Iossif
Strehin
a,
Dmitri
Gourevitch
b,
Yong
Zhang
b,
Ellen
Heber-Katz
b and
Phillip B.
Messersmith
*a
aBiomedical Engineering Department, Materials Science and Engineering Department, Chemical and Biological Engineering Department, Chemistry of Life Processes Institute, Institute for Bionanotechnology in Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL 60208, USA. E-mail: philm@northwestern.edu; Fax: +1 (847)491-4928; Tel: +1 (847)467-5273
bMolecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA 19104, USA
First published on 1st March 2013
Oxo-ester mediated native chemical ligation (OMNCL) is a variation of the more general native chemical ligation reaction that is widely employed for chemoselective ligation of peptide fragments. While OMNCL has been used for a variety of peptide ligations and for biomolecular modification of surfaces, it is typically practiced under harsh conditions that are unsuitable for use in a biological context. In this report we describe the use of OMNCL for polymer hydrogel formation, in vitro cell encapsulation, and in vivo implantation. Multivalent polymer precursors containing N-hydroxysuccinimide (NHS) activated oxo-esters and N-cysteine (N-Cys) endgroups were chemically synthesized from branched poly(ethylene glycol) (PEG). Hydrogels formed rapidly at physiologic pH upon mixing of aqueous solutions of NHS and N-Cys functionalized PEGs. Quantitative 1H NMR experiments showed that the reaction proceeds through an OMNCL pathway involving thiol capture to form a thioester intermediate, followed by an S-to-N acyl rearrangement to yield an amide cross-link. pH and temperature were found to influence gelation rate, allowing tailoring of gelation times from a few seconds to a few minutes. OMNCL hydrogels initially swelled before contracting to reach an equilibrium increase in relative wet weight of 0%. This unique behavior impacted the gel stiffness and was attributed to latent formation of disulfide cross-links between network-bound Cys residues. OMNCL hydrogels were adhesive to hydrated tissue, generating a lap shear adhesion strength of 46 kPa. Cells encapsulated in OMNCL hydrogels maintained high viability, and in situ formation of OMNCL hydrogel by subcutaneous injection in mice generated a minimal acute inflammatory response. OMNCL represents a promising strategy for chemical cross-linking of hydrogels in a biological context and is an attractive candidate for in vivo applications such as wound healing, tissue repair, drug delivery, and tissue engineering.
Scheme 1 Generalized reaction schemes for native chemical ligation (NCL) and oxo-ester mediated native chemical ligation (OMNCL). |
Several research groups in the biomaterials community have explored NCL for preparation of functional materials.6–12 These studies focused on the use of NCL for synthesis of collagen mimetic biomaterials,9 chemical modification of polymers10,11 and self-assembled peptide scaffolds,8 and modification of substrate surfaces.6 In principle, the chemoselectivity of NCL is attractive for in vitro and in vivo use, allowing chemical reactions to proceed with specificity in a complex biological milieu, preserving the bioactivity of endogenous compounds and facilitating the targeting of therapeutic or diagnostic molecules to specific biomolecular targets such as cell surface proteins and components of the extracellular matrix. Our group has been developing polymer hydrogels cross-linked via NCL,7,12 for potential in vitro and in vivo applications. The general strategy involves the reaction of a thioester-derivatized polymer with a second polymer containing N-terminal Cys residues. Mixing of the two polymer precursors under mild aqueous conditions led to gel network formation via NCL without the need for added catalysts.7 Later, we extended this strategy to the formation of gels for in vitro cell encapsulation, incorporating polymer-bound IL-1 receptor inhibitory peptides that provided an immunoprotective effect to entrapped insulin secreting cells.12
Despite these recent advances, several aspects of the NCL reaction remain challenging for use in a biological setting. For example, standard NCL conditions employ the use of strong reducing agents that may be harmful in living systems. Furthermore, the slow rate of NCL cross-linking,7 the hydrolytic instability of the thioester, and the adverse biological effects of the thiol leaving group13 remain obstacles to future in vivo applications of NCL.
