Branched multifunctional polyether polyketals: Variation of ketal group structure enables unprecedented control over polymer degradation in solution and within cells

Article abstract of DOI:10.1021/ja305080f

Multifunctional biocompatible and biodegradable nanomaterials incorporating specific degradable linkages that respond to various stimuli and with defined degradation profiles are critical to the advancement of targeted nanomedicine. Herein we report, for the first time, a new class of multifunctional dendritic polyether polyketals containing different ketal linkages in their backbone that exhibit unprecedented control over degradation in solution and within the cells. High-molecular-weight and highly compact poly(ketal hydroxyethers) (PKHEs) were synthesized from newly designed α-epoxy-ω-hydroxyl-functionalized AB₂-type ketal monomers carrying structurally different ketal groups (both cyclic and acyclic) with good control over polymer properties by anionic ring-opening multibranching polymerization. Polymer functionalization with multiple azide and amine groups was achieved without degradation of the ketal group. The polymer degradation was controlled primarily by the differences in the structure and torsional strain of the substituted ketal groups in the main chain, while for polymers with linear (acyclic) ketal groups, the hydrophobicity of the polymer may play an additional role. This was supported by the log a values of the monomers and the hydrophobicity of the polymers determined by fluorescence spectroscopy using pyrene as the probe. A range of hydrolysis half-lives of the polymers at mild acidic pH values was achieved, from a few minutes to a few hundred days, directly correlating with the differences in ketal group structures. Confocal microscopy analyses demonstrated similar degradation profiles for PKHEs within live cells, as seen in solution and the delivery of fluorescent marker to the cytosol. The cell viability measured by MTS assay and blood compatibility determined by complement activation, platelet activation, and coagulation assays demonstrate that PKHEs and their degradation products are highly biocompatible. Taken together, these data demonstrate the utility this new class of biodegradable polymer as a highly promising candidate in the development of multifunctional nanomedicine.

Full text of DOI:10.1021/ja305080f

Article
pubs.acs.org/JACS
Branched Multifunctional Polyether Polyketals: Variation of Ketal
Group Structure Enables Unprecedented Control over Polymer
Degradation in Solution and within Cells
Rajesh A. Shenoi,† Jayaprakash K. Narayanannair,‡ Jasmine L. Hamilton,† Benjamin F. L. Lai,†
Sonja Horte,† Rajesh K. Kainthan,‡ Jos P. Varghese,§ Kallanthottathil G. Rajeev,‡ Muthiah Manoharan,‡
and Jayachandran N. Kizhakkedathu*,†,⊥
†Centre for Blood Research and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver,
British Columbia, Canada V6T 1Z3
‡Alnylam Pharmaceuticals, 300 Third Street, Cambridge, Massachusetts 02142, United States
§Sanmar Speciality Chemicals Ltd., Chennai, Tamil Nadu, India
⊥Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
S
* Supporting Information
ABSTRACT: Multifunctional biocompatible and biodegradable nanoma-
terials incorporating specific degradable linkages that respond to various
stimuli and with defined degradation profiles are critical to the
advancement of targeted nanomedicine. Herein we report, for the first
time, a new class of multifunctional dendritic polyether polyketals
containing different ketal linkages in their backbone that exhibit
unprecedented control over degradation in solution and within the cells.
High-molecular-weight and highly compact poly(ketal hydroxyethers)
(PKHEs) were synthesized from newly designed α-epoxy-ω-hydroxyl-
functionalized AB2-type ketal monomers carrying structurally different
ketal groups (both cyclic and acyclic) with good control over polymer properties by anionic ring-opening multibranching
polymerization. Polymer functionalization with multiple azide and amine groups was achieved without degradation of the ketal
group. The polymer degradation was controlled primarily by the differences in the structure and torsional strain of the
substituted ketal groups in the main chain, while for polymers with linear (acyclic) ketal groups, the hydrophobicity of the
polymer may play an additional role. This was supported by the log P values of the monomers and the hydrophobicity of the
polymers determined by fluorescence spectroscopy using pyrene as the probe. A range of hydrolysis half-lives of the polymers at
mild acidic pH values was achieved, from a few minutes to a few hundred days, directly correlating with the differences in ketal
group structures. Confocal microscopy analyses demonstrated similar degradation profiles for PKHEs within live cells, as seen in
solution and the delivery of fluorescent marker to the cytosol. The cell viability measured by MTS assay and blood compatibility
determined by complement activation, platelet activation, and coagulation assays demonstrate that PKHEs and their degradation
products are highly biocompatible. Taken together, these data demonstrate the utility this new class of biodegradable polymer as
a highly promising candidate in the development of multifunctional nanomedicine.
targeted drug delivery devices. pH-degradable polymers are of
special interest due to their broad range of applications such as
tumor targeting, delivery of protein-based vaccines, nucleic acid
delivery, and treatment of acute inflammatory diseases.16 The
acidic nature of tumor tissues necessitates the need for pH-
responsive/degradable formulations for enhanced tumor
targeted therapy, while protein and nucleic acid delivery
requires the rapid endosomal degradation of the polymeric
carrier to generate osmotic imbalance and release of the
payload into the cytosol.17
1. INTRODUCTION
The availability of multifunctional biodegradable polymers with
defined structure, controlled degradation profiles, and bio-
compatibility is critical to the development of novel targeted
intercellular drug delivery systems, imaging agents, and
scaffolds for tissue engineering.1−3 Structurally different
biodegradable polymers with characteristic features such as
polyesters, polyorthoesters, polyanhydrides, polyurethanes,
polyketals, polyacetals, and disulfide-containing polymers
undergo degradation to various extents in vitro and/or in vivo
via hydrolysis, enzymatic degradation, pH changes, and redox
reactions.4−15 The presence of multiple reactive functionalities
on the polymer along with defined biodegradation profiles,
water solubility, and biocompatibility are critical for developing
Received: May 25, 2012
Published: August 20, 2012
© 2012 American Chemical Society
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Table 1. Structure and Characteristics of Synthesized α-Epoxy-ω-hydroxyl Ketal Monomers
a
Calculated using ALOGP program as per ref 29.
