Acid-triggered release via dePEGylation of fusogenic liposomes mediated by heterobifunctional phenyl-substituted vinyl ethers with tunable pH-sensitivity

Article abstract of DOI:10.1021/bc300266y

A new family of heterobifunctional phenyl-substituted vinyl ether (PIVE) coupling agents with tunable acid-sensitivity has been developed. The PIVE compounds are designed to hydrolyze under acidic conditions with hydrolysis rates that can be varied by rat

Full text of DOI:10.1021/bc300266y

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Article
Acid-Triggered Release via dePEGylation of Fusogenic
Liposomes Mediated by Heterobifunctional Phenyl
Substituted Vinyl Ethers with Tunable pH-Sensitivity
Hee-Kwon Kim, Jeroen Van den Bossche, Seok-Hee Hyun, and David H Thompson
Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 18 Sep 2012
Downloaded from http://pubs.acs.org on September 20, 2012
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Acid-Triggered Release via dePEGylation of Fusogenic
Liposomes Mediated by Heterobifunctional Phenyl
Substituted Vinyl Ethers with Tunable pH-Sensitivity
Hee-Kwon Kim,+ Jeroen Van den Bossche,+ Seok-Hee Hyun, and David H Thompson*
Purdue University, Department of Chemistry, 560 Oval Drive, West Lafayette, IN 47907, USA
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ABSTRACT
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A new family of heterobifunctional phenyl-substituted vinyl ether (PIVE) coupling agents with
tunable acid-sensitivity has been developed. The PIVE compounds are designed to hydrolyze
under acidic conditions with hydrolysis rates that can be varied by rational selection of the
phenyl ring substituent. These reagents were incorporated within 2-methoxypoly(ethylene
glycol) PEG-conjugated 1,3-dioctadecyl-rac-glycerol lipids to produce the acid-cleavable
lipopolymers mPEG-[H-PIVE]-DOG, mPEG-[F-PIVE]-DOG, mPEG-[Me-PIVE]-DOG, and mPEG-
[MeO-PIVE]-DOG. These lipopolymers were hydrolyzed under acidic conditions (pH 3.5 or 4.5)
at rates that were dependent on the electron donating or withdrawing character of the α-
phenyl vinyl ether substituent, while remaining stable at pH 7.4. Blending of these compounds
with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 10:90 mPEG-PIVE-Lipid:DOPE
ratio produced stable liposomes at neutral pH; however, acidification of the solution led to
dePEGylation and release of the liposomal cargo in a manner that correlated with the PIVE
proton affinity. Specifically, we observed 70 % calcein release within 12 h from mPEG-[MeO-
PIVE]-DOG-containing liposomes at pH 4.5, whereas only 22% calcein release was observed
from mPEG-[F-PIVE]-DOG:DOPE liposomes over this same timescale and pH. These results
indicate that dePEGylation following acidification is a triggering mechanism that can be
rationally designed and controlled through the appropriate selection of PIVE moieties.
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INTRODUCTION
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Many important physiological and pathological processes such as endosomal processing,
tumor growth and inflammation occur in acidic environments.1-2 For example, molecules
internalized within cells via the endocytic pathway encounter a significant pH drop from neutral
to pH 5.9-6.0 within 2 minutes after entry into this transient organelle.3 These compartments
can experience further reduction in pH from 6.0 to 4.0 during progression from late endosomes
to lysosomes.4 Solid tumors and inflammatory tissues are also known to be slightly acidic.5-6
Therefore, the design and development of materials that can respond to pH changes in a
measured and a priori predictable way remains an unmet goal for the realization of drug
delivery systems that are molecularly engineered to respond specific biological conditions and
kinetic time scales.
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The main challenge in the development of carrier systems with these characteristics is the
need for the vehicle to remain stable at physiological pH, but rapidly release its contents when
it is exposed to the decreased pH of the target site. Thus, there have been many reports of pH-
responsive carrier systems based on formulation approaches,7 manipulation of surface charge,8-
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as well as a wide variety of pH-sensitive linkages including acetals,10-12 ortho esters,13-18
hydrazones,19-20 cis-aconityl,21-23 silyl ether,24 and vinyl ethers,8, 25-26 that have been developed
for controlled drug release from macro- and nanoparticulate carriers.
