Two-stage enzyme mediated drug release from LMWG hydrogels

Article abstract of DOI:10.1039/b507157g

An enzymatically cleavable low molecular weight gelator-(model) drug conjugate system can be employed to effect a two-step enzyme mediated drug release, demonstrating the potential of LMWG systems for the development of drug delivery devices. The Royal So

Full text of DOI:10.1039/b507157g

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C O M M U N I C A T I O N
Two-stage enzyme mediated drug release from LMWG hydrogels
Kjeld J. C. van Bommel,*a Marc C. A. Stuart,b Ben L. Feringab and Jan van Esch*b
a Biomade Technology Foundation, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
E-mail: van.bommel@biomade.nl; Fax: +31 (0)50 3634429; Tel: +31 (0)50 3634599
b Department of Organic and Molecular Inorganic Chemistry, Stratingh Institute, University of
Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. E-mail: J.van.Esch@rug.nl;
Fax: +31 (0)50 3634296; Tel: +31 (0)50 3634428
Received 23rd May 2005, Accepted 17th June 2005
First published as an Advance Article on the web 8th July 2005
An enzymatically cleavable low molecular weight gelator–
(model) drug conjugate system can be employed to effect a
two-step enzyme mediated drug release, demonstrating the
potential of LMWG systems for the development of drug
delivery devices.
Results and discussion
Recently we reported the excellent gelation properties of a
novel class of cyclohexane trisamide-based hydrogelators with
a modular architecture and demonstrated that, through the
introduction of pH sensitive moieties onto the gelator scaffold,
the inherently thermoreversible gels could be made responsive
to changes in pH as well.4b For the two-step release system
described here, use was made of the cyclohexane trisamide
scaffold to which an L-phenylalanyl–amidoquinoline (L-Phe–
AQ) moiety as well as two ethylene glycol chains were con-
nected (Scheme 1). The L-Phe–AQ moiety can be enzymatically
cleaved by a-chymotrypsin (a-chy), resulting in the release of
the fluorogenic “model drug” 6-aminoquinoline (6-AQ).9 The
two ethylene glycol chains were introduced to give the overall
structure a minimum degree of water solubility, which improves
the gelation properties.
Introduction
One of the central problems in drug delivery today is striking
the balance between toxicity and therapeutic effect of phar-
maceuticals. By limiting the delivery to specific target sites,
possible toxic effects at non-target sites can be avoided and the
efficiency of the drug is increased. For this reason, smart drug
delivery systems have been a major focus of pharmaceutical
and materials research.1 Two-stage drug delivery systems are
particularly attractive as they allow an even higher degree of
selectivity, with release depending on the consecutive action of
two trigger mechanisms. One of the most interesting stimuli
that can be employed for drug release is the action of enzymes,
which allows the release of pharmaceuticals in very specific
locations. For instance, drug release in tumors as a result of the
enzymatic action of tumor-associated proteases (e.g., plasmin),2
or in designated areas of the GI tract under the influence of
digestive enzymes has been reported.3
Scheme 1 Enzymatically cleavable LMWG 1 and non-gelator 2.
In our search for smart, responsive drug delivery systems,4
that can be used as alternatives to polymer gel-based systems
we investigated two-step drug delivery hydrogels based on low
molecular weight gelator (LMWG)-drug conjugates.5 Hydrogels
of LMWGs6,7 can be made responsive to a variety of stimuli
to which they can respond by a fast gel to solution phase
transition. Hence, gels of LMWGs can have rapid response times
(in the order of a few seconds), not attainable by conventionally
studied polymeric systems.8 Here we present an enzymatically
cleavable LMWG–(model) drug conjugate system (Fig. 1) and
demonstrate that incorporation into the gel fibers protects
molecules from enzymatic cleavage. Upon applying a stimulus
(e.g., via a pH or temperature change, as in Fig. 1) these gel
fibers dissociate into individual molecules that can be cleaved
by the enzyme, resulting in a two-step release mechanism for
drugs.
Compound 1 indeed proved capable of gelating water with
a critical gelator concentration (CGC, i.e., the lowest con-
centration at which gelation occurs) at room temperature as
low as 0.45 mM (= 0.03 wt%), making it a so-called super
hydrogelator.10 All gels up to 0.75 wt% are highly transparent,
indicative of gelator aggregates with small diameters. This was
confirmed by cryo-transmission electron microscopy experi-
ments, which showed that hydrogels of LMWG 1 consist of
a dense network of unbranched, very long, relatively straight
tubular fibers (Fig. 2). Remarkably, all fibers were found to have
identical diameters of ca. 4.2 nm, corresponding to the length
of two molecules. Such monodispersity is rare and indicates that
fibers are formed under thermodynamic, rather than kinetic,
control.11
Fig. 1 Schematic representation of the enzymatic cleavage of a
gelator–drug conjugate and the influence of aggregation on enzymatic
cleavage; D = trigger for gel-to-sol transition (e.g., temperature, pH).
Fig. 2 Cryo-TEM images of a hydrogel of 1 (0.25 wt% = 4.77 mM).
Note the uniform fiber thickness (bars correspond to 100 nm).
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 9 1 7 – 2 9 2 0
2 9 1 7
©

