Tuning the molecular weight of polymeric amphiphiles as a tool to access micelles with a wide range of enzymatic degradation rates

Article abstract of DOI:10.1039/c8cc02415d

Enyzme-responsive polymeric assemblies hold great potential for biomedical applications due to the over-expression of disease-associated enzymes, which can be utilized to activate such systems only in afflicted tissues. Herein we demonstrate that the overall molecular weight of polymeric amphiphiles, which have the same hydrophilic/hydrophobic ratio, can be tuned to create polymeric micelles with an extreme range of degradation rates. This approach expands the available set of molecular parameters that can be adjusted to tune the degradation rate of polymeric assemblies, paving new possibilities for rational design of polymeric systems with controlled degradation rates.

Full text of DOI:10.1039/c8cc02415d

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DOI: 10.1039/C8CC02415D
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ARTICLE
Tuning the molecular weight of polymeric amphiphiles as a tool to
access micelles with a wide range of enzymatic degradation rates
Gadi Slor, a,b Nitsan Papo,a,b Uri Hananel,b,c and Roey J. Amir*,a,b,d
Received 00th January 20xx,
Accepted 00th January 20xx
Enyzme-responsive polymeric assemblies hold great potential for biomedical applications due to the over-expression of
disease-associated enzymes, which can be utilized to activate such systems only in afflicted tissue. Herein we demonstrate
that the overall molecular weight of polymeric amphiphiles, which have the same hydrophilic/hydrophobic ratio, can be
utilized to create polymeric micelles with an extreme range of degradation rates. This approach expands the available set of
molecular parameters that can be adjusted to tune the degradation rate of polymeric assemblies, paving new possibilities
for rational design of polymeric systems with controlled degradation rates.
DOI: 10.1039/x0xx00000x
www.rsc.org/
utilizing the high molecular precision of PEG-dendron hybrids,16
which were first introduced by Frechet, Gitsov, Hawker, and
Introduction
Wooley,17,18 we were able to recently show that small changes
in the amphiphilic ratio can lead to large differences in the
enzymatic degradation rates of these hybrid amphiphiles.
Tuning either the length of the PEG15 or the hydrophobicity of
the dendron, by changing the number19,20 or type of end-
groups,21 allowed us to prepare well-defined polymeric
amphiphiles with diverse degradation rates ranging from
rapidly degraded amphiphiles to highly stable assemblies that
show no apparent degradation or disassembly even after long
incubation periods with the activating enzyme.
Stimuli-responsive polymeric micelles have emerged as
promising candidates to serve as drug delivery systems, due to
their ability to encapsulate or covalently bind hydrophobic
drugs in their hydrophobic core and release them upon specific
cue.1–6 Among the various types of stimuli, enzymes are very
appealing for triggering drug release from nano, micro, and
macro delivery systems due to their high specificity and
overexpression of disease-associated enzymes in afflicted
tissues.7–9 Despite great potential, there are still significant
challenges to overcome in order to achieve efficient utilization
of enzymes as triggers, as it is often challenging to tune the
enzymatic degradation rates and many enzyme-responsive
assemblies show very limited degradability. These limited
responses are due to the lack of direct access of the activating
enzyme to its substrates, which are usually concealed inside the
hydrophobic core of the assembly. As suggested in previous
reports by Heise,10,11 Thayumanavan,12–14 and our group,15 it is
reasonable to assume the interaction between the enzyme and
its hydrophobic substrate occurs with non-assembled
amphiphiles that are present due to the monomer-assembly
equilibrium. This equilibrium-based mechanism gives rise to
non-linear dependency of the enzymatic degradation rates on
the hydrophilic/hydrophobic ratio, which governs the
thermodynamic and kinetic stability of the assemblies. By
Herein, taking advantage of the high molecular precision of
PEG-dendron amphiphiles, we studied the effect of the overall
molecular weight of these amphiphiles on their self-assembly,
a. Department of Organic Chemistry, School of Chemistry, Faculty of Exact Sciences,
Tel-Aviv University, Tel-Aviv 6997801, Israel.
b. Tel Aviv University Center for Nanoscience and Nanotechnology, Tel-Aviv
University, Tel-Aviv 6997801, Israel.
c. Department of Physical Chemistry, School of Chemistry, Faculty of Exact Sciences,
Tel-Aviv University, Tel-Aviv 6997801, Israel.
d. BLAVATNIK CENTER for Drug Discovery, Tel-Aviv University, Tel-Aviv 6997801,
Israel.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
Figure 1: Illustration of two types of enzyme-responsive PEG-dendron hybrids with
similar hydrophilic/hydrophobic ratio and two fold difference in total molecular
weight (hydrophilic = blue; hydrophobic = red)

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loading capacity, and enzymatic responsiveness of polymeric
amphiphiles with the same hydrophilic/hydrophobic ratios.