Thompson and coworkers have studied plasmenylcholines27 and other types of vinyl ether
lipids8, 25-26, 28 as potential vehicles for pH-sensitive drug delivery. Since the rate determining
step of acid-catalyzed vinyl ether hydrolysis is protonation of the β-carbon atom, followed by
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rapid hydration of the resulting transient, resonance-stabilized carbocation, the hydrolysis rate
of this motif can be controlled by varying the electronic character of the vinyl ether α-
substituent.29-30 We have recently reported studies showing a correlation between the proton
affinity of vinyl ether molecules determined by DFT calculations and their intrinsic second order
hydrolysis rate constants.28 Our initial studies guided the development of a novel acid-labile
PEG conjugated vinyl ether lipids (ST302, ST305, ST352, ST355) that have ability to accelerate
acid-triggered contents release from DOPE unilamellar liposomes when used as the minor
component in the membrane bilayer.8, 31 Calcein release rate data suggested that rate of
contents release was affected by the molar ratio of acid-labile PEG-Lipid, the PEG molecular
weight, and the proton affinity of the vinyl ether bond linking the PEG and lipid moieties.
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This work describes a new class of heterobifunctional compounds that utilize an α-phenyl
substituted vinyl ether (PIVE) bond as the modulating substituent for tuning the acid-sensitivity
of the construct (Scheme 1). These bifunctional PIVEs allow for the rational design of cleavable
linkers with predictable pH-sensitivity and facile synthesis of compounds with a diverse set of
substituents with varying electronic character. These features enable their facile incorporation
into a wide range of pH-sensitive materials, requiring effectively no major changes to the
composition of the delivery vehicle formulation.
Four PIVE heterobifunctional linkers were prepared and used to couple the same mPEG2000
water-soluble headgroup and 1,3-dioctadecyl-rac-glycerol lipid anchor to provide a homologous
series of lipopolymers for direct comparison of their activity as the critical components of pH-
sensitive liposomes for intracellular drug delivery (Figure 1). Proton affinities of mPEG-[H-PIVE]-
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DOG, mPEG-[F-PIVE]-DOG, mPEG-[Me-PIVE]-DOG, and mPEG-[MeO-PIVE]-DOG containing
different 4’-substituents on the α-phenyl vinyl ether bond were calculated using density
functional theory (DFT) methods. These target compounds were then synthesized and their
hydrolysis rates evaluated by HPLC analysis. Cargo release rates via dePEGylative activation of
10:90 mPEG-PIVE-Lipid:1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPE) liposomes also were
tested using a fluorescence dequenching assay and the change in liposome morphology
evaluated by cryogenic transmission electron microscopy (cryoTEM).
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EXPERIMENTAL PROCEDURES
Materials. All chemicals were obtained from Aldrich Chemical Company, except DOPE that
was obtained from Avanti Polar Lipids. Synthetic intermediates and final products were
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characterized by 300 MHz H NMR and MALDI MS using the Purdue University Center for
Cancer Research’s Interdepartmental NMR Facility and Campus-Wide Mass Spectrometry
Center, respectively. CryoTEM images of liposome samples were obtained via the Purdue
University Biological Electron Microscopy Facility.