Figure 1. Synthetic schemes for different α-epoxy-ω-hydroxyl ketal monomers.
Among the pH-degradable polymeric systems, polyketals are
of particular interest as they are neutral and generate nontoxic
neutral degradation products. Polymers with a main-chain
degradable dimethyl ketal group have been reported as delivery
vehicles for therapeutic agents.18−20 Murthy and co-workers
reported poly(1,4-phenyleneacetone dimethylene ketal) and
poly(cyclohexane-1,4-diyl acetone dimethylene ketal), both
containing a dimethyl ketal group, as a new class of pH-
sensitive drug delivery vehicle.18 Dimethyl ketal group-
containing polyurethane and linear poly(amido amines) have
been studied by the group of Frechet for applications such as
drug delivery and protein-based vaccines.19a,b Frechet’s group
also reported the enhanced cellular uptake of microparticles
based on a copolymer of a dimethyl ketal group containing
cross-linker with a cell-penetrating peptide containing acryl-
amide.19c Microparticles based on acetalated dextran containing
both linear and cyclic dimethyl ketal groups have been explored
as drug delivery vehicles for immunotherapy.19d There has been
a recent report on poly(β-amino ester ketal) nanoparticles that
contain both a β-amino ester and dimethyl ketal group in the
backbone as a dual pH-sensitive drug delivery vehicle.20
However, all these polyketal polymers are linear, have same
degradable dimethyl ketal linkages, have low molecular weights,
and lack the multifunctionality and water solubility desired for
several applications.21 Moreover, in most of the cases the
hydrolysis half-lives of these polymers were changed by altering
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Figure 2. General scheme for the synthesis of poly(ketal hydroxyethers). Scheme for the synthesis of copolymer of 2-(1-(2-(oxiran-2-
ylmethoxy)ethoxy)cyclohexyloxy)ethanol and glycidol by anionic multibranching ring-opening polymerization is shown. Other PKHEs were
synthesized by a similar method.
the diffusion of water to predominantly hydrophobic polymers,
which is very difficult to control. The hydrolysis rate also
depended on the physical nature of the polymeric systems; for
instance, nanoparticles degrade much rapidly than micro-
particles or a film of the polymer. The degradation rate also
depended on various processing conditions used.18−20 This
limitation is in fact true for many of other types of
biodegradable polymeric systems currently available and is a
major obstacle for their use in the enhanced delivery of
bioactive molecules within the cells.8,22 Hence there is a need
for developing new biocompatible biodegradable polymers that
possess the features of high pH sensitivity, multifunctionality,
increased/enhanced water solubility, and defined degradation
profiles for improved targeted drug delivery and enhanced rapid
endosomal escape/release of bioactive molecules.
Herein we report, for the first time, the design, synthesis, pH-
dependent degradation, and biocompatibility studies of a novel
class of well-defined branched multifunctional polyether
polyketals, poly(ketal hydroxyethers) (PKHEs), by anionic
ring-opening multibranching polymerization (ROMBP) of
newly developed functional AB2-type ketal monomers. The
PKHEs incorporate features such as high molecular weight,
water solubility, multifunctionality, excellent biocompatibility,
and defined pH-dependent degradability both in buffer
solutions and within the cells. We also demonstrate for the
first time that the biodegradability of the polyketals can be
controlled by incorporating structurally different ketal linkages
in their backbone, and PKHE polymers degrade with
unprecedented control to low-molecular-weight, neutral, non-
toxic, and easily excretable products. Our data demonstrate that
the new PKHE polymers have considerable potential in the
development of multifunctional drug delivery devices for
efficient endosomal escape and cytosolic delivery.
2. RESULTS AND DISCUSSION
2.1. Development of α-Epoxy-ω-hydroxyl-Function-
alized AB2-Type Ketal Monomers. To achieve controlled
pH degradability and multifunctionality of polymers, we first
designed and synthesized five novel heterofunctional AB2-type
monomers with structurally different ketal groups (Table 1)
that can be polymerized by ROMBP. These monomers are
DMKM (3), CHKM (6), CPKM (9), GHBKM (13), and
GCHKM (16), each containing an epoxide group, a hydroxyl
group, and a different type of ketal group per molecule.
New synthetic schemes were developed for the monomers as
shown in Figure 1. Monomers with linear (acyclic) ketal groups
(3, 6, 9) were synthesized starting from ethylene glycol. As
shown in Figure 1A, the monomer DMKM was synthesized
from ethylene glycol monoacetate (1),23 which was reacted
with 2-methoxypropene in the presence of p-toluenesulfonic
acid (PTSA) to obtain the corresponding dimethyl ketal
diacetate intermediate. The compound was then deacetylated
using potassium carbonate in methanol to obtain the dimethyl
ketal diol 2. Reaction of the ketalized diol with sodium/
isopropanol followed by reaction with epichlorohydrin afforded
the DMKM monomer 3. Synthesis of CHKM monomer
(Figure 1B) was started from the cyclohexanone enol ether
(4).24 Reaction of ethylene glycol monoacetate with 4 in the
presence of PTSA in anhydrous THF gave the corresponding
ketalized diacetate intermediate. Deprotection of acetyl group
from the diacetate with potassium carbonate in methanol
yielded the ketalized diol 5. Treatment of the diol 5 with
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Table 2. Characteristics of Poly(ketal hydroxyethers) Synthesized by ROMBP
a
b
c
d
PKHE
feed ratio TMP:ketal:glycidol (mmol)
conv (%)
Mn (Mw/Mn)
Rh (nm)
polymer composition ketal:glycidol
PDMKHE-1
PDMKHE-2
PDMKHE-3
PCHKHE-1
PCHKHE-2
PCHKHE-3
PCHKHE-4
PCPKHE-1
PCPKHE-2
PGHBKHE-1
PGCHKHE-1
0.357:7.73:2.61
0.357:15.91:5.38
0.357:31.8:10.8
0.357:13:4.2
64
60
70
54
63
62
55
45
60
62
60
8500 (1.6)
18900 (1.48)
28200 (1.44)
8300 (1.41)
18300 (1.44)
22000 (1.34)
56700 (1.6)
6200 (1.7)
2.8
3.3
3.2
3.4
3.8
4.2
5.2
2.6
3.5
3.2
3.3
52:48
56:44
66:34
54:46
53:47
60:40
65:35
52:48
62:38
57:43
0.357:19.2:6.1
0.268:22:7.2
0.224:30.8:10.5
0.357:7.32:2.4
0.357:32.5:11
0.357:32.1:10.8
0.357:28.7:9.6
14000 (1.65)
7000 (1.6)
8600 (1.58)
55:45
a
b
c
For all experiments, temperature was 95 °C and polymerization time was 24 h. Determined by GPC-MALLS analysis in chloroform. Determined
d
1
by QELLS analysis in chloroform. Calculated from H NMR spectra; feed composition of ketal:glycidol was 75:25 for all experiments.
