Photodegradable macromers and hydrogels for live cell encapsulation and release

Article abstract of DOI:10.1021/ja305280w

Hydrogel scaffolds are commonly used as 3D carriers for cells because their properties can be tailored to match natural extracellular matrix. Hydrogels may be used in tissue engineering and regenerative medicine to deliver therapeutic cells to injured or diseased tissue through controlled degradation. Hydrolysis and enzymolysis are the two most common mechanisms employed for hydrogel degradation, but neither allows sequential or staged release of cells. In contrast, photodegradation allows external real-time spatial and temporal control over hydrogel degradation, and allows for staged and sequential release of cells. We synthesized and characterized a series of macromers incorporating photodegradbale ortho-nitrobenzyl (o-NB) groups in the macromer backbone. We formed hydrogels from these macromers via redox polymerization and quantified the apparent rate constants of degradation (k_app) of each via photorheology at 370 nm, 10 mW/cm². Decreasing the number of aryl ethers on the o-NB group increases k_app, and changing the functionality from primary to seconday at the benzylic site dramatically increases k_app. Human mesenchymal stem cells (hMSCs) survive encapsulation in the hydrogels (90% viability postencapsulation). By exploiting the differences in reactivity of two different o-NB linkers, we quantitatively demonstrate the biased release of one stem cell population (green-fluoroescent protein expressing hMSCs) over another (red-fluorescent protein expressing hMSCs).

Full text of DOI:10.1021/ja305280w

Article
pubs.acs.org/JACS
Photodegradable Macromers and Hydrogels for Live Cell
Encapsulation and Release
Donald R. Griffin and Andrea M. Kasko*
Department of Bioengineering, University of California, Los Angeles, 410 Westwood Plaza, 5121 Eng V, Los Angeles, California
90095, United States
S
* Supporting Information
ABSTRACT: Hydrogel scaffolds are commonly used as 3D
carriers for cells because their properties can be tailored to
match natural extracellular matrix. Hydrogels may be used in
tissue engineering and regenerative medicine to deliver
therapeutic cells to injured or diseased tissue through
controlled degradation. Hydrolysis and enzymolysis are the
two most common mechanisms employed for hydrogel
degradation, but neither allows sequential or staged release
of cells. In contrast, photodegradation allows external real-time
spatial and temporal control over hydrogel degradation, and
allows for staged and sequential release of cells. We synthesized and characterized a series of macromers incorporating
photodegradbale ortho-nitrobenzyl (o-NB) groups in the macromer backbone. We formed hydrogels from these macromers via
redox polymerization and quantified the apparent rate constants of degradation (kapp) of each via photorheology at 370 nm, 10
mW/cm2. Decreasing the number of aryl ethers on the o-NB group increases kapp, and changing the functionality from primary to
seconday at the benzylic site dramatically increases kapp. Human mesenchymal stem cells (hMSCs) survive encapsulation in the
hydrogels (90% viability postencapsulation). By exploiting the differences in reactivity of two different o-NB linkers, we
quantitatively demonstrate the biased release of one stem cell population (green-fluoroescent protein expressing hMSCs) over
another (red-fluorescent protein expressing hMSCs).
reasonable (10 mW/cm2) intensity. This wavelength and dose
INTRODUCTION
■
of light has been shown to be compatible with live-cell
Hydrogels are commonly used as 3D carriers for cells, due to
encapsulation.6 However, long-wave UV may still interact with
their high water content, tunable mechanical properties, and the
and alter structures or chemicals within the cell7 and therefore
rapid diffusivity of nutrients through the network.1 Erodable
hydrogels are important in tissue engineering for creating
complex cell niches, and in therapeutics, where degradation
it is best to minimize cell exposure to it. Attenuation is also
significant due to the high molar absorptivity of this o-NB
linker. Finally, with only a single photolyzable linker, there is no
allows the predictable release of encapsulated cells and tethered
way to independently execute two separate events, such as the
therapeutics. Hydrolysis and enzymolysis are the two most
common mechanisms employed for hydrogel degradation. Both
mechanisms are effective for sustained hydrogel degradation;
however, the rate of degradation cannot be adjusted or arrested
after the hydrogel is fabricated, nor is degradation spatially
controlled. In contrast to enzymolyis and hydrolysis, photo-
degradation allows external real-time spatial and temporal
control over hydrogel properties. Kloxin et al. incorporated 2-
sequential release of different therapeutics or cells. Therefore,
new or additional o-NB groups with lower molar absorptivities
combined with more efficient degradation may improve cell
and tissue compatibility, and multiple photodegradable groups
may also allow multistage processes.