DFT Calculations. The proton affinity values of the vinyl ether series were determined by DFT
calculations using Gaussian 03 and the B3LYP hybrid functional.32-33 Neutral and protonated
structures were optimized using the 6-31G** basis set. Single point energies of the optimized
structures were obtained with the cc-pVTZ basis set.34
Synthesis of mPEG-PIVE-Lipids. Detailed methods for the preparation of 1,3-
bis(octadecyloxy)propan-2-yl(7-(((4-methoxypoly(ethylene
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glycol[MW2000])carbamoyl)oxy)butoxy)-7-phenylhept-6-en-2-yl)carbamate
(mPEG-[H-PIVE]-
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DOG), 1,3-bis(octadecyloxy)propan-2-yl(7-(4-fluorophenyl)-7-(((4-methoxypoly(ethylene glycol
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[MW2000])carbamoyl)oxy)butoxy)hept-6-en-2-y)carbamate
bis(octadecyloxy)propan-2-yl(7-(((4-methoxypoly(ethylene
(mPEG-[F-PIVE]-DOG),
1,3-
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glycol[MW2000])carbamoyl)oxy)butoxy)-7-(4’-methylphenyl)hept-6-en-2-yl)carbamate (mPEG-
[Me-PIVE]-DOG), and 1,3-bis(octadecyloxy)propan-2-yl(7-(((4-methoxypoly(ethylene
glycol[MW2000])carbamoyl)oxy)butoxy)-7-(4’-methoxyphenyl)hept-6-en-2-y)carbamate
(mPEG-[MeO-PIVE]-DOG) are described in Supplementary Information.
Hydrolysis Kinetics of mPEG-PIVE-Lipid Conjugates. Hydrolysis reactions were initiated by
dissolving 0.2 mg of the mPEG-PIVE-Lipid conjugate in 10 mL of 20 mM HEPES (pH7.4), 20 mM
citrate (pH 4.5), or 20 mM oxalate (pH 3.5) and incubating the mixture at 37 °C. Aliquots (200
µL) were periodically removed and the product mixture analyzed by HPLC at 40 °C (injection
volume = 40 µL) on an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA)
equipped with a Corona Charged Aerosol Detector and an Eclipse XDB-C8 column (4.6 x 150
mm, Agilent Technologies). Gradient elution was performed using a flow rate of 0.7 mL/min: 0-
20 min, 1:9 MeOH:H2O → 7:3 MeOH:H2O; 20-35 min, 7:3 MeOH:H2O → 7:3 MeOH:CH3CN; 35-
50 min, 7:3 MeOH:CH3CN → 1:9 MeOH:CH3CN; 50 min-60 min, 1:9 MeOH:CH3CN → 1:9
MeOH:H2O. The rate of hydrolysis was determined by monitoring the chromatographic peak
area using mPEG-OH RT = 22.9 min and mPEG-PIVE-Lipid conjugates RT = 43.5 min. The mPEG-
OH chromatographic peak was verified by comparison with the retention time determined for
commercial available standard samples. The observed first-order rate constants were obtained
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using the half-life method; the intrinsic second order hydrolysis rate constants were obtained
by correcting the observed first-order rate constants for the reactant in excess.
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Liposome Preparation. Stock solutions (20 mg/mL) of DOPE and the pH-sensitive mPEG-
PIVE-Lipid conjugates were prepared in 95:5 benzene:MeOH. The lipid stock solutions were
combined at the desired ratios (10 µmol total lipid) and vortexed for 2 min before flash freezing
in LN2 for 3 min and lyophilization (15 h) to produce a powder. The lyophilized lipid mixtures
were then hydrated in 1 mL of 50 mM calcein solution (dissolved in 20 mM 4-(2-hydroxyethyl)-
1-piperazine-ethanesulfonic acid (HEPES), 150 mM NaCl, pH 7.4) via ten freeze-thaw-vortex
cycles to produce multilamellar liposomes that were extruded ten times through 100 nm
polycarbonate membranes filters at 50 °C with ~200-250 psi N2. The liposome solutions were
passed through a 0.5 x 25 cm Sephadex G-50 column equilibrated with a 20 mM HEPES, 150
mM NaCl, pH 7.4 solution for removal of unentrapped calcein and the liposomes used
immediately after isolation.
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Dynamic Light Scattering. Liposome sizes were measured by dynamic light scattering using a
Malvern Nano ZS-90 instrument and the distributions calculated using the manufacturer’s
supplied software. Samples were prepared by diluting 50 µL liposome stock with 1 mL 18 MΩ
H2O. Experiments were run in triplicate.
