A Repertoire of Peptide Tags for Controlled Drug Release from

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A Repertoire of Peptide Tags for Controlled Drug Release from Injectable Noncovalent Hydrogel Robert Wieduwild,† Weilin Lin,† Annett Boden,† Karsten Kretschmer,‡ and Yixin Zhang*,† †

B CUBE Center for Molecular Bioengineering, Technische Universität Dresden, Arnoldstraße 18, 01307 Dresden, Germany CRTD/DFG-Center for Regenerative Therapies Dresden - Cluster of Excellence, Fetscherstraße 10, 01307 Dresden, Germany

‡

S Supporting Information *

ABSTRACT: A repertoire of conjugable tags for controlling the release of drugs from biomaterials is highly interesting for the development of combinatorial drug administration techniques. This paper describes such a system of 11 peptide tags derived from our previous work on a physical hydrogel system cross-linked through peptide−heparin interactions. The release kinetics of the tags correlate well with their affinity to heparin and obey Fick’s second law of diffusion, with the exception of the ATIII peptide, which displays a stable release profile close to a zero-order reaction. A system for release experiments over seven months was built, using the hydrogel matrix as a barrier between the reservoirs of tagged compounds and supernatant. The gel matrix can be injected without affecting the releasing properties. A tagged cyclosporin A derivative was also tested, and its release was monitored by measuring its biological activity. This work represents a design of biomaterials with an integral system of drug delivery, where both the assembly process of the matrix and affinity capture/release of tagged compounds are based on the noncovalent interaction of heparin with one class of peptides. both basic residues and heparin-induced α-helix formation of the peptides are important for the assembly process. A simple set of rules allows tuning of the matrix properties. For example, rheology studies have shown that the stiffness can be tuned through adjusting the number of (BA)n repeats or by changing the concentrations of heparin and peptide−polymer conjugate. The resulting hydrogels can also be used for cell encapsulation. Furthermore, the noncovalent polymer matrices are very stable. For example, the hydrogel made with (KA)7 peptide (KA7) is not only resistant to acid and basic treatments, but also stable over long period of time, while no erosion has been observed 2 years after the synthesis of the hydrogels. In this paper, a library of heparin-interacting peptides was investigated, whose sequences were derived from the noncovalent hydrogel system (Figure 1a). This work represents a design of biomaterials with an integral system of drug delivery, where both the assembly process of matrix formation and affinity capture/release of tagged compounds are based on the noncovalent interaction of heparin with one class of peptides. In addition to the peptide tags (BA)n, whose releases are governed by diffusion and decrease over time, the affinity tag ATIII has been discovered to allow a constant and stable release over a period of months.

1. INTRODUCTION There is a growing interest in the controlled release of therapeutics from biocompatible matrices.1,2 The challenges lie in the design and synthesis of biopolymers, and also in our understanding of drug−matrix interactions.3,4 An attractive solution would be to use a repertoire of structural motifs as tags, which would have different release kinetics from the matrix. These tags could be conjugated to drugs, allowing controlled release of the active compounds. In the emerging field of personalized medicine, a repertoire of tags that provide different release profiles would be very useful for the design of combinatorial drug releasing systems. Heparin, as part of the extra cellular matrix (ECM), binds to and controls the release of lots of proteins including several morphogens.5 Many hydrogel systems have been developed to resemble some ECM properties using heparin as a component. Covalent hydrogels were designed and synthesized to deliver growth factors like vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), bone morphogenetic protein 2 (BMP-2), and stromal-cell derived factor-1α (SDF-1α) to study vascularization, bone formation, and skin or kidney regeneration.6−14 Heparin can also be incorporated into hydrogels noncovalently through either conjugating heparin binding peptides (e.g., peptides derived from antithrombin III (ATIII) to the polymer matrices,15,16 or using physical hydrogels based on heparin−peptide interactions.17−19 In a previous study, we designed and screened simple peptide motifs, whose conjugates with poly(ethylene glycol) (PEG) interact and form physical hydrogels with heparin.20 The study revealed a simple structure−function relationship of the (BA)n (B is basic residue arginine or lysine, A is alanine) motifs, where © 2014 American Chemical Society