o-NB groups are widely used as photocages in the presence
of live cells,8−12 and also as degradable linkages in polymer
materials.13 Several different o-NB groups have been
methoxy-5-nitro-4-(1-hydroxyethyl)phenoxybutanoate (an
ortho-nitrobenzyl (o-NB) linker) into the backbone of a
reported,14−16 and small modifications to the structure of the
o-NB linker have a significant impact on the photolysis rate.14
The benzylic position (photodegradation site) of o-NB groups
can be primary or secondary (Chart 1a, R1 = −H or R1 =
−CH3). Additionally, the o-NB group may contain no other
aromatic substituents (R2 = R3 = −H), one or two aryl ethers,
or other substituents. Each of these structural changes can affect
poly(ethylene glycol) (PEG) macromer.2 Physical properties
of hydrogels formed from this macromer are temporally and
spatially controllable via light exposure (single and two-
photon),3,4 and photodegradation is compatible with live
cells.2,5 While photodegradation allows dynamic control of
hydrogel properties, there are practical limitations to the single
o-NB linker reported. The 2-methoxy-5-nitro-4-(1-
bromoethyl)phenoxybutanoate-based macromers degrade over
several minutes or longer when exposed to 365 nm light with
Received: May 31, 2012
Published: July 5, 2012
© 2012 American Chemical Society
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with 70% nitric acid alone.20 Adding sulfuric acid to the
reaction mixture catalyzes the substitution, but results in a
mixture of nitrated products. To properly guide nitration to the
ortho position, we react the phenol with oxalyl chloride to form
a dimer (Scheme 2) and then nitrate in a mixture of nitric and
sulfuric acid.21 This reaction is temperature sensitive; for
example, lowering the reaction temperature from −10 °C to
−20 °C results in a significant increase in yield (∼20% vs 85%)
and eliminates side-product formation. Once we obtain the
nitrated product, the rest of the reaction scheme follows the
above-described procedure.
Chart 1. (a) Structural Variables in o-NB Linkers; (b) Series
of o-NB Linkers Designed to Have Varying Degradation
Rates
the degradation rate. For example, the half-life of an o-NB
linker with a primary benzylic group is approximately 25 times
that of one with a secondary benzylic group (X = −NHR,
buffer, pH = 7).13 When X = RSO3−, the apparent rate
constant of photodegradation (kapp) is nearly 40 times higher
for a secondary benzylic group compared to a primary group
(solvent: CDCl3).17 Increasing the number of aryl ethers from
one (R1 = −CO2H, X = −SR, R2 = −OR, R3 = −H) to two (R2
= −OR, R3 = −OR) decreases the quantum yield of the o-NB
group at 365 nm by 30%.15
Scheme 2. Synthesis of 4-(3-(Hydroxymethyl)-4-
nitrophenoxy) Butanoic Acid and 4-(3-(1-Hydroxyethyl)-4-
nitrophenoxy) Butanoic Acid
Although general trends are clear, it is difficult to determine
which structural changes will have the greatest effect on the rate
of degradationprimary versus secondary benzylic group, or
the number of aryl ethers. In order to better understand the
effect of structural changes on the degradation of o-NB groups,
we developed a series of photodegradable o-NB based linkers
with varying functionality at the benzylic position (primary or
secondary) and varying number of aryl ether groups (Chart
1b). Each o-NB group contains two reactive handles, a benzylic
alcohol, and a carboxylic acid, to allow for selective conjugation
of each site to either a polymerizable group or therapeutic agent
(benzylic position), or to a polymer chain or macromer
(carboxylic acid).
To produce an o-NB linker with no aryl ether moieties, we
synthesized both 1,3-dihydroxymethyl-2-nitrobenzene (E) and
3-hydroxymethyl-2-nitrobenzoic acid. The synthesis of 1,3-
dihydroxymethyl-2-nitrobenzene (E) has already been reported
in the literature.22,23 To synthesize 3-hydroxymethyl-2-nitro-
benzoic acid, we first oxidize 2-nitro-m-xylene using potassium
permanganate to obtain 2-nitro-1,3-benzene dicarboxylic acid.
We then partially esterify the dicarboxylic acid in refluxing
ethanol overnight and isolate 3-(ethoxycarbonyl)-2-nitroben-
zoic acid by flash column chromatography (Scheme 3).