Toward this goal, we designed and synthesized two amphiphilic
hybrids based on 10-kDa monomethyl PEG as the hydrophilic
block and dendrons bearing eight amidase- or esterase-
cleavable end-groups. These new hybrids were designed to
have the same hydrophilic/hydrophobic ratio and twice the
molecular weight of our previously reported PEG-dendron
amphiphiles, which were based on 5-kDa PEG. As illustrated in
Scheme 1, the dendritic blocks of the higher molecular weight
amphiphiles contain exactly two copies of the dendrons that
were used to construct the smaller 5-kDa-based amphiphiles.
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DOI: 10.1039/C8CC02415D
Results and discussion
The synthesis of the amphiphiles starts from mono-amine or
diamine PEG, mPEG5k-NH2, or mPEG10k-(NH2)2 and required only
two high yielding synthetic steps to reach the final hybrids: In
the first step, the amine or amines of the mPEG were
conjugated with branching units that contained two propargyl
moieties. In the second step, the acetylenes were further
branched by using thiol-yne click reaction with a pre-made thiol
that contained the enzyme-responsive hydrophobic end-group
(Figures S12-S14, S17). The two larger amphiphiles have eight
phenylacetamide (mPEG10k-(dend-Ph4)2) or hexanoate ester
(mPEG10k-(dend-Hex4)2) end-groups, which can be cleaved by
penicillin G amidase (PGA) or porcine liver esterase (PLE),
respectively. The two smaller amphiphiles bear four amidase or
esterase-cleavable end-groups, respectively. The amphiphilic
hybrids were expected to self-assemble into micelles in aqueous
solutions. Upon enzymatic cleavage of the hydrophobic end-
groups, the original amphiphilic nature of the hybrid will be
altered, leading to fully soluble polymer that will no longer form
micelles (Figure 1).
Scheme 1: Chemical structures of amphiphilic PEG-dendron hybrids based on 5- or
10-kDa PEG bearing one or two identical dendrons, respectively.
DLS. Based on the molecular weights of the amphiphiles, it is
not surprising that both the DLS and TEM data showed that the
two 10-kDa-based hybrids formed micelles with larger
diameters than those of the 5-kDa-based ones.
Considering the differences in the diameters of the micelles and
the fact that at equal weight percent concentration of the 5- and
10-kDa-based hybrids the absolute amount of PEG and
dendrons in each solution is equal, we were interested to
evaluate the encapsulation capacity of hydrophobic cargo in the
four types of micelles. We chose camptothecin (CPT), an anti-
cancer drug, which has very low solubility in water, as a model
hydrophobic cargo.24 The encapsulation experiments were
conducted in aqueous solutions with similar weight percent of
the amphiphiles and commercial 5- and 10-kDa mPEG-OH were
used as control. Remarkably, solutions containing the micelles
of the larger amphiphiles encapsulated roughly two-fold higher
amounts of CPT than the solutions containing the smaller
micelles, although the total weight concentration of the
amphiphiles was kept constant (Figure 4). The higher loading
capacity can be attributed to the larger size of the dendrons,
resulting in significant increases in the polymer/CPT ratios for
the larger amphiphiles (Table 1).
Finally, we were interested to see how the molecular weight of
the amphiphiles influenced enzymatic degradability. Based on
the differences in the CMC values between the larger and
smaller amphiphiles, we were expecting the 10-kDa-based
hybrids to be somewhat more stable and to show longer
degradation times. The enzymatic degradation of the hybrids
was directly monitored using HPLC, and the analysis of the
results is presented in Figure 5. In the case of PGA-responsive
hybrids, when the kinetic experiments were conducted at low
All hybrids were synthesized through the new accelerated two-
step methodology in quantitative overall yield (>95%) and were
1
characterized by H and 13C NMR, HPLC, GPC, IR, and MALDI-
TOF. The experimental and expected theoretical data were in
good agreement (see supporting information).