Several modifications of the NCL reaction have been introduced in an effort to expand the utility of the method.14–16 Danishefsky and coworkers described the use of oxo-esters in NCL (Scheme 1), first through an indirect approach involving o-thiophenolic ester17 and followed later by a direct approach utilizing p-nitrophenyl (pNP) activated C-terminal ester.18 Termed oxo-ester mediated NCL (OMNCL), this approach enables high efficiency reactions even with bulky C-terminal amino acids, although disadvantages include hydrolytic susceptibility of the pNP ester and challenges associated with direct solid phase synthesis of pNP ester peptides.19 Weissenborn et al. described OMNCL on oxo-ester activated surfaces and found 2,3,4,5,6-pentafluorophenyl (PFP) to be more efficient than pNP and N-hydroxysuccinimide (NHS) activating agents.20
Here we describe polymer hydrogel formation via OMNCL between branched polymer precursors containing NHS activated ester and N-Cys endgroups.21 The focus of this initial report was to elucidate the cross-linking mechanism and to provide preliminary assessments of mechanical properties, cytocompatibility and in vivo acute inflammatory response. Mixing of NHS and N-Cys polymer precursors led to gel formation within seconds, and quantitative NMR studies revealed the cross-linking mechanism to be OMNCL. In addition to characterizing the bulk mechanical and adhesive properties of the hydrogels, we performed the first in vitro and in vivo studies of OMNCL hydrogels, showing favorable biological response in cytotoxicity assays and in a subcutaneous implant model. The OMNCL hydrogel strategy overcomes many of the earlier limitations of NCL, including cytotoxicity of thiol leaving groups and slow reaction kinetics, and represents a promising strategy for chemical cross-linking of hydrogels in a biological context.
The relative abundance (RA) of polymer species during the OMNCL reaction between P8NHS and L-Cys was determined by integrating the triplets associated with the protons bound to the C2 and C4 carbons of the terminal glutarate linker. In this reaction four polymer species are possible (P8NHS, P8G, P8G-TE-Cys, P8G-AM-Cys), each possessing slightly different chemical shifts of the C2 and C4 protons depending on the composition of the terminal group (Fig. S1 and Table S1†). The chemical shifts for the protons associated with the C2 and C4 carbons of glutarate of P8G and P8NHS were determined from spectra of the synthesized intermediate and final product (see above), whereas the chemical shifts of thioester (P8G-TE-Cys) and amide (P8G-AM-Cys) linked products were estimated from spectra obtained by reaction of P8NHS with N-acetyl-L-cysteine and S-methyl-L-cysteine, respectively. Integrated peak values were used in the equations below to calculate the relative abundance of each species present during the reaction of P8NHS and L-Cys.
(1) |
(2) |
(3) |
(4) |
(5) |
The adhesive strength of the hydrogel was measured using a lap shear test adapted from ASTM standard F2255-05. Unprocessed porcine pericardium (2.5 cm × 3 cm) was adhered to aluminum fixtures using a cyanoacrylate based adhesive and then covered with a PBS moistened paper. After 1 hour, 100 μL of hydrogel precursor was placed on one tissue surface and a second tissue surface brought into contact (2.5 cm × 1.25 cm overlap) such that the tissue sections were glued together. After 10 minutes of curing, the tissue and glue were covered with PBS moistened paper towel for an additional 50 minutes. The glued tissue was pulled apart at 5 mm min−1 in tensile shear, and the peak stress and tissue overlap area were used to calculate the adhesive strength of the material.
