Design of functionalized cyclic peptides through orthogonal click reactions for cell culture and targeting applications

Article abstract of DOI:10.1039/c8cc03218a

An approach for the design of functionalized cyclic peptides is established for use in 3D cell culture and in cell targeting. Sequential orthogonal click reactions, specifically a photoinitiated thiol-ene and strain promoted azide-alkyne cycloaddition, we

Full text of DOI:10.1039/c8cc03218a

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Design of functionalized cyclic peptides through
orthogonal click reactions for cell culture
and targeting applications†
Cite this:DOI: 10.1039/c8cc03218a
Received 27th April 2018,
Accepted 24th May 2018
a
a
a
a
Paige J. LeValley,‡ Elisa M. Ovadia,‡ Christopher A. Bresette, Lisa A. Sawicki,
b
c
ad
Emanual Maverakis, Shi Bai and April M. Kloxin
*
DOI: 10.1039/c8cc03218a
rsc.li/chemcomm
An approach for the design of functionalized cyclic peptides is of functional handles for facile incorporation of cyclized
established for use in 3D cell culture and in cell targeting. Sequen- peptides within materials for biological applications of interest.
tial orthogonal click reactions, specifically a photoinitiated thiol–
Several approaches have been established for the cyclization
ene and strain promoted azide–alkyne cycloaddition, were utilized of the peptides using chemical or enzymatic ligations, as nicely
1
3,14
for peptide cyclization and conjugation relevant for biomaterial and overviewed in recent reviews.
Most commonly, disulfide
biomedical applications, respectively. formation between two cysteines residues or carbodiimide
6
,7
couplings have been utilized. Although these methods are
Receptor binding peptides are widely used within the bio- effective for the formation of cyclized RGD, potential drawbacks
materials and biochemistry communities in the design of of these approaches include instability of disulfide bonds
1
in vitro cell culture models and targeted therapeutic agents.
in biological systems (e.g., reducing microenvironments like
1
5
In particular, the arginine–glycine–aspartate (RGD) peptide that found in tumors) and amide bonds impacting receptor
sequence broadly has been employed for promoting cell–matrix binding due to the loss of the N- and C-terminus, which can be
interactions within two- and three-dimensional (2D and 3D) important in protein binding and downstream signaling
1
6
culture models and for targeting cell surfaces for therapeutic events. Toward expanding the toolbox of chemistries avail-
2
–7
delivery or bioimaging.
The linear RGD sequence binds able for cyclization, on-resin cyclization has been demonstrated
, a , and a using select click reactions including photoinitiated thiol–
amongst others, whereas cyclization of RGD has been ene, thiol–maleimide, and azide–alkyne cycloaddition.
a range of different integrins, including a
v
b
3
b
v 5
5 1
b
2
17
18
19
observed to improve its binding affinity and specificity, espe- Once cyclization is achieved, conjugation of the resulting peptide
cially toward the avb3 integrin, by mimicking aspects of the to material systems of interest also must be considered.
helical structure in which RGD is presented within native Conjugation of cyclized peptides to a variety of systems, including
6
–8
20
20,21
proteins. With this increased specificity to a
v
b
3
, cyclic RGD coupling to therapeutics,
imaging agents,
typically has been achieved using either disulfide
and targeting of bond formation or carbodiimide coupling, where one reaction is
and 2D
3,10
has been used for the selective adhesion of specific cell types surfaces,
to substrates in 2D culture in vitro
therapeutics to tumor cells in vivo, which overexpress a
3
,7,9,10
5
,11
b
v 3
.
used for cyclization and the other for conjugation. While these
As use of cyclic peptides becomes more prevalent, improved methods have proven useful, inherent instability or cytotoxicity of
approaches are needed to allow for (1) stable cyclization of these coupling chemistries limits their applications. Few simple
1
2
RGD, as well as other peptide sequences, and (2) integration biocompatible methods exist for formation of cyclized peptides
and their subsequent conjugation to biomolecules or culture
substrates. We hypothesized that integration of reactive handles
22
for orthogonal click reactions into the peptide sequence design
would enable sequential cyclization and conjugation for investiga-
tions in cell culture and targeting.