Calcein Release Assay. An aliquot of the liposome stock solution (100 µL) was added to 1.3
mL of 20 mM HEPES (pH7.4), 20 mM citrate (pH 4.5), or 20 mM oxalate (pH 3.5) containing 150
mM NaCl and the solutions maintained at 37 °C for the duration of the experiment. At each
measurement point, 100 µL aliquots were taken and diluted with 2 mL HEPES-buffered saline.
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The fluorescence (λex = 490 nm, λem = 518 nm) was then measured before and after the addition
of 2 drops 10% Triton X-100 (solution gently shaken to ensure complete liposome disruption).
The percent release was quantified using the ratio method: % release = 100 x [(T - T0)/(1 - T0)],
where T = (fluorescence before Triton addition/fluorescence after Triton addition) at time T;
T0=fluorescence ratio at T = 0 min.35
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Cryogenic Transmission Electron Microscopy. A small drop of liposome solution was placed
on a bare Quantifoil grid by pipet and the excess liquid blotted away using filter paper under
humid environmental conditions to produce a thin (20-500 nm) sample film around the holes in
the polymer grid. The sample film was vitrified by rapid freezing in a liquid ethane slush cooled
by LN2 and transferred via cryotransfer device to a Philips CM200 transmission electron
microscope equipped with a LN2-cooled stage and a field-emission gun operating at 200 kV
accelerating voltage for observation. In order to minimize radiative sample damage, the
collection of high-resolution cryoTEM images was made at 50,000x magnification under low
dose conditions.
RESULTS AND DISCUSSION
PIVE Design Guided by DFT Calculations. A variety of groups have reported how electronic
effects can affect pH-sensitive drug delivery systems,8, 10, 20, 24, 36-37 including a recent study
describing a series of mPEG-Lipids linked by vinyl ethers with both electron donating and
electron withdrawing groups.28 The proton affinities were calculated using DFT methods,
producing a reasonably good correlation between predicted proton affinities and observed
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second order hydrolysis rate constants. Unfortunately, these compounds were limited by the
fact that a unique synthetic pathway was required for each member of the vinyl ether-linked
PEG-lipid series. Furthermore, those pathways restricted the scope of the methodology to the
synthesis of lipid-type structures. These limitations led us to seek a family of heterobifunctional
molecules with tunable hydrolysis rates that could all be produced via a common reaction
pathway to afford derivatives that could easily be incorporated into many pharmacologically
interesting constructs.
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DFT (B3LYP) calculations were initially performed to evaluate whether a broad range of
proton affinities could be expected from PIVEs using different substituents on the phenyl ring.
Neutral vinyl ether precursors and protonated intermediates were first optimized with the 6-
31G** basis set.32-33 The single point energies of the optimized structures then were obtained
with the cc-pVTZ basis set at the DFT level.34 Comparison of these parameters (Table 1) with the
intrinsic second order rate constants of similar vinyl ethers28 were then used to select targets
for synthesis that should display easily detected changes in reaction rate constants.
Synthesis of Acid Labile PEG-PIVE-Lipid Conjugates. Based on the findings of the DFT
calculations, we designed a series of pH-sensitive heterobifunctional PIVE reagents to
investigate the effect of α-phenyl ring substitution on acid-catalyzed hydrolysis rates. Four
different heterobifunctional PIVE crosslinking reagents (Scheme 2) were prepared to effect the
synthesis of the mPEG-PIVE-Lipid conjugates mPEG-[F-PIVE]-DOG, mPEG-[H-PIVE]-DOG, mPEG-
[Me-PIVE]-DOG, and mPEG-[MeO-PIVE]-DOG (Scheme 3).
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The heterobifunctional PIVE reagents were prepared from their corresponding
benzaldehydes in three steps as shown in Scheme 2. Para-substituted α-vinyl-benzyl alcohols
were prepared through a Grignard reaction of the benzaldehydes 2 with vinyl magnesium
bromide in THF at 22 °C. Williamson etherification with t-butyl(4-iodobutoxy)dimethylsilane in
the presence of NaH resulted in the allyl ethers 3 in 52-61% yield. The allyl ethers were treated
with sec-BuLi converting them to their corresponding lithioalkoxyallyl intermediates, which
were transmetallated using CuI in DMS. These organocopper reagents improved the coupling
efficiency with methyl vinyl ketone in the presence of trimethylsilyl chloride as previously
described by Berrien et al.,38 without the detectable formation of α-alkylation sideproducts.