Although the yield of the partial esterification reaction is low
(10%), we are able to recover a large portion of the starting
material (∼70%). Following purification, we selectively reduce
the carboxylic acid using 1 M borane in tetrahydrofuran to
produce a benzylic alcohol, and hydrolyze the ester to obtain
the heterobifunctional linker, 3-hydroxymethyl-2-nitrobenzoic
acid.
RESULTS AND DISCUSSION
■
We synthesized 4-(4-(hydroxymethyl)-2-methoxy-5-nitrophe-
noxy) butanoic acid (A) and 4-(4-(1-hydroxyethyl)-2-methoxy-
5-nitrophenoxy) butanoic acid (B) according to a modified
literature protocol (Scheme 1).2,18 First, we esterify the phenol
of either acetovanillone (R = −CH3) or vanillin (R = −H) with
ethyl-4-bromobutyrate under basic conditions. We use 70%
nitric acid to nitrate the ring ortho to the aldehyde/ketone. We
then hydrolyze the ester using aqueous trifluoracetic acid and
reduce the aldehyde/ketone using sodium borohydride. We
reversed the order of hydrolysis and reduction compared to the
reported procedure, which increases the overall yield from
these two steps from ∼36% to ∼80%.19
Scheme 3. Synthesis of 3-Hydroxymethyl-2-nitrobenzoic
Acid, and 1,3-Di(hydroxymethyl)-2-nitrobenzene (E)
We initially attempted to synthesize 4-(3-(hydroxymethyl)-4-
nitrophenoxy) butanoic acid (C) and 4-(3-(1-hydroxyethyl)-4-
nitrophenoxy) butanoic acid (D) using the same synthetic
route as above; however, the single aryl ether does not
sufficiently activate the ring toward electrophilic substitution
We measured the absorbance spectra of each linker and its
degradation product via UV/vis spectroscopy (Supporting
Information Figure S1). The molar absorptivities (ε) of each
linker and its degradation product at 365, 405, and 436 nm
(peak output wavelengths of a mercury vapor lamp) are
summarized in Table 1. Increasing the number of aryl ethers on
an o-NB linker results in a red-shift in the absorption spectrum,
as does going from a primary benzyl alcohol to a secondary
(although this effect is much less pronounced). The molar
absorptivity of each molecule decreases with increasing
wavelength. The molar absorptivity of the degraded products
Scheme 1. Synthesis of 4-(4-(Hydroxymethyl)-2-methoxy-5-
nitrophenoxy)butanoic Acid and 4-(4-(1-Hydroxyethyl)-2-
methoxy-5-nitrophenoxy)butanoic Acid
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Table 1. Molar Absorptivity of o-NB Linkers A−E and Degraded Products at λ = 365, 405, and 436 nm and kapp at λ = 370 nm
o-NB group
ε365 (M−1 cm−1
)
ε405 (M−1 cm−1
)
ε436 (M−1 cm−1
)
kapp/I0 × 104 (cm2/(mW·s))
τ, min
A
3420 165
1925 22
3500 57
2637 32
1975 299
2201 26
2509 93
4061 234
146.7 3.6
507.7 6.8
729 42
55.6 3.8
0.26 0.014
65 3.5
A degraded
1269
2
692
77.8 2.6
685
2
B
845 16
3.3 0.022
0.42 0.035
8.3 0.14
2.6 0.24
5.0 0.03
40 3.3
B degraded
1610 31
86.2 3.8
815.3 18.6
9
C
4.15 0.21
515.7 6.7
2.65 2.08
358 16
C degraded
D
161
6
2.0 0.03
6.5 0.6
D degraded
E
1608 59
4.70 0.26
166.7 6.1
0.35 0.21
84.7 4.2
E degraded
of each o-NB linker is generally higher than the undegraded
molecules, except for the degradation products of linkers A and
B at 365 nm, which exhibit lower molar absorptivity than
(undegraded) A and B.
1,3-di(hydroxymethyl)-2-nitrobenzene with acryloyl chloride
(limiting reagent). We used the remaining benzyl alcohol to
ring-open succinic anhydride, yielding a carboxylic acid, which
we converted to the acid chloride and coupled to PEG as
described for linkers A−D above (Supporting Information
Scheme S3).
Chart 2. PEG-Based Macromers Incorporating
We copolymerized each macromer (Mn ≈ 4500) with PEG-
375 acrylate, using redox initiation ammonium persulfate/
N,N,N′,N′-tetramethylethylene diamine (APS/TEMED) to
form a hydrogel (50 μm thickness) between the plates of a
rheometer modified to allow in situ light exposure, and
measured the decrease of the normalized elastic portion
(G′∼G) of the complex shear modulus of each hydrogel as a
function of exposure time at λ = 370 nm (UV-LED, Figure 1a).