2. MATERIALS AND METHODS 2.1. Peptide and Conjugate Synthesis. All peptides have been prepared using standard Fmoc chemistry on a solid-phase with HBTU Received: February 5, 2014 Revised: May 7, 2014 Published: May 13, 2014 2058

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Figure 1. Screening of peptides as a repertoire of releasing tags. A 10 μL hydrogel was prepared by mixing final concentrations of 2.5 mM KA7-starPEG (KA7 peptide coupled to four-arm poly(ethylene glycol)), 2.5 mM 14 kDa heparin and 2 mM fluorescently labeled peptide tag. (a) Scheme of peptidestarPEG/heparin hydrogel with compounds tagged with heparin-binding peptides. (b) Scheme of release of heparin-binding peptides using the hydrogel as storage. (c,d) Release analysis of fluorescently labeled peptides from hydrogel. The supernatant was changed every 3−4 days. Error bars indicate standard deviations (n = 4). 2.3. Release Experiment Setup. Ten-microliter hydrogels per well were prepared in a 6, 24, or 96 well plate and incubated overnight. For hydrogel injection, 10 μL of hydrogels were first formed in a microlitersyringe overnight and then injected into a 24-well plate. PBS or CO2 independent cell culture medium with 10% fetal bovine serum was added to each well. The well plates were sealed airtight using an adhesive black light absorbing film. The peptide release was analyzed by measuring the fluorescence intensity through the hydrogel-free region of the transparent bottom of the plate using a plate reader (PARADIGM Detection platform, Beckman Coulter, Brea, California, USA) or enzyme inhibition assay (ATIII-CsA) (see section 2.5.). For using the hydrogel as control barrier, 5 μL of hydrogel without peptide was injected at one end of a capillary before gelation. After hydrogel formation, 0.1 mL of 2 mM fluorescently labeled peptide was added onto the top of the hydrogel, and the other end of capillary was sealed. The capillaries were immersed in tubes of 50 mL PBS buffer. The amount of peptide in the 50 mL PBS was determined by fluorescence intensity measured in a plate reader. 2.4. Injection Model. The method was derived from ref 22. A solution of 3% (w/v) agarose in PBS was prepared and poured into heart shaped molds. Just prior to gelation, air bubbles were introduced with a syringe, and the agarose was incubated at 4 °C for an additional 30 min. Samples were equilibrated at 37 °C in PBS for 1 h, and then a syringe was used to inject hydrogel containing 2.5 mM KA7-Rho-starPEG, 2 mM ATIII-W and 2.5 mM 14 kDa heparin. The samples were incubated at 37 °C in PBS overnight and then dissected to examine gel formation. 2.5. ATIII-CsA Activity Assay. Recombinant His-Cyp18 was expressed in the Escherichia coli strain BL21(DE3) (see Supporting Information for details). The enzyme activity on the substrate Suc-AlaPhe-Pro-Phe-pNA was measured with a protease (α-chymotrypsin) coupled assay as described previously with some modifications.23 For