epichlorohydrin in the presence of potassium tert-butoxide in
tert-butanol afforded the epoxide-functionalized cylcohexyl
ketal monomer 6 (CHKM). The CPKM monomer 9 was
synthesized from cyclopentanone enol ether using a procedure
similar to that used for CHKM (Figure 1C).
feed composition, suggesting that ketal monomers were less
reactive than glycidol. The structure of the ketal group was also
found to have an effect on the reactivity of the monomers
toward ROMBP, as evident from the differences in the
copolymer compositions compared to initial feed ratio as well
as the relatively low conversion for certain monomers (Table
2). The monomers containing acyclic ketal groups (DMKM,
CPKM, and CHKM) were found to be slightly more reactive
than monomers containing cyclic ketal structures (GHBKM
and GCHKM). Also, at similar monomer-to-initiator ratios,
GHBKM and GCHKM gave lower molecular weights
compared to other monomers.
The monomers GHBKM and GCHKM were synthesized
from glycerol as the starting material. For the synthesis of
GHBKM (Figure 1D), glycerol was first converted into its α-
monoallyl ether 10.25 Reaction of 10 with ethyl acetoacetate in
the presence of PTSA in anhydrous ethylene dichloride yielded
ketal derivative 11, and subsequent reduction of the ester group
using lithium aluminum hydride in anhydrous THF yielded the
alcohol 12. In the final step, epoxidation of the double bond
was achieved by reacting with m-chloroperbenzoic acid in
dichloromethane to afford the GHBKM monomer 13.
Similarly, GCHKM monomer was synthesized by the reaction
of glycerol with 4-(2-propenyloxy)cyclohexanone (14).26
Ketalization followed by epoxidation of the double bond
afforded the required GCHKM monomer 16 as shown in
Figure 1E. All monomers were purified by column chromatog-
raphy and characterized by NMR and high-resolution mass
spectrometric analyses (Figures S1−S10, Supporting Informa-
tion).
2.2. Synthesis of PKHEs by Anionic ROMBP. After
synthesis of AB2-type monomers, we explored their anionic
ROMBP using trimethylol propane/potassium methoxide as
the initiator system at 95 °C. A representative synthetic scheme
for the polymerization of CHKM is shown in Figure 2.
Homopolymerization of the ketal monomers resulted in low
conversions (∼15−20%); however, the addition of glycidol as a
comonomer significantly improved the monomer conversions.
Thus, employing an initial feed ratio of ketal monomer to
glycidol in the molar composition 3:1 (Table 2), poly-
(dimethylketal hydroxyether) (PDMKHE), poly-
(cyclopentylketal hydroxyether) (PCPKHE), poly-
(cyclohexylketal hydroxyether) (PCHKHE), poly(glycerol
hydroxybutanone ketal hydroxyether) (PGHBKHE), and
poly(glycerol cyclohexanone ketal hydroxyether) (PGCHKHE)
having 52−66 mol % incorporation of ketal groups in the
polymer backbone were synthesized (Table 2). Polymers of
different molecular weights (6−57 kg/mol) and relatively low
molecular weight distributions were synthesized by varying the
monomer-to-initiator molar ratio. The polydispersity index of
the PKHE polymers was consistent with the reported values for
the multibranching polymerization of AB2-type monomers.27
The incorporation of ketal monomers was lower than the initial
All the PKHEs were soluble in organic solvents such as
methanol, chloroform, THF, and DMF; however, only
poly(dimethylketal hydroxyether) (PDMKHE) was soluble in
water. The GPC chromatograms of PKHE polymers showed
monomodal distribution of molecular weights (Figure S11). 1H
NMR spectra of the polymers showed characteristic peaks due
to the presence of ketal groups in the polymer (Figures S12−
S14). PDMKHE gave a signal at 1.25 ppm that corresponds to
the methyl resonance of the dimethyl ketal group. For
PCPKHE and PCHKHE, signals due to the methylene
resonance of the cyclopentyl and cyclohexyl ketal groups
respectively were observed in the region 1.3−1.8 ppm. Signals
at 1.2−1.3 ppm were found for PGHBKHE that correspond to
the methyl groups of the hydroxybutanone ketal group. NMR
spectra of the polymers together with the molecular weight
analysis in chloroform confirmed the formation of high-
molecular-weight multifunctional polymeric structure with
intact ketal moieties. A peak corresponding to −OH stretching
around 3300 cm−1 in the FTIR spectra of the PKHE polymers
and a characteristic signal between 4.5 and 4.8 ppm in the
NMR spectra of polymers in DMSO-d6 confirmed the presence
of hydroxyl groups in the polymers (Figures S12−S15).
The branched structure of the PKHE polymers was probed
by inverse-gated 13C NMR analysis. A representative NMR
spectrum for PDMKHE is shown in Figure S16. The signals at
25.37 and 101.33 ppm confirmed the presence of dimethyl
ketal groups in the polymer. The polymer has linear, dendritic,
and terminal structural units that are usually observed with
other hyperbranched polymers.28 The degree of branching
calculated from the spectra was in the range 0.50−0.60 for
different PKHE polymers and is very close to the values
reported for hyperbranched polymers obtained from AB2-type
monomers.28 The 13C NMR spectra also revealed that the ketal
groups were randomly distributed throughout the polymer. The
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Figure 3. Polymer modification and functionalization: scheme for PEG modification, azide and amine functionalization, and development of
fluorescently labeled polymers. Shown is an example for PCHKHE polymer functionalization. Other PKHE polymers were modified by similar
methods.
