A biocompatible oxidation-triggered carrier polymer with potential in therapeutics

Article abstract of DOI:10.1021/ja110468v

Dextran, a water-soluble, biocompatible polymer of glucose, was modified at its hydroxyls with arylboronic esters to make it soluble in common organic solvents, allowing for the facile preparation of oxidation-sensitive dextran (Oxi-DEX) carrier micropart

Full text of DOI:10.1021/ja110468v

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pubs.acs.org/JACS
A Biocompatible Oxidation-Triggered Carrier Polymer with
Potential in Therapeutics
Kyle E. Broaders, Sirisha Grandhe, and Jean M. J. Fr ꢀe chet*
Department of Chemistry, University of California, Berkeley, California 94720-1460, United States
S Supporting Information
b
Scheme 1. Synthesis and Degradation of Oxi-DEX
ABSTRACT: Dextran, a water-soluble, biocompatible poly-
mer of glucose, was modified at its hydroxyls with arylboronic
esters to make it soluble in common organic solvents,
allowing for the facile preparation of oxidation-sensitive
dextran (Oxi-DEX) carrier microparticles. These particles
were found to release their payload with a half-life of 36 min
at 1 mM H O , which can be compared with a half-life of
2
2
greater than 1 week in the absence of H O . When used in
2
2
a model vaccine application, Oxi-DEX particles loaded with
þ
ovalbumin (OVA) increased the presentation to CD8 T-cells
2
7-fold relative to OVA encapsulated in a classical vehicle
or rely on superoxide, a powerful but extremely short-lived cellular
oxidant.
not sensitive to oxidation. No presentation was observed
from cells incubated with unencapsulated OVA. Additionally,
Oxi-DEX was found to be nontoxic in preliminary in vitro
cytotoxicity assays. Because it is easy to prepare, sensitive to
biological oxidation, and biocompatible, this material may
represent an attractive new platform for selective delivery
applications.
11
Recently, significant advances in sensing oxidative stress have
been made by taking advantage of specific chemistry between
12,13
14
cellular oxidants such as H O2
or hypochlorous acid.
2
Here we demonstrate a biocompatible platform for delivery that
uses one such reaction to target delivery of therapeutic agents. To
accomplish this, we employed a solubility switching mechanism
in which a biocompatible, water-soluble polymer already approved
for some medical applications was reversibly modified to make it
insoluble in water but soluble in organic solvents, thus enabling
payload encapsulation. Particles made from the modified polymer
could then be returned to their original water-soluble state under
the specific conditions that reverse the original modification, with
concurrent release of their payload. We chose dextran, a poly-
saccharide consisting of R(1f6)-linked glucose repeat units, as the
polymer because it is biocompatible, biodegradable, widely avail-
able, and easily modified. We chose arylboronic esters as the
triggering groups because of their facile H O -mediated degrada-
ydrophobic biodegradable polymers such as polyortho-
1
2
3
H
esters, polyesters, and polyanhydrides have found
wide use in the biomedical field as sutures and scaffolds as well as
carriers for vaccine applications, gene delivery, and chemother-
apeutic agents. Their success is in part due to the fact that they
are degradable under biological conditions, making them both
biocompatible and resorbable. This degradation typically occurs
over the course of several months via surface erosion and
4
hydrolysis of the polymer backbone.
2
2
However, for many delivery applications, it is often desirable
to release an encapsulated payload rapidly and selectively by
taking advantage of a unique chemical environment. While many
tion at physiological pH and temperature.
Dextran was modified at the hydroxyls using imidazoyl
carbamate 1 (Scheme 1). Following attachment of some bor-
onate esters, the modified dextran could be precipitated into
water. Once isolated and lyophilized, the new oxidation-sensitive
dextran (Oxi-DEX) possessed only limited solubility in organic
solvents, including dimethyl sulfoxide, from which it was initially
precipitated. We hypothesized that this limited solubility was due
to transient cross-linking between polymer strands through
exchange of the boronic esters with remaining 1,2-diols on the
backbone, which took place upon concentration. The addition
of ∼10% MeOH to break these linkages restored the solubility of
Oxi-DEX in standard organic solvents such as dichloromethane,
N,N-dimethylformamide, tetrahydrofuran, and acetone. Oxi-DEX
is only sparingly soluble in MeOH, supporting the hypothesis that
5
6
carrier systems using acid- or reduction-mediated release have
been developed, relatively few systems that exploit biologically
relevant oxidative conditions for cargo release have been ex-
plored. Reactive oxygen species have been implicated in reperfu-
sion injury following cardiac arrest and are also heavily produced
in the phagosomes of antigen-presenting cells (APCs), which are
7
,8
critical initiators of the adaptive immune response. Recent
reports have indicated that the most effective APCs, dendritic
cells (DCs), may have phagosomes that are much more oxidizing
9
than they are acidic, having H O concentrations of up to 1 mM.
2
2
Despite these potential areas of use, very few drug delivery
systems that are sensitive to biologically relevant oxidative
conditions have been designed. At this time, the most promising
such systems may have either been tested only at H O con-
Received: November 21, 2010
Published: December 20, 2010
2
2
10
centrations far higher than would be encountered biologically
r 2010 American Chemical Society
756
dx.doi.org/10.1021/ja110468v
|J. Am. Chem. Soc. 2011, 133, 756–758