Photodegradable o-NB Linkers A−E
Figure 1. Kinetics of photodegradation. (a) Relative decrease in the
elastic portion of the shear modulus (G′) as a fraction of exposure time
for hydrogels formed from macromers containing linkers A−E. (b)
Consumption of the o-NB group as a function of exposure time for
linkers A−E. λ = 370 nm; I0 = 10 mW/cm2.
Figure 1b shows the consumption of photodegradable group as
a function of exposure time; the slope is used to determine kapp
.
Equation 1 gives kapp, where ϕ is quantum yield, ε is molar
absorptivity, I0 is light irradiance (intensity), λ is wavelength,
NA is Avogadro’s number, h is Planck’s constant, and c is the
speed of light.
ϕελI0(2.303 × 10−6)
kapp
=
We incorporated each linker into a PEG macromer (Chart
2); we first esterified the benzylic alcohol of linkers A−D using
acryloyl chloride and triethylamine in THF. We subsequently
converted the carboxylic acid of the o-NB to an acid chloride
using phosphorus pentachloride (PCl5), which we used to
esterify PEG 4K (Supporting Information Scheme S1−S2).
Although both 3-hydroxymethyl-2-nitrobenzoic acid and 1,3-
dihydroxymethyl-2-nitrobenzene (E) can be used to prepare
the final macromer, the overall yield using linker E is
substantially higher than that obtained using 3-hydroxymeth-
yl-2-nitrobenzoic acid (∼20% versus ∼5% over four steps). To
obtain the macromer incorporating linker E, we monoesterified
N hc
(1)
A
The variation in kapp/I0 with structure is summarized in Table 1.
As expected, decreasing the number of aryl ethers results in an
increase in kapp/I0. Changing the functionality at the benzylic
position from a secondary benzyl ester (B and D) to a primary
benzyl ester (A, C, and E) impacts kapp more significantly.
Linker D, with one aryl ether and a secondary benzyl ester,
exhibits the fastest degradation of the series. After normalizing
to intensity (kapp/I0) at a fixed wavelength (λ), the rate of
photodegradation should depend on the product of the molar
absorptivity at that wavelength and the quantum yield
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(quantum product, ϕε). Very low molar absorptivity at a
particular wavelength implies little to no photodegradation
unless the quantum yield is high. Linkers B and E have similar
kapp (3.3 × 10−3 and 2.6 × 10−3 s−1, respectively) but very
different ε (3500 and 146.7 M−1 cm−1, respectively), indicating
a significant difference in quantum yield for these two
structures.
Our series of photodegradable hydrogels incorporating o-NB
linkers offers several advantages to the previously reported
system utilizing linker B alone. For example, the dose of light
required to degrade the hydrogel may be substantially reduced
by using the faster degrading linker D. Additionally, the depth
of light penetration into the hydrogel increases if linker E is
used, as the molar absorptivity of this linker is an order of
magnitude lower than that of linkers A−D. Greater penetration
of light into the hydrogel allows for bulk erosion, in contrast to
the surface erosion achieved for linkers with higher absorptivity.
Moreover, combinations of these linkers can be used to achieve
degradation in an orthogonal manner. Recently, small-molecule
wavelength-orthogonal systems including surfaces24 and caged
neurotransmitters17 have been reported, but photoresponsive
biomaterials,25 particularly wavelength-orthogonal biomaterials,
are still an emerging research area.
One potential application of photodegradable hydrogels is
the encapsulation and on-demand release of therapeutic cells,
such as stem cells. Sequential cell introduction and growth is
essential in both embryo development26 and proper wound
healing.27,28 The ability to deliver or recruit multiple cell types
in a staged or sequential manner is therefore critical to wound
healing and tissue regeneration, yet remains a major challenge.
Many researchers have reported the combined29 and/or
sequential30,31 delivery of growth factors, which represents an
indirect route to generating the different cells. That is, the
growth factors must arrive at the site and act on progenitor cells
or otherwise recruit host cells at an appropriate time to
generate the parenchymal cells. There are some limitations to
this approach. For example, growth factors may degrade during
delivery, and/or may act on cells other than their intended
targets in vivo. Delivering cells directly to the injury site is an
alternative, however intravenously delivered cells are associated
with low engraftment rates, limiting the effectiveness of such
procedures. Therefore, there is a need for new cell delivery
therapies.