Table 3. Characteristics of pPKHEs and Their pH-Dependent Hydrolysis in Various Buffer Solutions
hydrolysis half-life (h)
pH 5.5
37 °C
pH 6.0
pH 6.5
pH 7.4
a
b
polymer
mPEG content (mol %) Mn (Mw/Mn)
Rh (nm) 25 °C
25 °C 37 °C 25 °C 37 °C
25 °C
21.6
37 °C
3.9
c
pPDMKHE-3
pPCPKHE-2
pPCHKHE-3
pPGHBKHE-1
pPGCHKHE-1
28
30
30
25
30
38000 (1.3)
29000 (1.7)
37800 (1.5)
13800 (1.4)
22000 (1.8)
5.4
3.3
4.5
3.8
4.2
0.3
very fast
0.18
1.1
2.2
8.7
0.16
0.5
2.5
nd
3.5
6.9
15.9
nd
0.43
1.4
9.0
nd
0.8
69.3
17.3
2.5
0.82
187
79
d
e
e
f
f
25300
21120
3650
2950
nd
nd
very slow
very slow
very slow
very slow
nd
nd
nd
a
b
c
Determined by GPC-MALLS in 0.1 M NaNO3 at pH 8.5. Determined by QELLS in 0.1 M NaNO3 at pH 8.5. Hydrolysis was 100% at the first
d
e
f
time point of NMR analysis (30 min). nd = not determined. No measurable hydrolysis observed in 400 days. 5% hydrolysis observed in 400 days.
spectra showed characteristic signals for the presence of linear
(L), dendritic (D), and terminal (T) units for hyperbranched
structures. Since the terminal structural units (signal at 64−65
ppm) come primarily from glycidol component of the
copolymer, the NMR data support the random distribution
of ketal groups within the polymer. Evidence for the compact
nature of the polymers was also obtained from their relatively
small hydrodynamic radii (2.6−5.2 nm, Table 2) measured by
dynamic light scattering.
in the monomers and the polymers makes them susceptible to
cleavage under acidic conditions. In order to compare the
degradation of the polymers in aqueous buffer conditions, it is
necessary that all the polymers have similar water solubility. To
achieve this, a portion of the hydroxyl groups of PKHEs was
deprotonated with sodium hydride followed by reaction with α-
methoxy-ω-epoxy polyethylene glycol (mPEG400-epoxide) to
generate PEGylated PKHEs (pPKHEs) (Figure 3). The
polymers PDMKHE-3, PCHKHE-3, PCPKHE-2, PGHBKHE-
1, and PGCHKHE-1 were selected for modification using
mPEG400-epoxide. Detailed characteristics of the modified
2.3. Effect of Ketal Structure on Polymer Degradation
in Aqueous Buffer solutions. The presence of ketal groups
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1
Figure 4. Comparison of degradation of ketal monomers and polymers in aqueous phosphate buffer at pH 5.5 in D2O at 25 °C. (A) H NMR
spectra showing the hydrolysis of DMKM. (B) Comparison of hydrolysis kinetics plots of various ketal monomers. The percentage of hydrolysis is
1
1
calculated from the H NMR integration data. (C) H NMR spectra showing the degradation of pPCHKHE-3. (D) Comparison of hydrolysis
1
kinetics plots of different pPKHEs. The percentage of hydrolysis was calculated from the H NMR integration data.
PKHEs are given in Table 3. The polymers have 25−30 mol %
of mPEG and were found to be highly water-soluble. The
presence of mPEG in the polymers was confirmed by 1H NMR
analysis and also from the increase in the absolute molecular
weights of the modified PKHEs compared to the original
polymers (Tables 2 and 3). For example, the Mn of PDMKHE-
3 was increased from 28 200 to 38 000 g/mol upon
incorporation of ∼28 mol % of mPEG chains. The ketal
groups were stable during the modification as evident from
NMR and molecular weight analyses. The very small
hydrodynamic radii (3.3−5.4 nm) of pPKHEs (Table 3)
along with high branching density confirm the highly compact
structure of these polymers in water without aggregation. The
water solubility, compact structure, polyether backbone, and
multifunctionality differentiate the current class of ketal
polymers from previously reported polyketal polymers.18−20
2.3.1. Degradation of α-Epoxy-ω-hydroxyl Ketal Mono-
mers at pH 5.5. Initially, the degradation kinetics of the
monomers was studied by dissolving them in phosphate buffer
at pH 5.5 prepared in D2O and recording the 1H NMR spectra
at 25 °C at different time intervals. The percent of hydrolysis
was calculated from the disappearance of signals corresponding
to the ketal functionality and the simultaneous appearance of
signals due to the formation of the corresponding ketone. Thus,
for the monomer DMKM, the peak at 1.39 ppm due to the
dimethyl ketal moiety gradually disappeared and a new peak
appeared at 2.19 ppm due to the formation of acetone (Figure
4A). Similarly other monomers also exhibited characteristic
signals for the ketal and ketone moieties. The new monomers
exhibited ketal group structure-dependent hydrolysis; the
monomers with acyclic ketal structure (DMKM, CPKM, and
CHKM) were hydrolyzed much faster compared to those with
cyclic structure (GHBKM and GCHKM) (Figure 4B). The
hydrolysis half-lives (t1/2) at pH 5.5 were 6.3, 11, and 19.8 min
respectively for the monomers DMKM, CPKM and CHKM,
while GHBKM and GCHKM were not hydrolyzed for several
days at this pH (Table 1).