Deprotection of TMS with NaHCO3 in MeOH produced the PIVE linkers (4) in 55-79% yield.
Overall yields for the synthesis of F-PIVE, H-PIVE, Me-PIVE, and MeO-PIVE from their
corresponding benzaldehyde precursors were 22.7%, 30.8%, 23.2%, and 23.6%.
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Ketones 4 then were converted to primary amines via reductive animation in 62-83% yields
(Scheme 3) to produce intermediates that enable facile incorporation into a wide variety of pH-
sensitive constructs. To generate the target series of pH-sensitive mPEG-PIVE-Lipids,
Intermediates 5 were treated with the 4-nitrophenylcarbonate-activated 1,3-dioctadecyl-rac-
glycerol intermediate, BOP-PNP, in DCM at 22 °C to give 6, followed by TBDMS deprotection to
give alcohols 7. Treatment of this intermediate with 4-nitrophenyl chloroformate in DCM at 22
°C for 18 h gave activated carbonate 8 that was subsequently coupled with mPEG2000-NH2 for
3 d at 22 °C to afford the desired mPEG-PIVE-Lipids 9 in 71-91 % yield.
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Hydrolysis of mPEG-PIVE-Lipid Conjugates. The hydrolysis kinetics of mPEG-PIVE-Lipid
derivatives were evaluated as a function of pH at 37 °C via HPLC-CCAD by monitoring both the
disappearance of the starting materials and the appearance of the hydrolysis product, 1-
methoxypoly(ethylene glycol), MW2000 (mPEG-OH). The extent of lipid hydrolysis was
determined by integrating the areas under the chromatographic peaks for mPEG-PIVE-Lipid and
mPEG-OH. Under all hydrolysis conditions, a decrease in mPEG-PIVE-lipid peak area was
accompanied by a proportional increase in the peak area of the degradation product, mPEG-OH,
which was verified by injection of a commercial standard.
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The HPLC data (Figure 2) indicated that mPEG-PIVE-lipid hydrolysis rates are strongly pH-
dependent. Substituent effects on mPEG-PIVE-Lipid hydrolysis rates were also compared for
electron-donating (4’-methyl, 4’-methoxy) and electron-withdrawing (4’-fluoro) substituents on
the phenyl ring relative to the unsubstituted PIVE (Table 1). The hydrolysis of PEG-[H-PIVE]-DOG
after 2 h was 19 % at pH 4.5 with t1/2 = 5.46 h, whereas negligible hydrolysis was observed after
3 d at pH 7.4. The hydrolysis rate increased slightly for mPEG-[Me-PIVE]-DOG (24% hydrolysis
after 2 h at pH 4.5; t1/2 = 4.04 h), but much more for mPEG-[MeO-PIVE]-DOG (42% hydrolysis
after 2 h at pH 4.5; t1/2 = 2.64 h). Conversely, the hydrolysis rate of the electron deficient PEG-
[F-PIVE]-DOG congener was significantly slower at 37 °C (16 % hydrolysis after 2 h at pH 4.5; t1/2
= 7.5 h). These results are consistent with the hypothesis that the rate of PIVE hydrolysis can be
influenced by varying the electronics of the α-phenyl ring.
Liposome Preparation and Analysis of Acid-Triggered Calcein Release from mPEG-PIVE-
Lipid:DOPE Dispersions. Liposomes were prepared via the thin film extrusion method using
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mPEG-PIVE-lipid:DOPE ratios of 5:95 and 10:90. These dispersions had average diameters of
~170 nm as measured by dynamic light scattering and were stable for at least one week.