Journal of the American Chemical Society
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Figure 1. Characterization of particles by (a) SEM and (b) DLS con-
firmed that particles made from Oxi-DEX were spheroidal with sizes
of ∼100 nm.
Figure 2. (a) Degradation of Oxi-DEX microparticles with and without
1
mM H O as measured by loss of scattering at 550 nm. (b) Appearance
2 2
of NMR signals from degradation byproducts of Oxi-DEX microparti-
cles incubated in 1 mM D
the solubilization is due to the methanolysis of the boronic ester
cross-links. Although this yields a mix of pinacol and dimethoxy
boronic esters, the desired oxidative degradation reaction should
occur in both cases.
2 2
O .
Scheme 2. Proposed Pathway of Particle Uptake, Degrada-
tion, and Presentation
Particles could be prepared from Oxi-DEX using standard
15
emulsion techniques. Scanning electron microscopy (SEM)
micrographs revealed that these particles were roughly spherical
with sizes on the order of 100-200 nm (Figure 1a). This size
distribution was corroborated in bulk suspension by dynamic
light scattering (DLS) measurements in water, which indicated
that the particles had an average diameter of 100 nm. Chicken
egg albumin (OVA) was encapsulated in Oxi-DEX particles as
a model hydrophilic biomacromolecular payload. Analysis of
degraded particle suspensions using fluorescamine revealed that
the protein loading was 1.6 ( 0.1 wt %.
oxidation of the boronic ester is rate-limiting for the degradation
of the particles. Alternatively, incomplete aromatic group re-
moval via the quinone methide rearrangement could also be
sufficient to resolublize the degrading polymer. It should be
noted that no pinacol or pinacol borate was observed over this
At physiologically relevant neutral aqueous H O concentra-
2
2
tions, the arylboronic esters are expected to be oxidized to
1
6
phenols, which should then undergo a quinone methide
rearrangement, unmasking the hydroxyl groups of dextran. The
complete degradation of Oxi-DEX should result in the release of
pinacol borate, dextran, and p-quinone methide. The p-quinone
methide should then be trapped by water to form p-hydroxy-
time in the absence of D O .
2 2
We have previously shown that acid-sensitive particles en-
hance protein-based vaccine efficacy in cancer treatment by
17
þ
methylphenol (HMP). To confirm degradation, particles were
suspended in pH 7.4 phosphate buffer with or without 1 mM
H O . The degradation of particles at 20 °C was observed by
enhancing MHC class I presentation and CD8 T-cell activ-
18
ation. We wanted to test whether oxidation was also an effec-
tive method of increasing MHC I presentation from DCs
(Scheme 2). To assess the biocompatibility of Oxi-DEX particles,
we compared them to particles prepared from poly(lactic-co-
glycolic acid) (PLGA), an FDA-approved and widely medically
used polymer. In in vitro cytotoxicity assays, we found no
significant difference in toxicity between the two materials in
both HeLa human cervical epithelial cells and RAW 264.7
murine macrophages (Figures S2 and S3). PLGA has also been
2
2
measuring light scattering over time (Figure 2a). The suspen-
sions became completely transparent within 2 h in the pre-
sence of H O , while peroxide-free buffer led to only a slight
2
2
decrease in scattering over 6 h due to particle settling. The
degradation was found to follow first-order kinetics with a
half-life of 36 ( 1 min.
To investigate how the degradation of the polymer relates to
the particle dissolution, NMR spectroscopy was employed.
Particles were suspended in 1 mM D O at pD 7.4, and the
1
9
successfully used in model microparticulate vaccines. In order
to assess the feasibility of using Oxi-DEX-based materials for
vaccine applications, OVA-loaded Oxi-DEX particles, OVA-
loaded PLGA particles, or free OVA was incubated with DC
2.4 murine dendritic cells. After 6 h of incubation, Oxi-DEX
particles led to robust MHC class I presentation of the OVA-
2
2
appearance of dextran and other polymer degradation peaks was
observed (Figure 2b and Figure S1 in the Supporting In-
formation). Dextran slowly became soluble at roughly the same
rate as HMP. The half-life of appearance of each of these
molecules was roughly 4 h. On the basis of the relative quantities
after degradation, Oxi-DEX contained 1.15 modifications for
each anhydroglucose unit. The final quantity of pinacol boronate
indicated that 55% of the pinacol boronic ester was removed
from the polymer prior to particle formation. This could occur
via a combination of hydrolysis on washing, competition from
the diols on dextran, and methanolysis upon resolubilization in
organic solvent. Interestingly, pinacol borate was released from
the particles at a relatively much higher rate than the other
degradation byproducts, with a half-life of ∼0.5 h. This is similar
to the rate of dissolution of particles, which may indicate that
þ
0,21
derived CD8 T-cell epitope, SIINFEKL, as measured by the
2
B3Z assay
(Figure 3). In contrast, no presentation was
observed from cells incubated with free OVA at matching protein
concentrations. Additionally, Oxi-DEX particles outperformed
similarly loaded PLGA particles by over 27-fold. This drastic
increase in presentation indicates that these particles may be
promising materials for vaccines against tumors and certain
viruses where MHC I presentation is crucial for the activation
þ
and proliferation of CD8 T-cells.
In conclusion, we have described the preparation of a bio-
compatible modified biopolymer carrier that selectively releases
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Journal of the American Chemical Society
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8
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2
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2 2
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’
ASSOCIATED CONTENT
(
3
S
Supporting Information. Figures S1-S3, experimental
b
methods, general procedures, and materials. This material is
available free of charge via the Internet at http://pubs.acs.org.
’
AUTHOR INFORMATION
Corresponding Author
frechet@berkeley.edu
’
ACKNOWLEDGMENT
We thank the various Fr ꢀe chet Gift Funds and the donors of the
Henry Rapoport Chair of Organic Chemistry at UC Berkeley for
the support of this research and Ann Fisher in the MCB Cell
Culture Facility for her expertise and help.
’
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