Herein, we report a unique approach using orthogonal
click reactions for the synthesis of a cyclic RGD peptide with
a chemically active functional handle that allows integration
within materials systems for 2D and 3D cell culture and cell-
labeling applications. Specifically, photoinitiated thiol–ene click
chemistry was used to cyclize a linear RGD peptide containing an
a
b
c
Department of Chemical and Biomolecular Engineering, University of Delaware,
Newark, DE 19716, USA. E-mail: akloxin@udel.edu
Department of Dermatology, School of Medicine, University of California Davis,
Davis, CA 95616, USA
Department of Chemistry and Biochemistry, University of Delaware, Newark,
DE 19716, USA
d
Department of Material Science and Engineering, University of Delaware, Newark,
DE 19716, USA
†
‡
Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc03218a
These authors contributed equally.
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azide functional handle for subsequent conjugation to materials
of interest by an azide–alkyne cycloaddition. The utility of this
synthetic scheme was demonstrated by the facile incorporation of
the peptide into poly(ethylene glycol) (PEG)-based hydrogels for
3
D cell culture and attachment to a fluorescent dye for cell
targeting. These material systems may prove useful in future
studies to investigate the role of a in cancer progression or
v 3
b
to target diagnostic or therapeutic agents to tumor sites for
improved treatment strategies. Further, the orthogonal click
reaction scheme established here may be easily modified to
cyclize and conjugate various peptide motifs for a range of Fig. 2 Characterization of RGD cyclization. No mass gain was observed
through ESI+ MS with the transition from the (A) linear peptide to the (B)
cyclized peptide. (C) With H NMR, for the linear RGD (pink), the H peaks
applications.
1
1
To synthesize cyclic peptides with an active chemical handle
we combined two orthogonal click reactions: (i) a photoinitated
thiol–ene reaction and (ii) an azide–alkyne cycloaddition.
for the free thiol of the Cys (d = 1.36 ppm) and the alloc (d = 4.45, 5.17,
5.26, and 5.9 ppm) were present (arrows) and, upon cyclization (blue),
disappeared or shifted. (D) With COSY 2D NMR, the alkyl–sulfide bond
The peptide sequence chosen for this demonstration was formed upon cyclization was observed: orange and green lines indicate
the COSY correlations associated with the Cys and the alloc-group
cGRGDdvK(alloc)DRK(Az), which was inspired by a sequence
previously determined to have high binding affinity to a b .
v 3
ÀÀ À
6
attached to the Lys.
D-Cysteine (c) and allyloxycarbonyl (alloc)-protected lysine
(
K(alloc)) were integrated to flank the binding sequence such low dose of long wavelength UV light for peptide cyclization
À2
that a radically-initiated thiol–ene click reaction could be used (20 mW cm
at 365 nm for 10 minutes). Dilute solution
to form the cyclized peptide (Fig. 1A). Additionally, an conditions were used to mitigate oligomer formation. After
azide functionalized lysine (K(Az)) was incorporated at the purification by HPLC, this cyclization method afforded product
C-terminus to impart a functional handle for subsequent con- yields of approximately 70%, producing sufficient peptide for
jugation reactions (Fig. 1B and C).
use in 3D hydrogel culture systems or conjugation to other
The linear cGRGDdvK(alloc)DRK(Az) sequence was success- molecules of interest. Peptide cyclization was confirmed with a
fully synthesized by solid phase peptide synthesis, using an combination of MS and NMR spectroscopy (Fig. 2). With MS, we
automated peptide synthesizer and standard Fmoc chemistry, observed the same molecular weight for the linear peptide
purified by reverse phase high performance liquid chromato- sequence and the cyclic peptide sequence, supporting peptide
graphy (HPLC), and characterized with electrospray ionization cyclization and retention of the azide during the cyclization
(ESI+) mass spectrometry (MS) (Fig. 2A). Initially, the peptide process (Fig. 2A and B). Importantly, no higher molecular
cyclization was attempted on-resin; however, in our hands, this weights were observed, indicating that no peptide oligomers
solid-phase cyclization resulted in yields of product that were were present in the purified product. Next, the conversion
low and not easily scalable for use in the formation or of the thiol and alloc groups after photoinitiated thiol–ene
1
modification of bulk materials. To improve yield and scalability, reaction was confirmed using H NMR (Fig. 2C). Here, we
a solution-phase approach was adopted: linear RGD functiona- observed the loss of proton signals associated with the Cys
lized with a pendant thiol, alloc, and azide (5 mM) was dissolved (d = 1.36, 4.06 ppm) and the Lys-alloc (d = 4.45, 5.17, 5.26, and
in deionized (DI) water with the photoinitiator lithium acylpho- 5.9 ppm) upon cyclization.