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Content release rates of mPEG-PIVE-Lipid:DOPE liposomes as a function of pH were
conducted by using a calcein fluorescence dequenching assay. As calcein encapsulated at self-
quenching concentration leaks into the extraliposomal solution, fluorescence dequenching
occurs to produce an increase in calcein fluorescence.35 Liposomes containing calcein (50 mM)
were incubated in buffer solutions of either pH 3.5, 4.5, or 7.4 at 37 °C and the time-dependent
change in calcein fluorescence monitored in HEPES buffer at 518 nm. The calcein release
percentage at each time point was calculated as a ratio versus 100% release as determined by
addition of Triton X-100 to the liposome sample.
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Figure 3 shows the calcein release kinetics at 37 °C of 10:90 mPEG-[H-PIVE]-DOG:DOPE
liposomes at pH 4.5 and 7.4, with observed release rates of 36% at pH 4.5 after 12 h and less
than 10% over 24 h at pH 7.4. These data show that faster calcein release can be achieved at
lower pH values; we infer from these results that the rate of mPEG-PIVE-lipid hydrolysis
controls the rate of calcein release. Consistent with this interpretation is the observation that
increased rates of calcein release occur under acidic conditions when the membrane
composition of mPEG-[H-PIVE]-DOG is reduced from 10 to 5 mol% (Supplemental Information).
Calcein release rates also were found to depend on the electronic properties of the PIVE
conjugate. Liposomes composed of 10:90 mPEG-[Me-PIVE]-DOG:DOPE produced 42 % calcein
release at pH 4.5 within 24 h at 37 °C, whereas mPEG-[MeO-PIVE]-DOG-containing DOPE
liposomes at the same molar ratio liberated 86% of their calcein cargo under the same
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conditions. Notably slower was the 10:90 PEG-[F-PIVE]-DOG:DOPE liposome dispersion, that
released only 24 % of the encapsulated calcein within 12 h at pH 4.5 (37 °C). All mPEG-PIVE-
Lipid:DOPE liposomes dispersions were stable (i.e., < 10% release over 2 d at pH 7.4) under
normal physiological conditions. These results parallel the findings of the mPEG-PIVE-Lipid
hydrolysis studies in that the electronic character of the 4’-substituent on the phenyl group also
produces a marked influence on calcein release rates, via modulation of the PIVE hydrolysis rate
and accompanying rate of liposome dePEGylation.
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The structural consequences of PIVE linker hydrolysis on mPEG-PIVE-Lipid:DOPE liposomes
was evaluated using cryoTEM. Dispersions comprised of 10:90 mPEG-[H-PIVE]-DOG:DOPE were
stable liposomes at pH 7.4, appearing as spherical vesicle-like structures; however, after
acidification to pH 4.5, dense collapsed structures and small vesicle fragments were observed.
We infer from these results that the individual mPEG-PIVE-BO:DOPE liposomes had collapsed
into small HII phase lipidic structures upon exposure to pH 4.5, due to a Lα - HII phase transition
that is induced by acid-catalyzed dePEGylation of the mPEG-PIVE-Lipid:DOPE liposome
dispersion. We infer from these observations that hydrolyzed mPEG may be weakly adsorbed
onto the small HII phase clusters, thus preventing the contact and coalescence to form larger HII
phase aggregates, in a manner similar to that reported for hydrolyzed mPEG-vinyl ether-
cholesterol:DOPE dispersions at low lipid concentrations.26
CONCLUSIONS
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A new class of heterobifunctional reagents with tunable acid-sensitivity that can be used to
couple two different functional components in a drug delivery formulation been developed.
These compounds are based on a phenyl substituted vinyl ether (PIVE) motif that can provide
rational control over the design of pH-sensitive drug delivery systems based on the electronic
effects exerted on the phenyl substituent(s) of the PIVE using DFT calculations. A short, facile
synthetic pathway was developed for this new family of vinyl ether derivatives. Degradative
analysis showed that these linkers hydrolyzed under acidic conditions while remaining stable at
pH 7.4. As anticipated by the DFT calculations, the rates of acid-catalyzed hydrolysis were
dependent on the electron donating or withdrawing character of the PIVE substituents. Taken
together, these results suggest that it is possible to design pH-responsive delivery vehicles on
an a priori basis to provide a level of functional control that is difficult to achieve with standard
formulation methodology. These results also suggest that PIVE linkers can be readily and
reliably incorporated into a variety of materials to enable more precisely controllable acid-
triggered drug delivery.
