresponds to the absorption peak of curcurnin. To
determine the role of the enzyme on hydrogel degradation,
similar experiments were performed by adding only buffered
solution without an enzyme. As we expected curcumin
encapsulated gel was still intact over a month, there was no
absorbance peaks corresponding to the curcurnin were
observed suggesting that the enzyme is required for gel
degradation. Interestingly, increasing either the concentration
of enzyme or incubation temperature to 45 °C doubled the rate
of drug release.
a) 4
~
2
III
.0
1
c
(5
-4hr
8 hr
12hr
16hr
20 hr
24hr
0
2~OO----3~00---400
~~~500~
J.. (nm)
C) 100..----------""C-"...,-J- - - - ,
~
75
~
(j)
<.)
50
"0
~
<II
.r:
25
"0
«
0
20
;i
80
.~
:.0
60
100
~
III
til
~
br
r :E-
X
I:
IiII
II
II
PBS treated
:>
Apn
Apn·7·COOH
I
X
[ 1
PBS treated
Apn
c::J
Apn·7·COOH
40
20
0
d) 30
'b
T""
CJ
I
:?:!.
20
~
15
(j)
~---
25
.
:::::--- - -
- - - Apn
_ _ PBS treated
<.)
~- Apn ·7·C OOH
'0
0
0
2
4
days
6
8
Figure 2. a) Absorption spectra curcumin released from degraded hydrogel of
Apn-7-COOH at different time points. Examining the effect of Apn-7COOH prodrug on MSCs cellar properties such as b) viability, c) adhesion
and d) proliferation.
To test the cytocompatibility of prodrug-based amphiphiles,
and their effect on cell characteristics such as viability,
proliferation, and adhesion; a series of experiments were
performed with MSCs. In these experiments unmodified drug
(Apn) and modified drug Apn-7-COOH were examined with
trypan blue assay. The presented results (Fig. 2b) suggest that
there was no significant decrease in the viability of the MSCs
after 48 hours incubation with prodrug-based amphiphile.
These results clearly show that Apn-derived prodrug
amphiphiles did not induce cell toxicity. In addition, data
showed in Fig. 2 c and d clearly suggesting that Apn-7COOH amphiphile did not affect the inherent cell properties
such as adhesion and ability to proliferate.
IV. CONCLUSIONS
In conclusion, Apn-based amphiphilic prodrugs have
showed excellent hydrogelation ability and could encapsulate
curcumin. Delivery of single and multiple drugs has been
achieved upon enzyme-triggered gel degradation at
physiological condition. Newly synthesized prodrugs were
highly cytocompatible in nature.
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