2.3.2. Degradation of Modified pPKHEs at pH 5.5. We then
studied the hydrolysis of the pPKHEs at pH 5.5 in a similar
manner to that of the monomers by monitoring the decrease in
the intensity of the signals due to the ketal groups and
corresponding appearance of ketone group. Representative
NMR spectra showing degradation profiles of pPCHKHE-3 is
shown in Figure 4C. The cyclohexyl group of the ketal moiety
in pPCHKHE-3 polymer had a broad characteristic signal
between δ 1.39 and 1.75 ppm. Upon degradation, the intensity
of this peak gradually decreased, and new sharp characteristic
NMR signals appeared at δ 1.85 and 2.34 ppm due to
formation of cyclohexanone (Figure 4C). The percentage of
degradation was calculated from the ratio of the intensities of
the NMR signals corresponding to the cyclohexyl ketal group
within the main chain and the cyclohexanone. Similar
observations were seen for other pPKHE polymers (Figures
S17−S19). The polymers exhibited a similar trend in the
hydrolysis as was observed with the corresponding monomers;
however, the hydrolysis rate was slower. The hydrolysis rate
followed the order pPDMKHE-3 > pPCPKHE-2 >
pPCHKHE-3 ≫ pPGHBKHE-1 ≈ pPGCHKHE-1 (Figure
4D). As shown in Table 3, the hydrolysis half-lives at pH 5.5 for
pPDMKHE-3, pPCPKHE-2, pPCHKHE-3, pPGHBKHE-1,
and pPGCHKHE-1 were 0.3, 0.8, 2.5, 25 300, and 21 120 h,
respectively. In contrast to pPDMKHE-3, pPCPKHE-2, and
pPCHKHE-3 with acyclic ketal structures, the polymers
containing cyclic 1,3-dioxolane (pPGHBKHE-1) and spiro
ketal groups (pPGCHKHE-1) were more stable towards
hydrolysis.
The observed differences in the hydrolysis rates of the
monomers and polymers may be attributed to several factors:
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Figure 5. Comparison of pH-dependent degradation of pPKHEs at 25 °C at different pH values in aqueous buffer solutions measured by NMR
analysis. pH-dependent degradation of (A) pPDMKHE-3, (B) pPCHKHE-3, and (C) pPCPKHE-2. (D) Degradation of pPGHBKHE-1 at pH 1.1.
The pH values of the solutions are shown in the graph.
(i) the differences in hydrophobicity of the groups surrounding
the ketal moiety, (ii) the ring strain of cyclic ketal groups, and
(iii) the differences in the ketal structure. All these factors can
alter their reactivity toward acids during hydrolysis.
copolymers, the I1/I3 value reported was between 1.1 and 1.2 in
aqueous conditions.30c Pyrene fluorescence spectra in the
presence of PEGylated PKHEs at two different concentrations
(0.4 and 4 mg/mL) were obtained. The I1/I3 values for
different polymers at 0.4 mg/mL concentration was 1.80, 1.55,
1.64, and 1.74 respectively for pPDMKHE, pPCPKHE,
pPCHKHE, and pPGHBKHE (Table S1 and Figure S20)
and followed a trend similar to that of the hydrophobicity
values calculated from log P values for the corresponding
monomers. Though the I1/I3 values suggest a slightly
hydrophobic environment for these polymers, they were higher
than the range (1.0−1.4) reported for micellar systems30a−c and
very close to that reported for PEG (1.58−1.60).30d The data
suggest that the pPKHEs may have a highly hydrated core−
shell structure in water. Since the hydrophobicity values do not
correlate with the degradation profile (Figure 4D), we can
confirm that, globally, the hydrophobicity differences of the
polymers do not have a significant role in controlling the
polymer degradation. However, in the case of acyclic ketal
polymeric structures (pPDMKHE, pPCPKHE, and
pPCHKHE), the hydrophobicity trend measured by pyrene
fluorescence followed the same trend as the log P values
calculated for different monomers. The polymer degradation
also followed a similar trend. Thus, the influence of
hydrophobicity changes on polymer degradation cannot be
completely ruled out for acyclic ketal polymeric structures.
These results are also supported by the literature evidence on
small organic molecules containing ketal groups, where it has
been shown that the groups attached to the ketal structure can
influence the reactivity of ketal groups.31,32 Significant differ-
ences in the hydrolysis rates were observed between diethyl
ketals of cyclopentanone and cyclohexanone, with the rate of
hydrolysis for cyclopentanone ketal being 3 times faster than
that of cyclohexanone ketal.31 The decrease in the torsional
strain energy for the cyclopentanone derivative as a result of the
In order to verify whether the observed differences in the
hydrolysis rate of the monomers is due to the differences in
hydrophobic character of the monomers, we calculated the
log P value (which is a measure of the hydrophobicity of the
molecule) using the ALOGP program.29 The total number of
carbon atoms in the current monomer set ranged from 10 to
13, and all the monomers had the same number of oxygen
atoms per molecule (Table 1). The CHKM monomer had the
highest number of carbon atoms per molecule among the
monomers. The calculated log P values were −0.94, 0.0, 0.46,
−0.79, and −0.39 for monomers DMKM, CPKM, CHKM,
GHBKM, and GCHKM, respectively, which suggest that their
hydrophobicity was in the order CHKM > CPKM > GCHKM
> GHBKM > DMKM (Table 1). The degradation of the
monomers followed the order DMKM > CPKM > CHKM ≫
GHBKM ≈ GCHKM (Figure 4B), which suggests that the
hydrophobicity of monomers may not be influencing the
degradation.