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Gel formation by 1 is completely thermoreversible, with gel–
sol transition temperatures4b ranging from 52 ◦C for a 0.03 wt%
gel, to 114 ◦C for a 0.55 wt% gel. Furthermore, owing to the
presence of the basic quinoline moiety, gels of 1 can be reversibly
switched to solutions by decreasing the pH (between pH 3–5,
depending on the concentration of the LMWG). Several cycles of
temperature- or pH-induced dissolution-gelation can be carried
out without any loss in gelation capability, demonstrating the
excellent responsiveness, as well as the robustness, of our system.
The first step in demonstrating this two-step drug release
system is to prove that incorporation into gel fibers protects
molecules from enzymatic cleavage. To this end, enzyme kinetics
for the cleavage of LMWG 1 and non-gelating model compound
29 were studied by means of fluorescence spectroscopy. In order
to prevent the diffusion of the enzyme from being rate limiting,
a-chy needs to be included homogeneously into gels of 1, rather
than being injected into or added as a solution on top of
a gel. As a-chy is sensitive to large changes in temperature12
and the experiments necessitated the use of buffered solutions
(pH 7.75), enzyme incorporation via temperature or pH induced
dissolution–gelation was not possible. A solution was found in
the use of mixed solvent systems. The rapid addition of a buffer
solution of a-chy to a solution of LMWG 1 in a small amount
of DMSO resulted in the instantaneous formation of a clear,
homogeneous gel suitable for fluorescence experiments (buffer :
DMSO = 9 : 1).
substrate 2 (both present in 7.54 mM). The observed value for
V0 (13.7 lmol min−1) is well above the maximum observed value
for the pure gel samples of 1 (=1.8 lmol min−1; see Fig. 3) and
close to the value observed for an equimolar solution sample
of 2 (=13.8 lmol min−1; see Fig. 3), demonstrating that a-chy
included in the gel matrix retains an activity level similar to that
in solution.
As more gel fibers of 1 disassociate at higher temperatures,
this should result in an increased release of 6-AQ. In order to
demonstrate that the rate of enzymatic cleavage is controllable
by altering the gel–sol equilibrium, hydrolysis experiments were
carried out over the temperature range 25–45 ◦C. Studying
the formation of 6-AQ in solutions of 2, demonstrates that
the increase in the rate of hydrolysis (V) as a result of
the temperature-induced increase in enzymatic activity only
corresponds to ca. 100% over a 20 ◦C interval (Fig. 4, lower
data points). Conversely, in the gel sample of LMWG 1, V
increased by nearly 1200% over a 20 ◦C interval (upper data
points), confirming that increasing the temperature results in an
augmented release of 6-AQ as a result of the two-step mechanism
of gel fiber dissolution followed by enzymatic cleavage.
The initial rates of hydrolysis (V0) of the non-gelating
substrate 2 as a function of the concentration (S) are accurately
described using standard enzyme kinetic models (Fig. 3). The
maximum initial rate Vmax (22.4 lmol min−1) and Michaelis
constant Km (4.8 mM) were calculated by means of an Eadie–
Hofstee plot.
Fig. 4 Temperature dependent increase in enzymatic hydrolysis (V)
with respect to V(25), in 10 mM gels of 1 (ꢁ) and 10 mM solutions
of 2 (᭜). Error bars: standard deviation over three independent
measurements. For experimental conditions see Fig. 3.
In conclusion, a simple model system based on hydrogels from
a LMWG–(model)drug conjugate has successfully been used to
demonstrate the concept of two-stage enzyme mediated drug
release. It was shown that LMWG–(model) drug molecules are
protected from enzymatic cleavage by incorporation into the
gel fibers, although the enzyme included in the gel is still fully
functional. By raising the temperature of the LMWG system,
the gel–sol equilibrium is altered and more gelator becomes
available for enzymatic cleavage, resulting in a dramatic increase
in the rate of release of the model ‘drug’. The results reported
here demonstrate the potential of LMWG gels for controlled
drug release systems and we plan to use such systems for the
development of drug delivery devices.
Fig. 3 Initial rate of hydrolysis of LMWG 1 (᭹) and model substrate 2
(ꢀ) as a function of the substrate concentration. Solid lines: theoretical
curves based on the calculated Vmax and Km values. Dashed line:
experimental curve. Inset: enlarged view of data points and theoretical
curve for LMWG 1. Conditions: 25 ◦C, [a-chy] = 40 lM, buffer
(tris–HCl, 0.1 M, pH 7.75) : DMSO = 9 : 1.
The behavior observed for LMWG 1 is in total contrast
to that of 2. At low concentrations of 1 the values of V0
increase as a function of the substrate concentration (Vmax = 4.1
lmol min−1, Km = 1.8 mM).13 However, V0 levels off abruptly at a
concentration of ca. 1.5 mM (inset of Fig. 3), which corresponds
to the CGC in this solvent mixture.14 Since the fraction of
gelator molecules in excess of the CGC is incorporated in the gel
fibers, the CGC is also the maximum concentration of gelator
in solution. As the value for V0 no longer increases once the
concentration of LMWG 1 has reached the CGC, this strongly
indicates that only the molecules in solution are cleaved, and
molecules that are incorporated into the gel fibers are protected
from enzymatic cleavage. The inclusion of the a-chy in the gel
did not lead to an inhibition of its activity, as was shown in a
reference experiment using a gel sample of LMWG 1 containing
Experimental
Materials
a-Chymotrypsin (a-CT) from bovine pancreas (MW 40.0 kDa)
was purchased from Sigma and used without further purifica-
tion. a-CT was Type II Sigma preparation, 3 times crystallized,
dialyzed, and lyophilized. All other chemicals were purchased
from Aldrich or Fluka and used without further purification.
Doubly-distilled, deoxygenated water was used for all gelation
tests.
2 9 1 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 9 1 7 – 2 9 2 0