activation on an automated solid-phase peptide synthesizer (ResPep SL, Intavis, Cologne, Germany). 5(6)-Carboxyfluorescein, 5(6)-carboxytetramethylrhodamine (TAMRA), or COOH-CsA (synthesized as described earlier21) was coupled to the N-terminus of the peptides on the resin. The peptide was cleaved from the resin with TFA/TIS/water/ DTT (90(v/v):5(v/v):2.5(v/v):2.5(m/v)), and the product was precipitated and washed with ice-cold diethyl ether. Peptide purification was performed via reverse-phase high pressure liquid chromatography (HPLC) on a preparative HPLC (ProStar, Agilent Technologies, Santa Clara, Unites States) equipped with a preparative C18 column by applying an isocratic gradient. Purity was confirmed by analytical reverse phase ultra HPLC (UPLC Aquity with UV Detector, Waters, Milford Massachusetts, USA) equipped with an analytical C18 column using an isocratic gradient and electrospray ionization mass spectrometry (ESI-MS) (ACQUITY TQ Detector, Waters, Milford Massachusetts, USA) (Supporting Information Figure S1). The synthesis of the KA7-starPEG conjugate utilized in hydrogel assembly was conducted via Michael addition reactions between maleimide-terminated four-arm PEG and cysteine-terminated KA7 peptide (see Supporting Information for details). Rhodamine-labeled KA7-starPEG (KA7-Rho-starPEG) consisted of 90% KA7 peptide and 10% KA7-Rho peptide (N-terminal TAMRA labeled KA7 peptide) mixed prior to the Michael addition reaction. 2.2. Hydrogel Formation. Fourteen kilodalton heparin, fluorescein-labeled peptide, and KA7-starPEG conjugate were dissolved in phosphate buffered saline (PBS) and filtered through a 0.22 μm centrifuge tube filter. These solutions were mixed (by vortexing) in a volume ratio of 1:1:3 (heparin: fluorescein labeled peptide: KA7starPEG conjugate) to yield 1.25, 2.5, or 5 mM 14 kDa heparin, 2.5 mM KA7-starPEG conjugate and 0.5, 1, or 2 mM labeled peptide. 2059

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the inhibition of Cyp18, different concentrations of ATIII-CsA were used to determine the IC50 value of inhibition. The inhibition curve was then used to determine the concentration of released ATIII-CsA from the hydrogel in the supernatant. 2.6. Immunosuppressive Assay Using Flow Cytometry. Singlecell suspensions from mesenteric lymph nodes (mLNs), subcutaneous lymph nodes (scLNs) and spleens of BALB/c.Foxp3IRES‑GFP mice were prepared using 70 μm cell strainers.24 Samples were enriched for CD4+ cells after sequential staining with biotinylated anti-CD4 antibodies, magnetic streptavidin-coated microbeads, and Pacific Blue-conjugated streptavidin using automagnetically activated cell sorting (AutoMACS system, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). An LSRII (BD, Le Pont de Claix Cedex, France) was used for analytical flow cytometry. For analyzing T cell proliferation, 1.5 × 107 cells/mL were suspended in PBS (with 0.1% BSA) and labeled for 12 min at 37 °C with eFluor670 proliferation dye at a final concentration of 5 μM. Cells were treated with 1 μM of CsA or ATIII-CsA or DMSO (control) and stimulated with anti-CD3/CD28-coated beads for 3 days in the presence of 100 U/ml recombinant human IL-2. Foxp3 expression and dilution of eFluor670 at the single-cell level were analyzed at day three (after initiation of cultures) for viable proliferating cells.