Light exposure has no apparent effect on hMSC viability, as 91
2% hMSCs were viable postrelease, successfully demonstrat-
ing that this system can be used for on-demand delivery of
therapeutic cells.
While the entrapment and on-demand release of cells has
many applications in tissue engineering and regenerative
medicine, the controlled release of two or more cell populations
in a staged manner may provide synergistic behavior and
therapeutic benefit not induced by the release of a single cell
type or the simultaneous release of multiple cell types.
Exploiting the differences in reactivity of the o-NB groups
should allow biased release of a cell population encapsulated in
one gel over another gel. To demonstrate this, we chose the
two o-NB linkers with the fastest degradation times, B and D,
which is the most challenging system in which to observe bias.
We encapsulated hMSCs expressing red fluorescent protein
(RFP-hMSCs) in hydrogels incorporating macromer B and
green fluorescent protein (GFP-hMSCs) into hydrogels
incorporating macromer D. The hydrogels were fabricated in
direct contact with one another (Figure 2a). The interface
where the two hydrogels meet is directly observable, as is the
partitioning of RFP-hMSCs and GFP-hMSCs (Figure 2b). The
entire construct was exposed to light (λ = 365 nm, I0 = 10
mW/cm2) for three intervals of 10 minutes, and the released
cells collected and counted after each interval (Figure 2c).
GFP-hMSCs are released at a faster rate than RFP-hMSCs,
demonstrating the biased release of one cell population over
another in a system exposed to a single light program. The ratio
of the GFP-hMSC to RFP-hMSC release rates, RGFP/RRFP
=
2.36
0.68, matches well with the ratio of the apparent
degradation rate constants of the two linkers, D and B, kappD
kappB = 2.47.
/
To address this need, researchers have developed tech-
nologies such as erodible hydrogels32 and stem cell patches.33
Despite these advances, no material-based platform for
sequentially delivering stem cells in response to environmental
or external triggers exists. Several groups have reported
techniques for the release of cells from a surface, such as
photoablation of a substrate to release adherent cells.34 Kloxin
et al. reported the migration of encapsulated fibrosarcoma cells
within channels photopatterned into a hydrogel; however, no
cells were released from the gel nor was cell viability
determined.2
To demonstrate the utility of our system to deliver stem cells,
we copolymerized macromer B with PEG-375 acrylate (as
above) in the presence of human mesenchymal stem cells
(hMSCs). We quantified the viability of the encapsulated cells
via a LIVE/DEAD assay; 87 3% cells survive encapsulation.
We exposed the hydrogels to light (λ = 365 nm, I0 = 10 mW/
cm2) for three intervals of 10 minutes, allowing the gels to
equilibrate in media after each interval. We collected the media
after each interval and assessed the viability of the released cells.
Figure 2. Wavelength-biased release of encapsulated cells. (a)
Macromers containing either B (left of interface) or D (right of
interface) were used to encapsulate RFG-expressing hMSCs or GFP-
expressing hMSCs. (b) The interface between the two gels is directly
observable in optical microscopy. (c) The rate of release of GFP cells
from gel containing linker D is faster than the release of RFP-
expressing cells from a gel containing linker B: this is consistent with
ratio of the apparent rate constants of degradation (kappD/kappB = 2.47;
RGFP/RRFP = 2.36 0.68).
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We have synthesized and characterized a series of macromers
incorporating o-NB groups that photolyze over a broad range of
rates. Decreasing the number of aryl ethers on the o-NB group
increases kapp, and changing the functionality at the benzylic site
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ASSOCIATED CONTENT
* Supporting Information
Experimental details for synthesis and characterization of
linkers A−E and their macromers, degradation kinetics, cell
encapsulation and release, reaction schemes for macromer
synthesis, absorbance spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
akasko@ucla.edu
■
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Notes
The authors declare the following competing financial
interest(s): A.K. is a co-inventor on U.S. Patent Application
No. 11/374,471 which includes compounds described in this
report.
ACKNOWLEDGMENTS
■
This work was funded by UCLA HSSEAS Start-up funds and
the National Institutes of Health through the NIH Director’s
New Innovator Award Program, 1-DP2-OD008533. We
gratefully acknowledge J. Huynh, M. Mattson, and Prof. J. A.
Kornfield for access to and assistance with photorheology
experiments. Some of the materials employed in this work were
provided by the Texas A&M Health Science Center College of
Medicine Institute for Regenerative Medicine at Scott & White
through a grant from NCRR of the NIH, Grant No.
P40RR017447.
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