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Scheme 2 Synthesis of LMWG 1.
Characterization
after which the acetone was slowly evaporated, resulting in the
formation of a precipitate that was collected by filtration and
subsequently dried to give pure 4 as a light orange solid. Yield:
2.95 g (6.03 mmol = 60.3%). 1H NMR: d 12.2 (bs, 2 H, COOH),
10.45 (s, 1 H, NH-Qui), 8.76 (d, 1H, J = 3.4 Hz, Qui-H), 8.35–
8.26 (m, 3H, Qui-H + NH), 7.96 (d, 1H, J = 9.0 Hz, Qui-H),
7.77 (d, 1H, J = 9.0 Hz, Qui-H), 7.45 (m, 1H, Qui-H), 7.24 (m,
5H, ArH), 4.71 (m, 1 H, NHCH), 3.05 (m, 2H, CH2Ar), 2.31
(bm, 3H, CHex), 2.03 (m, 2H, CHex), 1.75 (m, 1H, CHex), 1.23
(bm, 3H, CHex). 13C NMR: d 174.7, 173.1, 169.8, 148.1, 143.7,
136.6, 135.7, 134.5, 128.5, 128.2, 127.2, 127.0, 125.3, 122.3,
120.7, 114.2, 53.8, 40.8, 39.8, 37.2, 36.6, 30.1, 29.4. EI-MS m/z
490.1 [M + H]+, calcd. for C27H27N3O6: 489.2.
NMR experiments were performed using a Varian Gemini
NMR spectrometer operating at 200 MHz, or a Varian VXR
NMR spectrometer operating at 300 MHz. All spectra were
recorded in DMSO-d6 unless stated otherwise. MS-spectra were
measured on a JEOL JMS-600H or a Science API 3000 mass
spectrometer. All gel-to-sol transition temperatures (Tgs) were
determined using the “dropping ball” method,15 which consists
in carefully placing a stainless steel ball (65 mg, 2.5 mm in
diameter) on top of a gel that had been prepared 16 h earlier
in 2 mL glass vials and subsequently placing these vials in
a heating block where the gels can be monitored by means
of a CCD camera. The temperature of the heating block is
increased by 5 ◦C h−1 and the Tgs is defined as the temperature
at which the steel ball reaches the bottom of the vial. pH values
(pHgs) were determined by dissolving a weighed amount of the
gelator in acidic water (HCl). Subsequently, small volumes of
a slightly basic solution were added until the onset of gelation
could be observed (either a thickening of the solution or the
appearance of small “gel flakes”), upon which the pH of the
solution was measured. The total volume of the gelator solution
was then used to calculate the exact gelator concentration. The
pH measurements were carried out with a WTW inoLab pH
Level 1 meter equipped with a Hamilton minitrode that was
calibrated before use. Cryo-transmission electron microscopy
(CryoTEM): A few microliters of suspension were deposited
on a bare 700 mesh copper grid. After blotting away the
excess of liquid the grids were plunged quickly in liquid ethane.
Frozen-hydrated specimens were mounted in a cryo-holder
(Gatan, model 626) and observed in a Philips CM 120 electron
microscope, operating at 120 KV. Micrographs were recorded
under low-dose conditions on a slow-scan CCD camera (Gatan,
model 794). Enzyme kinetics studies were conducted using 1 mL
samples that were prepared by rapid addition of enzyme stock