Although we anticipated similar hydrophobicity for the
polymers prepared from these monomers, we investigated the
hydrophobic character of the modified polymers by fluo-
rescence spectroscopy using pyrene as the probe. The
vibrational spectrum of pyrene is known to be sensitive to
the hydrophobicity of its environment and can be measured
from the ratio of the intensities of the first (I1 = 372 nm) and
third (I3 = 384 nm) peaks in the fluorescence spectrum.30a For
instance in aqueous solutions, the I1/I3 values will be in the
range 1.9−2.0, and in the hydrophobic nonpolar environments,
they will be 0.55−0.6.30a Kalyanasundaram et al. also reported
that for micellar systems the I1/I3 value ranges from 1 to 1.4,
depending on the nature of the micelle.30a For PS-PEG block
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Figure 6. Comparison of pH-dependent degradation of pPKHEs at 37 °C at different pH values in aqueous buffer solutions measured by NMR
analysis. pH-dependent degradation of (A) pPDMKHE-3, (B) pPCHKHE-3, and (C) pPGHBKHE-1. The pH values of the solutions are shown in
the graph.
activation process involved in hydrolysis was attributed to this
observation. It has been also shown that a slight increase in the
torsional energy strain for the cyclohexanone ketal results in
slower hydrolysis rates.31 In the case of GHBKM and GCHKM,
the ketal group is part of the ring, and cleavage of the ring
(endocyclic cleavage) must take place during hydrolysis, which
occurs at much slower rates.32 Based on this evidence from the
low-molecular-weight molecules, we attribute the differences in
the hydrolysis rates of the PKHE polymers primarily to the
differences in the ketal group structure and ring strain of the
groups with minor dependence on the polymer hydrophobicity.
In the pyrene fluorescence measurements, slightly lower I1/I3
values (1.41−1.55) were obtained at higher polymer concen-
tration (4 mg/mL) compared to 0.4 mg/mL (Table S1). These
data suggest that a slightly more hydrophobic environment for
pyrene exists at higher polymer concentrations. To investigate
whether such differences influence pPKHE degradation, we
studied the degradation at two different polymer concen-
trations, 4 and 20 mg/mL. Our results (Figure S21) showed
that polymer degradation was independent of polymer
concentration. These data also support the insignificant role
of hydrophobicity in polymer degradation.
It is also important to understand if there is any broadening
or attenuation of the signals in the NMR spectrum of the
polymers due to the formation of unimolecular micelles, as the
pPKHEs have a hydrated core−shell structure. This may affect
the accuracy of the degradation kinetics since the polymer
degradation was measured from the integration of the peaks of
the polymer and the ketones resulting from degradation. To
probe this, initially the NMR spectra of pPKHEs were recorded
in D2O and CDCl3. In both solvents, the ratio of the intensities
of peaks from polyether backbone and PEG chains to ketal
groups remained similar (Figures S22−S24), which suggests
that solvents did not have any effect on the NMR peak
intensity. If there was any attenuation of the signals due to the
micelle formation in water, quite different behavior would have
been observed, as these polymers were not expected to adopt a
micellar structure in chloroform. Moreover, the ratio of the
total intensity of the signals from the ketal and ketone groups
to that of the polyether backbone and PEG chains remained
constant throughout the degradation. In the case of the
polymers pPGHBKHE-1 and pPGCHKHE-1, the ketone
structure remains as part of the degraded polymer, and no
small molecule was formed in this case. Taken together with
polymer degradation studied at different polymer concen-
trations (Figure S21), these data support the fact that the
degradation rate of the polymers could be accurately measured
from the signals corresponding to the ketal and ketone groups
in the NMR spectra. Similar methods have been reported by
Broaders et al. previously.19d
2.3.3. Effect of pH on the Degradation of pPKHEs. We
further investigated the degradation kinetics of pPKHE
polymers at different pH values (1.1−8.2) at 25 °C. The
hydrolysis profiles for the polymers shown in Figure 5
demonstrate that pPDMKHE-3, pPCHKHE-3, and
pPCPKHE-2 with linear (acyclic) ketal groups were completely
hydrolyzed at pH 1.1 and 4.1 within a few minutes (Figure 5A−
C). A more controlled degradation was observed in the pH
range 5.5−8.2, with the hydrolysis rates being slower at higher
pH values. For instance, the hydrolysis half-lives (t1/2) for
pPCHKHE-3 were 2.5, 8.7, 15.9, and 187 h at pH 5.5, 6.0, 6.5,
and 7.4, respectively (Table 3), and the polymer was relatively
stable at physiological pH and above. A similar trend was
observed for the hydrolysis of polymers containing dimethyl
and cyclopentyl ketal groups (Figure 5A,C, Table 3). The
hydrolysis of pPGHBKHE-1 and pPGCHKHE-1 were much
slower even at acidic pH values (Figure 5D). While the
polymers with linear ketal groups were hydrolyzed within a few
minutes at pH 1.1 and 4.1, the hydrolysis half-life of
pGHBKHE-1 at these pH values at 25 °C was 0.5 and 1400
h, respectively. Both pPGHBKHE-1 and pPGCHKHE-1 were
very stable at pH above 5.5; pPGHBKHE-1 showed only 30%
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Figure 7. Polymer degradation studied by GPC analysis. PKHE polymers were degraded in 0.1 N HCl, neutralized, and analyzed. GPC
chromatograms of pPKHEs before and after the degradation are shown for (A) pPDMKHE-3, (B) pPCHKHE-3, and (C) pPGHBKHE-1.
degradation in 400 days at pH 5.5 and 25 °C. Thus we have
shown that a range of hydrolysis half-lives can be achieved by
changing the bonding structure of the ketal groups in the
polymer backbone and the pH of the solution.
2.4. Functionalization and Cellular Uptake of PKHEs.
2.4.1. Functionalization of PKHEs. Polymer functionalization
with reactive groups such as azides or amines will facilitate the
conjugation of fluorophores or other bioactive molecules to the
polymer. In order to incorporate various functionalities in the
polymer, initially the hydroxyl groups of pPKHEs were reacted
with sodium hydride and α-azido-(PEG)8-ω-epoxide to
synthesize azido-functionalized polymers (Figure 3). The
azide groups were then reduced with triphenylphosphine/
water to generate the amine-functionalized polymers. The ketal
groups were found to be stable under the reaction conditions
developed. In order to investigate the cellular uptake of
pPKHEs, fluorescently labeled polymers (FL-pPKHEs) were
prepared by the reaction of the amine-functionalized polymers
with Alexa-488 carboxylic acid N-hydroxysuccinimidyl ester
(Figure 3). The unreacted fluorophore was removed by dialysis
of the polymer solutions in phosphate buffer at pH 9. The
conjugation of the fluorophore to the polymers was confirmed
by fluorescence emission spectra at 515 nm.