sphinate (LAP, 5 mM), and the solution was irradiated with a
To further confirm the structure of the peptide, 2D NMR was
used to observe the alkyl–sulfide bond formed upon successful
execution of the thiol–ene reaction. First, the amide peaks
associated with the individual amino acids along the peptide
backbone were identified using the NOESY cross-peaks of the
1
linear peptide followed by H assignment using a combination
of TOCSY and COSY spectra (Fig. S7–S9, ESI†). Once the amino
acids and their side groups were assigned for the linear
1
peptide, H signals that appeared after the cyclization process
were identified, and COSY was used to identify the proton
correlations relevant to the formation of the alkyl–sulfide bond
(Fig. 2D). Neighboring proton correlations were observed
Fig. 1 Synthesis and use of cyclic RGD. (A) Linear RGD was cyclized using between the alpha proton (d = 3.91 ppm, peak 1) and the beta
a photoinitiated thiol–ene click reaction between the free thiol of the Cys
proton (d = 1.77 ppm and d = 1.65 ppm, peaks 2a and b) for the
(red) and the alloc protecting group of the Lys (blue). An azide functiona-
Cys. The up-field shift observed for the Cys beta protons is
speculated to be caused by the ring strain upon cyclization
and loss of the proton on the thiol upon bond formation.
Similarly, COSY correlations were observed between the three
lized Lys (green) provided a facile handle for incorporating cyclic RGD into
various systems using a SPAAC reaction, including (B) hydrogels for cell
culture and (C) conjugation to a fluorophore for cell labeling applications
(
see Fig. S4 for chemical structures, ESI†).
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ethyl groups associated with the Lys alloc-protecting group. morphology, which is the typical mesenchymal phenotype of these
These peak shifts were identified to be at d = 2.60 ppm (peak 3), TNBC cells (Fig. 3D). We hypothesize that the increased affinity and
d = 1.80 ppm (peak 4), and d = 3.99 (peak 5). No other protons specificity for integrin binding to the cyclic RGD peptide, relative to
24
were observed to interact with these protons, which is expected the linear RGD sequence, promoted a more spread morphology at
owing to the presence of the sulfur and the amide linkages in this early culture time point. In sum, these 2D culture studies
the peptide backbone adjacent to the alkyl protons. Taken supported the successful incorporation of pendant peptides within
1
together, these analyses from H NMR, MS, and 2D NMR hydrogels and the capacity of the cyclic RGD sequence for promoting
support that the thiol–ene reaction was successfully executed cell binding and adhesion after cyclization and immobilization.
to form the desired cyclized product.
Our approach for peptide synthesis and immobilization allows
To demonstrate the utility of having a cyclic peptide with a not only presentation from surfaces but also immobilization within
functional handle, we first incorporated the cyclic azido-RGD hydrogels for 3D cell culture. Indeed, well-defined, 3D culture
into a poly(ethylene glycol) (PEG)-based hydrogel culture system systems are of continued and growing interest for studying breast
using strain promoted azide–alkyne cycloaddition (SPAAC) cancer amongst other disease and regenerative processes. In
chemistry (Fig. 1B). Cell attachment to the cyclized sequence in particular, within the tumor microenvironment, extracellular
2
D culture initially was assessed. Previously, fibroblast, endothelial, matrix interactions are known to be regulated by binding of the
2
3,24
and cancer cells have been shown to have good attachment to
cyclic RGD presented from surfaces in 2D culture.
a
v
b
3
integrin amongst others.