After completion of the synthesis, we studied the self-assembly
of the four hybrids in aqueous media (phosphate buffer, pH
7.4). First the CMC values of the amphiphiles were determined
using the Nile Red method;22 the values are shown in Table 1.
Although each of the two pairs of our hybrids has the same
hydrophilic/hydrophobic ratio, we expected that the larger
amphiphiles, mPEG10k-(dend-Ph4)2 and mPEG10k-(dend-Hex4)2,
would have lower CMC values than the smaller ones, mPEG5k-
dend-Ph4 and mPEG5k-dend-Hex4, respectively. The obtained
results indeed showed the expected trend, which correlates
well with previous publications showing that increasing the
molecular weight of amphiphilic diblock copolymers while
keeping the hydrophilic/hydrophobic ratio the same led to
some decrease in the CMC values.23 Next, the micellar
diameters were determined using dynamic light scattering
(DLS), which indicated the formation of structures with sizes of
18-34 nm (Figure 3 solid lines and table 1). Transmission
electron microscopy (TEM) images confirmed the formation of
spherical structures (Figure 2) and sizes were in agreement with
Figure 2: TEM images of micellar solutions of the four amphiphiles. Arrows indicate
micellar structures. Scale bars, 50 nm.
2 | J. Name., 2012, 00, 1-4
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DOI: 10.1039/C8CC02415D
Figure 3: DLS of micellar solutions before (solid lines) and after (dashed) 24 hours
incubation with the activating enzymes ([PLE] = 14nM, [PGA] = 5 nM).
concentration of the enzyme (5 nM), we saw that mPEG5k-dend-
Ph4 was fully degraded in approximately 16 hours. Although we
expected slower degradation of mPEG10k-(dend-Ph4)2, we were
surprised to see almost no degradation of the larger amphiphile
under these conditions. In order to evaluate the extent of the
stability difference, we significantly increased the PGA
concentration by 100 fold to 500 nM. Under this relatively high
concentration of activating enzyme, the smaller amphiphile
mPEG5k-dend-Ph4 was fully degraded in less than 30 minutes,
and we also observed substantial degradation the larger
Figure 5: HPLC-based enzymatic degradation profiles of (a) amidase-responsive
amphiphiles at enzyme concentration of
5 and 500 nM (solid and dotted,
respectively) and (b) esterase-responsive amphiphiles at enzyme concentration of
14 and 1400 nM (solid and dotted lines, respectively) ([hybrid] = 1 mg/ ml).
In addition to directly monitoring the degradation of the hybrids
by HPLC, we also used DLS to monitor the presence of self-
assembled or disassembled structures in the solution (Figure 2,
dashed lines). As expected, the two 5-kDa-based amphiphiles
showed no traces of the assembled structures after the time at
which degradation was confirmed by HPLC, and new peaks
appeared indicative of smaller structures that corresponded to
the degraded hybrids. On the other hand, the 10-kDa-based
hybrids showed structures of similar sizes to the ones measured
before the addition of the enzyme. When the concentrations of
the enzymes were increased, we still observed micelles and also
smaller structures that corresponded to both the non-
amidase-responsive
amphiphile,
mPEG10k-(dend-Ph4)2,
although this occurred significantly slower with nearly 75%
degraded at 24 hours. As mentioned before, the inverse
correlation of higher stability with lower CMC values for
amphiphilic
di-block
copolymers
with
same
hydrophilic/hydrophobic ratio and larger molecular weights
was previously reported.23 However, our results demonstrate
that the effect of the total molecular weight on enzyme-
responsiveness seems to be much more substantial than the
difference in the CMC values for the two amidase-responsive
amphiphiles. Intrigued by this extreme stability difference, we
conducted enzymatic degradation experiments on PLE-
responsive mPEG10k-(dend-Hex4)2 and mPEG5k-dend-Hex4. By
now, we were not surprised to find that at low PLE
concentration (14 nM) the 5-kDa-based hybrid was degraded
completely in 16 hours and the 10-kDa-based amphiphile
showed nearly no degradation at this time point. For these
esterase-responsive amphiphiles, the stability difference was so
dramatic that even after 100-fold increase in PLE concentration
only 25% of mPEG10k-(dend-Hex4)2 was degraded after 24 hours.
assembled
hydrolyzed
hybrids
that
have
higher
hydrophilic/hydrophobic ratios and the activating enzymes,
which were used at relatively high concentrations in order to try
and push the degradation of the larger amphiphiles.