2.4.2. Cellular Uptake and Degradation of pPKHEs.
Controlled intercellular degradation is an important criterion
for the development of drug delivery devices for efficient rapid
endosomal escape and cytosolic delivery.22a It has been shown
that, during endocytosis and endosomal maturation, there is a
gradual change in the pH from 6.0−6.2 in early endosome to
5.5 in late endosome to 4.5−5 in lysosome.33 Thus, developing
polymeric systems that can rapidly degrade in a controlled way
could possibly disrupt the endosome due to osmotic imbalance
and help endosomal escape and cytosolic delivery of
therapeutic agents.34−43 Thus, we investigated the degradation
of pPKHE polymers in cells using fluorescent derivatives. The
cellular uptake and subsequent trafficking of fluorescent-labeled
polymers were examined using confocal microscopy at various
time intervals. The CHO cells were incubated with FL-pPKHEs
(10 mg/mL) for 1 h at 37 °C and were washed with Dulbecco’s
phosphate-buffered saline before placing them in fresh media at
37 °C, and the cells were viewed at various time intervals. The
uptake of all three polymers, FL-pPDMKHE-3 (rapidly
degrading), FL-pPCHKHE-3 (slow degrading), and FL-
pPGHBKHE-1 (stable at physiological pH), was observed
2.3.4. Effect of Temperature on the Degradation of
pPKHEs. In order to simulate the hydrolysis behavior of the
polymers under physiological conditions and within cells, the
polymer degradation was studied at 37 °C. A 3- to 4-fold
increase in hydrolysis rates was observed at 37 °C compared to
those at 25 °C for a given pH (Figure 6 and Table 3). For
example, the t1/2 of pPDMKHE-3 at pH 6.0 decreased from 1.1
to 0.16 h when the temperature was increased from 25 °C to
37 °C (Figure 6A, Table 3). Similarly, pPCHKHE-3 gave a
hydrolysis half-life of 2.5 h at 37 °C compared to 8.7 h at 25 °C
(Figure 6B, Table 3) at pH 6.0. The hydrolysis rates were also
dependent on the type of ketal structures present. The
hydrolysis half-life of pPGHBKHE-1 at pH 4.1 and 5.5 was 3
days and 152 days, respectively, at 37 °C (Figure 6C, Table 3).
There was no significant hydrolysis (<5%) observed for
pPGHBKHE-1 and pPGCHKHE-1 at pH 7.4, even after 400
days at 37 °C. All these data support the fact that degradation
of PKHE polymers can be also modulated by changing the
temperature apart from the ketal group structure.
2.3.5. Degradation of pPKHE Polymers Determined by
GPC Analysis. Information regarding the molecular weights of
degradation products of the polymer and the uniformity of
ketal monomer incorporation within the polymers was obtained
from GPC analysis. The GPC chromatograms for pPDMKHE-
3, pPCHKHE-3, and pPGHBKHE-1 before and after
degradation are shown in Figure 7. All the polymers showed
a monomodal distribution of chains before hydrolysis with no
evidence of low-molecular-weight fractions in the sample. Upon
hydrolysis, a shift in the chromatogram towards higher elution
volume was observed for all the polymers, indicating that they
were completely degraded to low-molecular-weight fragments.
Together with 13C NMR data (section 2.2), the GPC data
confirmed that the ketal groups are uniformly incorporated in
the polymer backbone. Similar results were obtained for the
other pPKHE polymers.
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Figure 8. Confocal microscopy images of Chinese hamster ovarian (CHO) cells incubated with fluorophore labeled polymers 2, 4, and 24 h post-
treatment. The polymers are labeled with Alexa-488 (green). The nuclei are stained blue using DAPI. Diffused fluorescence observed for
pPDMKHE-3 at 4 and 24 h post-treatment and pPCHKHE-3 at 24 h post-treatment suggests degradation of the polymers within the cells and
delivery of fluorescent markers into the cytosol. Fluorescence for the slowest degrading polymer pPGHBKHE-1 appeared as distinct points even
after 24 h, suggesting that these polymers are still within the various compartments in the cells.
(Figure 8). At 2 h post-incubation, all the polymers were
observed as punctate dots, indicating that they are entrapped
within the endosomes as reported.22a,44 At 4 h post-treatment,
the rapidly degrading FL-pPDMKHE-3 was visible as diffused
green fluorescence and as punctate dots in distinct compart-
ments within the cells (Figures 8 and S25). Both the slowly
degrading FL-pPCHKHE-3 and the stable FL-pPGHBKHE-1
polymers presented mostly as punctate dots, indicating that
they are still entrapped within the endosomes. At 24 h, the
discrete points of the fluorescence were not visible for both FL-
pPDMKHE-3 and FL-pPCHKHE-3; instead a diffused intra-
cellular fluorescence was observed, suggesting the degradation
of the polymers and release of low-molecular-weight polymer
fragments along with fluorescence marker to the cytosol. For
the slowest-degrading polymer FL-pPGHBKHE-1, fluorescence
was confined to discrete points even 24 h post-incubation,
indicating that this polymer was still retained in the endosomes
or lysosome without noticeable degradation or excretion from
cells.45 At 4 h post-treatment, the fast-degrading FL-
pPDMKHE-3 has the lowest number of punctate fluorescent
dots and relatively more diffused fluorescence compared to
other polymers. These data together with the results obtained
after 24 h suggest that the intracellular degradation of PKHE
polymers in the cells may follow a similar trend as observed in
the NMR degradation studies in solution (Figures 4−6). The
decrease in the number of punctate fluorescent dots and
increase in the diffused fluorescence for rapidly degrading
pPDMKHE-3 and slowly degrading pPCHKHE-3 in the cells
with time may not be due to the exocytosis or the loss of the
polymer from cells. This is supported by the fact that the cells
treated with equal concentrations of stable pPGHBKHE-1
polymer have considerable amount of punctate fluorescent dots
at treatment intervals identical to those of pPDMKHE-3 and
pPCHKHE-3 (Figures 8 and S25). The data from pPDMKHE-
3 and pPCHKHE-3 also confirmed the delivery of fluorescent
molecule (a surrogate drug) into cytosol. Taken together, these
results support the fact that the PKHE polymers may have
similar degradation profiles in cells as seen in solution, which
makes them highly suitable for intercellular drug delivery
applications.