In the second viability assay, cells were suspended in the P8Cys component dissolved in PBS and mixed with the P8NHS component dissolved in PBS to yield a 7% (w/v) hydrogel containing 1:1 (w/w) ratio of P8Cys to P8NHS. The hydrogels were allowed to set for 1 to 2 minutes and then incubated in culture medium at 37 °C and 5% CO2. After 24 hours, cell viability was quantified using calcein AM and ethidium homodimer-1. Cells were stained for 15 minutes in culture medium substituted with 4 μM calcein AM and 4 μM ethidium homodimer-1. Images were acquired using a fluorescent microscope equipped with a 485 ± 10 nm optical filter for calcein AM (live cells) and a 530 ± 12.5 nm optical filter for ethidium homodimer-1 (dead cells). The images were merged and processed using ImageJ (National Institute of Health, Bethesda, MD).
Tissue sections were cleared, rehydrated, and then stained with hematoxylin and eosin or with picro-sirius red, dehydrated, cleared with xylene and coverslipped with Permount mounting media. Staining was visualized using an Olympus (AX70) microscope (Olympus America, Center Valley, PA, USA) in bright field for H&E and under polarized light for Picro-Sirius Red. Images were recorded using a Spot camera with bounded software.
Fig. 1 The two polymer precursors P8NHS and P8Cys react in aqueous solution via OMNCL to yield polymer hydrogels with network cross-links as shown at bottom right. |
Fig. 2 Quantitative 1H NMR analysis of the model reaction between P8NHS and L-Cys in D2O. Chemical structures (A) and relative abundance (B) of polymer species observed during the reaction. |
Upon mixing P8NHS and L-Cys, rapid disappearance of P8NHS was accompanied by rapid emergence of the thioester intermediate P8G-TE-Cys. P8G-TE-Cys reached a maximum value after several minutes and slowly started to disappear thereafter and was found in only trace amounts after 75 h (Fig. 2B). Concomitant with disappearance of P8G-TE-Cys, the amide linked product P8G-AM-Cys emerged slowly over the first hour and represented over ∼80% of species present in the reaction mixture after 75 h. P8G appeared in minor but detectable amounts, present at less than ∼20% at all time points. Similar trends were observed when the reaction of P8NHS and L-Cys was performed in the presence of phosphate buffer at pH 6, albeit with significantly faster kinetics (Fig. 3A and Fig. S3†). At pH 7, the reaction was essentially complete within 5 minutes (Fig. 3B). Under the same conditions, control experiments showed that hydrolysis of P8NHS was insignificant within this timeframe (Fig. S4†). At pH 7, when L-Cys was replaced with S-methyl-L-cysteine, the reaction was significantly slower (Fig. 3C) despite the lower pKa of the amino group of S-methyl-L-cysteine (8.7523vs. 10.78 for L-Cys23). In a separate experiment at pH 7, P8NHS was reacted with equal concentrations of both L-Cys and L-Gly (amine pKa 9.623), yielding 80% P8G-AM-Cys, 15% P8G-AM-Gly and 5% P8G (Fig. S5†). Interestingly, mixtures of 10% (w/v) P8NHS (38 mM NHS ester) and 19 mM L-Cys were observed to form stable gels after 2 hours of incubation (100 mM PBS, pH 7.0), although these gels liquefied in the presence of β-mercaptoethanol but not PBS (Fig. S6†), implying cross-linking via disulfide bond formation.
Fig. 3 Quantitative 1H NMR analysis of the reaction between P8NHS and L-Cys in buffered D2O. (A, B) Relative abundance of polymer species formed during reaction of P8NHS with L-Cys at (A) pH 6.0 and (B) pH 7.0, indicating that the reaction proceeds more quickly at higher pH. (C) Relative abundance of polymer species formed during the reaction of P8NHS with S-methyl-L-cysteine at pH 7.0, illustrating significantly slower reaction kinetics when the thiol group is protected. |
These findings led us to select a buffer concentration of 100 mM for gel kinetic studies under different pH conditions. The pH dependence of gelation time for a 10% w/v mixture of P8NHS and P8Cys in 100 mM PBS is shown in Fig. 4. It can be seen in this figure that gel time changed significantly within the pH range 6–8, ranging from ∼50 s at pH 6 to <10 s at pH 8. Finally, we determined the temperature dependence of gelation time, which showed that gel formation was accelerated by ∼9 s when the temperature was changed from room to body temperature (Fig. S9†).