It is important to note that although the differences in CMC
values and sizes of the micelles were anticipated based on
previous reports in the literature,23 the significant effect on the
loading capacity and the exceptionally large differences in
degradation rates, illustrate the importance that overall
molecular weight has in determining the stability and enzymatic
degradability of polymeric assemblies. The increased stability
may also be attributed to steric hindrance due to the longer PEG
chains and larger dendrons. The results of this work deepen the
understanding of the different structural parameters that
govern enzymatic degradability of polymeric amphiphiles.
Furthermore, the higher loading capacity and enhanced
micellar stability of the larger amphiphiles make them highly
promising for the construction of stable delivery systems with
adjustable release rates. Cell studies (internalization and
toxicity) of these micellar systems are currently under further
investigation.
Figure 4: Encapsulation capacity of CPT (in µM and wt%) in micelles formed by the
different amphiphiles ([hybrid] = 1 mg/ ml). PEG 5 kDa and 10 kDa were used as
controls.
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Dend.
Mn
[Da]
Hydrophilic/h
ydrophobic
[%]a
Micellar
diameter
[nm]d
polymer
DOI: 10.1039/C8CC02415D
/CPT mol.
PEG Mn
[kDa]
Theoretical
Mn [kDa]
Mn
[kDa]b
Mw/
Mnb
Mn
[kDa]c
CMC [µM
/ ng/ml]
Hybrid
ratio
mPEG5k-(dend-
Ph4)
24 ± 1 /
145 ± 6
5.0
10.0
5.0
1112
2180
1036
2028
82/18
82/18
83/17
83/17
6.1
12.2
6.0
6.2
12.8
6.2
1.06
1.18
1.06
1.13
6.2
11.5
6.1
18 ± 2
31 ± 6
20 ± 2
34 ± 6
2.7 ± 0.8
mPEG10k
-
5 ± 2 /
61 ± 24
0.6 ± 0.1
2.1 ± 0.1
0.6 ± 0.1
(dend-Ph4)2
mPEG5k-(dend-
Hex4)
10 ± 2 /
60 ± 12
mPEG10k
(dend-Hex4)2
3 ± 1 /
36 ± 12
10.0
12.0
12.5
11.9
Table 1: Characterization table of the two series of PEG-dendron hybrids. aweight ratio of PEG/dendron. bmeasured by GPC. c measured by MALDI-TOF dmeasured by DLS.
F. Winnik, S. Zauscher, I. Luzinov and S. Minko, Nat. Mater.,
2010, 9, 101–113.
3
M. Karimi, A. Ghasemi, P. Sahandi Zangabad, R. Rahighi, S. M.
Moosavi Basri, H. Mirshekari, M. Amiri, Z. Shafaei Pishabad, A.
Aslani, M. Bozorgomid, D. Ghosh, A. Beyzavi, A. Vaseghi, A. R.
Aref, L. Haghani, S. Bahrami and M. R. Hamblin, Chem. Soc.
Rev., 2016, 45, 1457–1501.
Conclusions
To summarize, we prepared two pairs of enzyme-responsive
PEG-dendron
hybrids
with
nearly
identical
hydrophilic/hydrophobic ratios that differ in their total
molecular weights. Each of the pairs of amphiphiles was
designed to respond to a different enzyme: an amidase and an
esterase, based on the type of cleavable end-groups. The
amphiphiles were synthesized through high yielding step-
efficient synthesis by a combination of amidation and thiol-yne
reactions. As expected, the 10-kDa-based hybrids had lower
CMC values and formed micelles of larger diameters than the
smaller hybrids. Interestingly, although we kept the overall
polymer concentration (weight %) similar, the larger micelles
could encapsulate approximately twice the amount of CPT,
which was used as model hydrophobic cargo, in comparison
with the smaller micelles. Most importantly, there was a
tremendous difference between the enzymatic responsiveness
and degradation rates for the larger and smaller hybrids for
both enzymes (amidase and esterase) that were tested. The
observed kinetic trends indicate that the absolute molecular
weight of the hydrophobic block strongly affects the enzymatic
responsiveness of the amphiphiles and their assembled
structures. Our results demonstrate the potential utilization of
the overall molecular weight of amphiphiles as a modular tool
for tuning their enzymatic degradation rates, extending the
molecular tool box beyond the more frequently used approach
of adjusting the hydrophilic/hydrophobic ratio.