2.5. Biocompatibility of PKHEs and Their Degradation
Products. Biocompatibility of polymers and their degradation
products is critical for their use in various biomedical
applications.46 Therefore, cell and blood compatibilities of the
newly synthesized pPKHEs and their degradation products
were studied by measuring the cell viability, blood coagulation,
complement activation, and platelet activation in their presence.
2.5.1. Cell Viability against Human Umbilical Vein
Endothelial Cells. The cell viability of pPKHEs and their
degradation products was assessed by incubating human
umbilical vein endothelial cells (HUVEC) with increasing
polymer concentrations for 48 h and measuring the viable cells
by MTS assay. Results are shown in Figure 9A; the polymers
showed high cell viability up to a concentration of 5 mg/mL.
The degradation products of pPKHEs were also found to be
nontoxic to the cells (Figure S26). These results demonstrate
the excellent cell compatibility of the new polyketal polymers.
2.5.2. Blood Compatibility. 2.5.2.1. Blood Coagulation.
The interaction of polymers with blood components could alter
the blood coagulation and may lead to thrombotic
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Figure 9. Biocompatibility of pPKHEs and their degradation products. (A) Cell viability of human umbilical vein endothelial cells measured by MTS
assay. (B) Activated partial thromboplastin time (APTT) in human plasma measured at 37 °C. HEPES buffer was used as normal control. (C)
Platelet activation in human platelet-rich plasma by measuring the expression of activation marker CD62P as measured by flow cytometry. Bovine
thrombin and HEPES buffer were used as positive and normal controls, respectively. (D) Complement activation in human serum measured by a
complement consumption assay (CH50) using antibody-sensitized sheep red blood cells. The percentage of complement proteins consumed is
given. All the blood compatibility tests were performed at a polymer concentration of 1 mg/mL. The data given are from triplicate measurements
using three different donors.
complications upon intravenous administration.46 Such in-
formation is critical for the development of drug delivery
vehicles for intravenous applications. So the blood coagulation
in the presence of pPKHEs and their degradation products was
studied by measuring the blood clotting time using clinical
coagulation assays.47 The polymer solutions (1 mg/mL) were
incubated with human platelet-poor plasma (PPP) at 37 °C,
and the clotting time was measured by activated partial
thromboplastin time (APTT), which represents the intrinsic
pathway of blood coagulation. APTT is the time (in seconds)
taken for a fibrin clot to form after the addition of partial
thromboplastin reagent (actin) and calcium chloride. As shown
in Figure 9B, pPKHEs and their degradation products did not
show significant change in the APTT values compared to that
of the buffer control, which indicated that these polymers may
not cause any adverse effect on blood coagulation.
2.5.2.2. Platelet Activation. Platelet activation on interaction
with polymers can also lead to several adverse effects, such as
thrombotic complications and arterial embolization.46a Platelet
activation results in the expression of the glycoprotein P-
selectin CD62 on the surface of platelets, and this was
measured by flow cytometry analysis. Polymer solutions (1 mg/
mL, final concentration) were incubated with platelet-rich
plasma (PRP) for 30 min, and the platelet activation was
measured from the expression of platelet activation marker
CD62P using monoclonal anti-CD62P-FITC antibody. Platelet
activation by pPKHEs was compared with that of buffer control
and a positive control (thrombin) and is expressed as the
percentage of platelets that are positive for CD62P. The extent
of platelet activation by the PKHE polymers and their
degradation products was very similar to that of the buffer
control (Figure 9C), indicating that the new polymers did not
induce any platelet activation.
2.5.2.3. Complement Activation. The complement system
consists of a series of proteins circulating in the blood as
precursors and is part of the innate immune system. Activation
of the components of the complement system upon interaction
with a foreign material can produce anaphylotoxins, which can
result in cellular responses such as histamine release and
induction of inflammation.46a It has been shown that hydroxyl-
carrying polymers such as starch, poly(vinyl alcohol), and
dextran can activate the complement system.48 However,
hydroxyl-containing polymers such as hyperbranched poly-
glycerol49 and some glycopolymers50 did not induce comple-
ment activation. To investigate whether the new PKHE
polymers activate the complement system, the level of
complement activation by pPKHE polymer was studied by
antibody-sensitized sheep erythrocyte complement lysis
(CH50) assay that measures the total complement activity
from the ability of the serum sample to lyse sheep red blood
cells coated with IgM antibody.51 The polymer samples were
incubated at 37 °C for 1 h with human serum to which the
antibody-sensitized sheep erythrocytes were added. Lysis of the
sheep erythrocytes indicative of unactivated complement and
the RBC lysis were quantified by measuring the amount of
hemoglobin released. Thus, the total amount of complement
consumption by the polymer was measured and compared with
positive control (immunoglobulin G, IgG), negative control
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(EDTA-incubated serum), and a buffer control. Results shown
in Figure 9D confirm that the new polyketal polymers do not
activate the complement system.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details and additional characterization (1H and
13C NMR, IR, and GPC of the monomer and polymers),
polymer degradation, and cellular uptake data. This material is
available free of charge via the Internet http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
jay@pathology.ubc.ca
■
Notes
The authors declare the following competing financial
interest(s): R.A.S., J.K.N., K.G.R., M.M., and J.N.K. have filed
a PCT application based on this work.
ACKNOWLEDGMENTS
■
This research was funded by Alnylam Pharmaceuticals and
Canadian Institutes of Health Research (CIHR). The Macro-
molecular Hub at the UBC Centre for Blood Research was
funded by Canada Foundation for Innovation and Michael
Smith Foundation of Health Research (MSFHR). J.N.K. is a
recipient of a CIHR/CBS new investigator in Transfusion
Science and MSFHR career Investigator Award.
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