Fig. 4 The effect of initial pH on gelation time of 10% w/v hydrogels prepared in 100 mM PBS at room temperature with P8Cys and P8NHS (1:1 w/w). |
Several types of mechanical characterizations were undertaken on hydrogels formed from P8NHS with P8Cys. Swelling experiments were performed by immersing hydrogel samples in excess PBS and measuring the weight changes as a function of time. OMNCL hydrogels increased in relative wet weight by approximately 21% in the first several hours and then slowly contracted over a period of many hours to a final increase in relative wet weight of approximately 0% (Fig. 5A). The young's modulus of the hydrogel was measured at 5, 75 and 150 h and was found to increase from 128 to 182 kPa during this time (Fig. 5B). The latent modulus increase is unlikely to be attributed to additional cross-linking by the OMNCL mechanism, as model 1H NMR studies showed that at pH 7 the reaction was mostly complete after 5 minutes (Fig. 3B). The observed swelling and modulus changes are unlikely to reflect mass changes induced by hydrolytic degradation of the gels, as NMR analysis of ester hydrolysis (Fig. S10†) and a preliminary evaluation of gel degradation at pH 7.0 showed little mass loss over a 12 week period (Fig. S11†).
Fig. 5 Physical characterization of OMNCL hydrogels formed by mixing equal volumes of 10% (w/v) P8NHS and 10% (w/v) P8Cys in PBS. (A) Swelling of OMNCL hydrogels in 10 mM PBS (closed symbols) or 10 mM PBS substituted with 0.2 M β-ME (open symbols). The two sets of hydrogels (diamonds and circles, n = 5 per set) varied by the sequence in which they were incubated in PBS or β-ME. In one case (circles), the hydrogels were incubated in PBS followed by β-ME and then PBS again. In the second case (diamonds), the hydrogels were incubated in PBS for the first few hours and thereafter in β-ME. (B) Young's moduli (n = 4) at various time points for OMNCL hydrogels incubated in PBS. *p < 0.05, ***p < 0.001. |
Suspecting therefore that gel shrinkage was the result of disulfide bond formation, fully equilibrated hydrogels (>300 hours swelling in PBS) were transferred into 0.2 M β-mercaptoethanol in PBS, whereupon the hydrogels increased in relative wet weight by approximately 27% (Fig. 5A). After approximately 140 hours in reducing agent, swollen samples were transferred back into PBS and swelling was observed to decrease once again, implying the re-formation of disulfide bonds.
Finally, the adhesive strength of the OMNCL hydrogel to unprocessed porcine pericardium was measured in lap shear similar to the protocol described in ASTM standard F2255-05. Tissue surfaces were glued together using the OMNCL hydrogel and then pulled apart after one hour post gelation, yielding a lap shear adhesive strength of 46 ± 8 kPa.