solution (900 lL, a-chy in 0.1 M Tris–HCl buffer, pH 7.75) to a
DMSO solution of 1, 2 or 1 + 2. Enzyme and substrate solutions
were prepared immediately before their use in experiments.
The enzyme concentration after mixing with the DMSO was
40 lM. Substrate concentrations ranged between 0.09 and
15.49 mM. No cleavage of substrates 1 and 2 was observed in the
absence of a-chy (t = 5000 sec). Fluorescence measurements were
carried out on a Sim-Aminco SPF-500C spectrofluorometer
equipped with a thermostated cell holder controlled at 25, 30,
CHex(AmPhe–6AQ)(AmEtOEtOH)2 (1).
A solution of
compound 4 (2.80 g, 5.73 mmol), 2(-2-aminoethoxy)-1-ethanol
(1.36 g, 12.94 mmol), and DMT-MM (3.58 g, 12.94 mmol) in
MeOH (100 mL) and DMSO (150 mL) was stirred overnight at
RT. After completion of the reaction H2O (300 mL) was added
and the resultant precipitate was filtered off, washed with H2O
(3 × 100 mL), and dried. The crude product was purified by
column chromatography (SiO2, CH2Cl2 : MeOH = 9 : 1–8 : 2)
to give pure 1 as a light yellow solid. Yield: 1.60 g (2.41 mmol =
42.1%). 1H NMR: d 10.50 (s, 1H, NH), 8.80 (d, 1H, J = 4.4 Hz,
ArH), 8.37 (s, 1H, ArH), 8.28 (m, 2H, ArH), 7.98 (d, 1H, J =
9.2 Hz, ArH), 7.82 (m, 3H, ArH), 7.49 (m, 1H, ArH), 7.35–7.17
(m, 5H, PhH), 4.74 (m, 1H, CH), 4.59 (m, 2H, OH), 3.5–3.35 (m,
12H, CH2OCH2CH2N), 3.19 (d, 4H, J = 5.5 Hz, CH2OH), 3.2–
3.0 + 3.0–2.9 (m, 2H, CH2Ph), 2.35–2.15 (m, 3H, CHex), 1.70
(m, 2H, CHex), 1.56 (m, 1H, CHex), 1.45–1.30 (m, 3H, CHex).
13C NMR: d 173.3, 173.2, 169.8, 148.0, 143.6, 136.6, 135.7, 134.5,
128.6, 128.1, 127.2, 127.0, 125.3, 122.3, 120.7, 114.1, 71.0, 68.0,
59.1, 53.7, 41.4, 41.1, 39.7, 37.4, 37.2, 36.5, 30.4. EI-MS m/z
664.2 [M + H]+, 686.2 (M + Na)+ calcd for C35H45N5O8: 663.3.
Anal. calcd. for C35H45N5O8 + H2O: C, 61.66; H, 6.95; N, 10.27;
found: C, 61.6; H, 6.9; N, 10.3.
Acknowledgements
Dr Menno R. de Jong is kindly acknowledged for helpful
discussions.
References
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Synthesis
Compounds 2 and 3 were synthesized according to a literature
procedure.9 The synthesis of compounds 4 and 1 is depicted in
Scheme 2.
CHex(AmPhe–6AQ)(COOH)2 (4). To a solution of cis,cis-
1,3,5-cyclohexanetricarboxylic acid (6.48 g; 30.0 mmol) and
HOBT (2.55 g, 18.87 mmol) in DMSO (200 mL) was added
CDI (1.62 g, 10.0 mmol). After stirring for 2 h at RT, 3 (4.51 g,
10.0 mmol) and Et3N (4.04 g, 40.0 mmol) were added and
stirring was continued overnight after which the solution was
poured into H2O (600 mL). The solid that was collected by
filtration was dissolved in DMSO–H2O–acetone and filtered,
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 9 1 7 – 2 9 2 0
2 9 1 9

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ˇ
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2 9 2 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 9 1 7 – 2 9 2 0