Materials to mimic these
6,7,9
Notably, interactions could enable the development of improved culture
MDA-MB-231 cells, an aggressive metastatic triple negative breast models for studying complex processes, such as the mechanisms of
23
cancer (TNBC) cell line, highly expresses the a b integrin. Here, cancer progression, metastasis, and recurrence. Here, to demon-
v
3
we used these MDA-MB-231 cells as a model cell type to examine strate the relevance of the azido-cyclic peptide for 3D culture, we
attachment of cells to hydrogels incorporating the synthesized encapsulated MDA-MB-231s within hydrogels presenting cyclic
cyclic RGD. MDA-MB-231s were seeded onto hydrogels with no RGD and measured cell viability and proliferation over one week
peptide (negative control), linear RGD (positive control), and cyclic in 3D culture (Fig. 4). A live/dead cytotoxicity assay was used to
RGD (Fig. 3). The hydrogels were gently rinsed with culture measure the viability of cells (Fig. 4A). Initial cell viability was
medium 8 hours after seeding to remove any unadhered cells, observed to be typical for encapsulation within PEG-based hydro-
25
and cells were subsequently fixed at 24 hours in culture to assess gels formed using SPAAC chemistry (viability 78 Æ 4%). After one
initial cell attachment. MDA-MB-231s exhibited uniform attachment week in culture, overall cell viability increased to 87 Æ 5%,
À2
to hydrogel surfaces presenting cyclic RGD (7921 Æ 1433 cells cm ), supporting the potential growth and proliferation of cells in these
which was significantly higher than the few cells attached to the no 3D culture environments. Cell viability and proliferation were
À2
peptide negative control (895 Æ 81 cells cm ) (p-value o 0.05) examined further with measurements of metabolic activity
(
Fig. 3A). As expected, MDA-MB-231s also exhibited good attachment (Fig. 4B) and immunostaining for Ki-67 (Fig. 4C), a nuclear protein
À2
to hydrogels presenting linear RGD (8382 Æ 1169 cells cm ), associated with cell proliferation that is highly expressed in breast
26
statistically similar to the cyclic RGD condition. Cell morphology cancer and proliferating breast cancer cells. Increased metabolic
also was examined by staining for the cytoskeletal protein F-actin activity and consistent expression of Ki-67 were observed over
(red) and cell nuclei (blue) (Fig. 3B–D). We observed a rounded 7 days, further supporting proliferation of cells in these 3D cultures.
morphology of MDA-MB-231s for the few cells attached to the no Overall, these studies have demonstrated high viability, growth,
peptide control and for cells attached to hydrogels presenting linear and proliferation of human cells in 3D hydrogel-based cultures
RGD (Fig. 3B and C). In contrast, MDA-MB-231s seeded onto presenting cyclic RGD. To our knowledge, this is the first demon-
hydrogels presenting cyclic RGD exhibited a spread and elongated stration of culturing cells in three dimensions in the presence of a
Fig. 3 MDA-MB-231 attachment to cyclic RGD hydrogels. (A) Cell attach-
ment to hydrogels presenting cyclic RGD (CYC) and linear RGD (LIN) (positive
control) was significantly higher than without pendant peptide (CTL) (negative
control), demonstrating the capacity of the cyclized RGD to promote cell
binding and adhesion. Representative images shown for (B) no peptide,
Fig. 4 3D Culture of MDA-MB-231s in hydrogels presenting cyclic RGD.
Cells were encapsulated within hydrogels containing cyclic RGD using
SPAAC chemistry: (A) good viability was observed with a live/dead cyto-
toxicity assay, and (B) increased metabolic activity and (C) positive Ki-67
staining further support cell viability and proliferation in these 3D cultures
with cyclic RGD (F-actin (red), DAPI (blue), proliferation marker Ki-67
(green); scale bars, 50 mm) (**p-value o 0.01).
(C) cyclic RGD, and (D) linear RGD (F-actin (red), DAPI nuclei (blue); scale
bars, 100 mm) (*p-value o 0.05).
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This study was supported by the Delaware COBRE programs,
with grants from the NIGMS (P20GM104316 and 5 P30
GM110758-02), the Susan G. Komen Foundation made possible
through funding from American Airlines (CCR16377327), the
NCI (P30CA093373), the Burroughs Wellcome Fund (1006787),
and the CIRM (RN3-06460).
Conflicts of interest
Fig. 5 Fluorescent cyclic RGD labeling of MDA-MB-231 cells. AF488-DIBO
was conjugated to the azide handle presented by the cyclic RGD through a
SPAAC reaction. (A) Cyclic RGD-AF488 (CYC) labeled a significant portion of
There are no conflicts to declare.
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v 3
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5
23
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8
4
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1
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