Fig. 6 In vitro cytocompatibility of OMNCL hydrogels. (A) Quantitative analysis of 3T3 fibroblast viability after 24 hours in conditioned medium, conducted in accordance with ISO standards 10993-05 and 10993-12. Cell culture medium included either extract from P8NHS/P8Cys hydrogel or 5% w/v P8NHS. (B) 3T3 fibroblasts encapsulated in OMNCL hydrogels and stained with calcein AM (green, live cells) and ethidium homodimer-1 (red, dead cells). Image analysis indicated 87 ± 7% of cells remained viable after 24 hours of encapsulation. |
Fig. 7 In vivo subcutaneous characterization of OMNCL hydrogel. (A) H&E stained tissue section at 20× magnification with gel associated with the outer skin. The gel is stained blue and surrounding tissue stained blue and red (an overview of the skin-gel injection area is shown on the right at 4× magnification). (B) H&E stained tissue section at 40× magnification from sequentially obtained tissue sections, showing the gel (blue body at the bottom), subtle fibrous capsule (marked with black arrows) and supra-capsular muscle layer (purple-red, marked with green arrows). (C) Picro-Sirius Red stained section (40× magnification) obtained from the same area as B. Hydrogel is at the bottom; the capsule surrounding the hydrogel is a bright-red fibrous structure (marked with white arrows) and muscle mass shown in brown-red (marked with green arrows). The scale bars indicate length in micrometers (mc). |
Implementation of NCL in a biological context can be problematic due to the adverse biological effects of the buffers and reducing agents typically employed in NCL reactions. Furthermore, we have shown that the small molecule thiol leaving group liberated during NCL can be toxic to cells.13 OMNCL, on the other hand, is often practiced in solutions containing concentrated guanidine (5–6 M),18–20 which would have adverse effects in a biological system because of its strong denaturing potential. Our results show that hydrogel formation by OMNCL proceeds well in phosphate buffer, achieving rapid gel formation without the use of catalysts or additives. In the pH range of our experiments (6–7.3), the rate of gel formation by OMNCL was adjustable through pH control and ranged from several seconds to less than a minute. This is in contrast to gels formed by NCL chemistry,7,12 which have slower gelation rates at physiological pH.
We elucidated the mechanism of cross-linking by following the reaction of P8NHS and L-Cys by 1H NMR (Fig. 2, 3A,B), taking advantage of chemical shift differences to reveal the temporal evolution of reactants, intermediates and products. Several lines of evidence from these studies point to cross-link formation via OMNCL. First, the rapid increase in thioester cross-link in parallel with the rapid decrease in P8NHS at the beginning of the reaction is indicative of thiol capture. The thioester intermediate reaches as high as >50% relative abundance within the first few minutes but then decreases until it is no longer detectable. This decrease cannot be explained by thioester hydrolysis, as the hydrolysis product (P8G) was never present at greater than ∼10% relative abundance at pH 6 or 7. Second, the kinetics of thioester cross-link disappearance was roughly matched with the kinetics of amide cross-link emergence, which is a strong indicator of S-to-N rearrangement. It should be noted that at neutral and acidic pH the thiol should be considerably more reactive than the primary amino group due to the high pKa of the terminal amine of L-Cys (pKa ∼ 10.7823). As evidence of this, in the reaction between P8NHS and L-Cys at neutral pH, conversion to P8G-AM-Cys and P8G-TE-Cys was 83% within the first minute. However, reaction of P8NHS with S-methyl-L-Cys yielded only 20% conversion under the same conditions, despite the significantly lower pKa of the S-methyl-L-Cys amino group (pKa ∼ 8.75)23 compared to L-Cys. Finally, further support for OMNCL pathway was provided by a competitive reaction between P8NHS, L-Cys and L-Gly, where 80% of the reaction proceeded with L-Cys and only 15% with L-Gly despite the lower amino pKa of L-Gly (pKa ∼ 9.623).
Taking these findings into consideration, we therefore propose the OMNCL reaction pathway shown in Scheme 2 for the gel-forming reaction between P8NHS and P8Cys. For reasons elaborated above, we consider the hydrolysis of P8NHS and the thioester intermediate as minor competing reactions under our conditions. Both of these reactions would produce acid terminated polymer endgroups that would not be capable of contributing to gel formation. It is impossible to exclude some contribution from direct amide bond formation between the terminal amine of P8Cys and P8NHS. However, it should be noted that the pKa value of the N-terminal amino of Cys is 10.78 whereas that of the thiol side chain is 8.33.23 Thus, at the pH values of 6–7.3 employed in gel formation, most N-terminal amino groups of Cys would be rendered inactive toward reaction with the NHS activated ester by protonation. In support of this, mixtures of P8NHS and 8-arm PEG-amine form gels more than 10 times slower in PBS buffer.