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ACKNOWLEDGMENTS
We gratefully acknowledge financial support by National Institutes of Health (GM087016).
We would like to thank Valorie Bowman (Purdue University, Department of Biological Sciences)
for her assistance in the collection of cryoTEM images of the liposome dispersions and the
Purdue University Center for Cancer Research Interdepartmental NMR Facilities and Campus-
Wide Mass Spectrometry Center (supported by NCI CCSG CA23168 to Purdue University Center
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for Cancer Research). We also thank Prof. Shin-ichi Fujiwara for fruitful discussions and initial
assistance with the proton affinity calculations.
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Supporting Information. DFT calculations, detailed synthetic procedures of PIVE and mPEG-
1
PIVE-Lipid conjugates, H NMR and 13C NMR spectra, and calcein release kinetics from 5:95
mPEG-PIVE-Lipid:DOPE liposomes. This material is available free of charge via the Internet at
http://pubs.acs.org.
Corresponding Author
davethom@purdue.edu
+ These authors contributed equally to this work.
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Table 1. Proton affinities determined by
DFT (CC-PVTZ) calculations and measured
intrinsic second order hydrolysis rate
constants for a related series of α-phenyl
vinyl ether (PIVE) derivatives.
Measured k2
ΔHP.A.
(kcal/m)
Substitution
(M-1s-1)
R1,3,5 = OMe; R2,4 = H
R1,3,5 = CF3; R2,4 = H
R3 = OMe; R1,2,4,5 = H
R2,4 = OMe; R1,3,5 = H
R3 = Me; R1,2,4,5 = H
R3 = H; R1,2,4,5 = H
R3 = F; R1,2,4,5 = H
R3 = CF3; R1,2,4,5 = H
R2,4 = CF3; R1,3,5 = H
-249.2
-243.3
-235.7
-233.9
-233.7
-230.1
-227.9
-222.6
-217.2
n.d.
n.d.
2.8
n.d.
1.4
1.0
0.68
n.d.
n.d.
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Scheme 1. PIVE hydrolysis mechanism
under acidic conditions in water. A – E:
Electron
donating
or
withdrawing
substituents.
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Scheme 2. General synthesis pathway for
the preparation of heterobifunctional PIVE
coupling reagents.
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Scheme 3. Synthesis pathway for pH-
sensitive mPEG-PIVE-Lipid conjugates from
heterobifunctional PIVE reagents.
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Figure 1. Schematic of acid-catalyzed
DePEGylative triggering from PEG-PIVE-
Lipid:DOPE liposomes through lamellar (Lα)
to hexagonal (HII) phase transition.
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mPEG[MeO-PIVE]-DOG
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mPEG[Me-PIVE]-DOG
mPEG[H-PIVE]-DOG
mPEG[F-PIVE]-DOG
Figure 2. Hydrolysis rates of mPEG-PIVE-
Lipids at pH 4.5 and pH 7.4, 37 °C. Aliquots
were taken at each time point and the
degree of hydrolysis was analyzed by HPLC-
CCAD.
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10:90 mPEG[MeO-PIVE]-DOG:DOPE
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10:90 mPEG[Me-PIVE]-DOG:DOPE
10:90 mPEG[H-PIVE]-DOG:DOPE
10:90 mPEG[F-PIVE]-DOG:DOPE
Figure 3. Calcein release kinetics from
10:90 mPEG-PIVE-Lipid:DOPE liposomes at
pH 4.5 and pH 7.4 at 37 °C. Aliquots were
taken at each time point and the
fluorescence (λex = 490 nm, λem = 518 nm)
was then measured before and after the
addition of 2 drops 10% Triton X-100.
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Figure 4. Cryogenic transmission electron
microscopy images of 10:90 PEG-[H-PIVE]-
DOG:DOPE liposomes before (A) and after
(B) 24 h exposure at pH 4.5. Liposomes had
converted to small dark fragments after
dePEGylation at pH 4.5.
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