4
5
6
S. Mura, J. Nicolas and P. Couvreur, Nat. Mater., 2013, 12,
991–1003.
P. Theato, B. S. Sumerlin, R. K. O’Reilly and T. H. Epps, III,
Chem. Soc. Rev., 2013, 42, 7055–7056.
W. L. A. Brooks, G. Vancoillie, C. P. Kabb, R. Hoogenboom and
B. S. Sumerlin, J. Polym. Sci. Part A Polym. Chem., 2017, 55,
2309–2317.
R. de la Rica, D. Aili and M. M. Stevens, Adv. Drug Deliv. Rev.,
2012, 64, 967–978.
S. Samarajeewa, R. P. Zentay, N. D. Jhurry, A. Li, K. Seetho, J.
Zou and K. L. Wooley, Chem. Commun. (Camb)., 2014, 50,
968–70.
M. Zelzer, S. J. Todd, A. R. Hirst, T. O. McDonald and R. V. Ulijn,
Biomater. Sci., 2013, 1, 11–39.
7
8
9
10 G. J. M. Habraken, M. Peeters, P. D. Thornton, C. E. Koning and
A. Heise, Biomacromolecules, 2011, 12, 3761–9.
11 P. D. Thornton and A. Heise, Chem. Commun. (Camb)., 2011,
47, 3108–3110.
12 K. R. Raghupathi, M. a. Azagarsamy and S. Thayumanavan,
Chem. - A Eur. J., 2011, 17, 11752–11760.
13 J. Guo, J. Zhuang, F. Wang, K. R. Raghupathi and S.
Thayumanavan, J. Am. Chem. Soc., 2014, 136, 2220–3.
14 M. A. Azagarsamy, P. Sokkalingam and S. Thayumanavan, J.
Am. Chem. Soc., 2009, 131, 14184–14185.
15 A. J. Harnoy, I. Rosenbaum, E. Tirosh, Y. Ebenstein, R.
Shaharabani, R. Beck and R. J. Amir, J. Am. Chem. Soc., 2014,
136, 7531–7534.
16 I. Gitsov, J. Polym. Sci. Part A Polym. Chem., 2008, 46, 5295–
5314.
17 I. Gitsov, K. L. Wooley and J. M. J. Frechet, Angew. Chemie Int.
Ed. English, 1992, 31, 1200–1202.
Conflicts of interest
There are no conflicts to declare.
18 I. Gitsov, K. L. Wooley, C. J. Hawker, P. T. Ivanova and J. M. J.
Fréchet, Macromolecules, 1993, 26, 5621–5627.
19 A. J. Harnoy, M. Buzhor, E. Tirosh, R. Shaharabani, R. Beck and
R. J. Amir, Biomacromolecules, 2017, 18, 1218–1228.
20 A. J. Harnoy, G. Slor, E. Tirosh and R. J. Amir, Org. Biomol.
Chem, 2016, 14, 5813–5819.
Acknowledgements
21 M. Segal, R. Avinery, M. Buzhor, R. Shaharabani, A. J. Harnoy,
E. Tirosh, R. Beck and R. J. Amir, J. Am. Chem. Soc., 2017, 139,
803–810.
22 E. R. Gillies, T. B. Jonsson and J. M. J. Fréchet, J. Am. Chem.
Soc., 2004, 126, 11936–11943.
RJA thanks the ISF (Grant No. 966/14) for the financial support.
Notes and references
23 V. P. Torchilin, J. Control. Release, 2001, 73, 137–172.
24 C. J. Thomas, N. J. Rahier and S. M. Hecht, Bioorganic Med.
Chem., 2004, 12, 1585–1604.
1
2
V. P. Torchilin, Adv. Drug Deliv. Rev., 2012, 64, 302–315.
M. a C. Stuart, W. T. S. Huck, J. Genzer, M. Müller, C. Ober, M.
Stamm, G. B. Sukhorukov, I. Szleifer, V. V Tsukruk, M. Urban,
4 | J. Name., 2012, 00, 1-4
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