Scheme 2 OMNCL cross-linking of P8Cys and P8NHS. Fast reaction pathways are indicated by solid arrows, slow pathways by dashed arrows. Thiol capture followed by S-to-N acyl rearrangement results in polymer cross-linking. Secondary cross-links arise through the formation of disulfide bonds among network-bound Cys residues. P1 = P8NHS; P2 = P8Cys. |
Most polymer hydrogels typically absorb water under physiologic conditions, leading to a significant increase in volume. The results of our gel swelling experiments were notable for two reasons. First, the increase in relative wet weight (0%) for OMNCL hydrogels is considerably lower than many experimental and clinically approved PEG-based hydrogels. For example, studies of PEG based hydrogels cross-linked via amide or thioester bonds report increasing relative wet weight values of 50%25 to 400%26 within the first 24 hours. In some cases, swelling of implanted materials may lead to complications such as nerve compression27–31 or other serious problems requiring intervention.32 Secondly, the swelling experiments provided important evidence of the latent formation of disulfide bonds, and insight into the mechanical consequences of this secondary cross-linking mechanism. Exposure of OMNCL gels to reducing agent resulted in an increase in swelling that was reversible upon removal of reducing agent, implying the formation of disulfide bonds within the gel network. This can be understood to be a result of the S-to-N acyl rearrangement step that releases the thiol side chain of Cys, which then becomes available for disulfide bond formation with other network-bound thiols. Through comparison of the kinetics of the OMNCL reaction (Fig. 3A,B) and the swelling results (Fig. 5), we surmised that the reduction in swelling observed after ∼5 h results from disulfide bond formation. Thus, we conclude that the hydrogels form initially by OMNCL and are later further cross-linked through the formation of disulfide bonds.
Our in vitro cell experiments demonstrated low cytotoxicity of OMNCL hydrogel extracts and high viability of encapsulated cells (Fig. 6). This led us to undertake an initial in vivo evaluation of OMNCL hydrogels in a subcutaneous implant model. Gels were formed by injection of precursor solutions and then evaluated at 6 weeks. Histological sections of explants showed low-level acute inflammatory response to implanted gels and deposition of a thin fibrous capsule surrounding the implant (Fig. 7). These findings confirm the potential of OMNCL hydrogels for in vivo applications.
Activated ester PEG polymers are currently approved by the FDA in the form of the medical sealants DuraSeal™ and COSEAL™. An important distinction between these existing materials and our OMNCL hydrogels relates to the pH range of the cross-linking reaction. Both DuraSeal™ and COSEAL™ are deployed at highly alkaline pH (typically pH 9–10),33 whereas the OMNCL hydrogels reported here are capable of rapid gel formation at neutral pH. In our studies, PEG was chosen as the backbone for both components because it is non-cytotoxic and has demonstrated favorable results in previous in vivo studies. PEG is also attractive as a simple platform for quantitative NMR studies and is amenable towards chemical modification. However, it is important to note that the OMNCL hydrogel chemistry described here can be easily adapted for use with other suitable polymer platforms.
In summary, we have detailed the synthesis and characterization of a two-component hydrogel formulation that sets rapidly at physiologic pH and ionic strength in the absence of catalysts or other additives. The cross-linking mechanism was revealed by NMR studies to be primarily OMNCL, proceeding by thiol capture to form a thioester intermediate followed by a S-to-N acyl rearrangement to generate amide cross-links. OMNCL hydrogels exhibit attractive mechanical properties that include high compressive moduli and good adhesion to tissue, and low equilibrium swelling due to the latent formation of secondary disulfide cross-links. The biological performance of OMNCL hydrogels was assessed, showing high in vitro cytocompatibility and low acute inflammatory response in vivo. OMNCL hydrogels represent attractive candidates for in vivo applications such as wound repair and sealing, drug delivery, and tissue engineering.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3bm00201b |
This journal is © The Royal Society of Chemistry 2013 |