1) were synthesized using Fmoc solid phase peptide chemistry. The N-terminals of the peptides were conjugated to fluorescein or rhodamine (Figure 1a). As a model system, the release characteristics of conjugates from hydrogel were investigated (Figure 1b,c). KA7-starPEG/heparin hydrogel was chosen for this study for its high stability, making it an ideal candidate for long-term drug release studies and applications.20 The CWGG(KA)7 (KA7) peptide was conjugated to 10 kDa four-arm maleimide PEG polymer (starPEG) through Michael addition as described earlier.20 The resulting conjugate (KA7-starPEG) was premixed with peptide−fluorescein conjugates and used to form hydrogels with 14 kDa heparin (Figure 1a). After hydrogel formation, each 10 μL hydrogel was incubated with 0.2 mL PBS buffer at room temperature (Figure 1b). The release of fluorescent compounds was monitored using fluorescence spectrometry (Figure 1c). The release of molecules from a stable bulky matrix is controlled by diffusion inside the hydrogel, which is dependent on many factors including pore size and affinity capture of molecules by the matrix.12,14−16,25−33 The amount of released peptide often decreases continuously over time.25−28 This is mainly caused by the increase of diffusion length as the release proceeds, which can be described by Fick’s second law of diffusion. The binding of tagged molecules to heparin limits their diffusion in matrices. As expected, the (KA)n and (RA)n tags showed a continuous decrease in peptide release. The different fluorescein conjugated peptide tags exhibited release profiles ranging from minutes to weeks. As shown in Figure 1c, when n of (BA)n is smaller than 5, the peptide-fluorescein conjugates were released quickly from the hydrogel. When n is 5, the peptides exhibited significantly slower release from hydrogel than the shorter analogues. Increasing n can further reduce the release. When n is constant, the (KA)n tags showed stronger retention than the (RA)n tags. The release of KA7-F is the slowest among the (KA)n and (RA)n conjugates analyzed in this study. Interestingly, a subtle difference between the (KA)n and (RA)n motifs has been observed. For example, the initial release profile of RA7-F was slower than that of KA6-F, while at the end of the experiment the release of RA7-F was higher. A similar difference was also been observed for KA5-F and RA6-F. Interestingly, ATIII-F was released constantly throughout the experiment. To further demonstrate that the release of tagged compounds is mainly controlled by the peptide-heparin interaction, we analyzed the release of immunoglobulin G (IgG) from hydrogels. IgG is a 150 kDa protein, while its release from hydrogels is complete in 24 h, significantly faster than those of small molecules KA4-F, KA5-F, KA6-F, KA7-F, RA5-F, RA6-F, RA7-F, and ATIII-F (Supporting Information Figure S2). This result has shown that the slow release of the tagged compounds is not caused by the hydrogel porosity or the free diffusion of compounds in solution. 3.2. Kinetics and Dynamics of ATIII Tag Release. Different from the other tags used in this study, the ATIII peptide does not possess the (KA)n or (RA)n motif and is derived from the heparin-binding antithrombin III protein.34,35 Interestingly, the release of ATIII-F does show the behavior expected for an affinity tag. The ATIII-F conjugate showed a continuous and stable release for over a month (Figure 1c). A similar release profile of ATIII peptide from a heparin containing hydrogel has also been reported by Seal et al.36 As the hydrogel system is mechanically very stable, not only can long-term experiments be designed and performed, but also frequent change of super-

3. RESULTS AND DISCUSSIONS 3.1. Design and Screening of Peptide Tags. Eleven peptides derived from the noncovalent hydrogel system (Table Table 1. Synthesised Peptide Library name

peptide sequence

molecular weight [Da]

ATIII-F KA3-F KA4-F KA5-F KA6-F KA7-F RA3-F RA4-F RA5-F RA6-F RA7-F

fluorescein-GGKAFAKLAARLYRKA fluorescein-GGKAKAKA fluorescein-GGKAKAKAKA fluorescein-GGKAKAKAKAKA fluorescein-GGKAKAKAKAKAKA fluorescein-GGKAKAKAKAKAKAKA fluorescein-GGRARARA fluorescein-GGRARARARA fluorescein-GGRARARARARA fluorescein-GGRARARARARARA fluorescein-GGRARARARARARARA

2079.18 1087.96 1287.21 1486.46 1685.71 1884.96 1172.02 1399.29 1626.56 1853.83 2081.10

Shown are the sequences, abbreviations, and molecular weights of the peptides used in this study. ATIII is a peptide derived from the antithrombin III protein.

Figure 2. Release of ATIII-F from hydrogel with different heparin concentrations. Hydrogel (10 μL) was prepared by mixing final concentrations of 2.5 mM KA7-starPEG, 1.25 mM or 5 mM 14 kDa heparin and 2 mM fluorescently labeled peptide tag ATIII-F. Half of the supernatant (0.1 of 0.2 mL) was changed every day. Error bars indicate standard deviations (n = 4).

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Figure 3. Analysis of release and affinity capture of fluorescent peptide tags. Ten microliter hydrogels consisting of 2.5 mM KA7-starPEG and 14 kDa heparin each were loaded with 2 mM ATIII-W. Fourteen micromolar ATIII-F in PBS buffer was added as supernatant (0.2 mL). (a) Affinity capture and release of ATIII-F (black). After 6 days, PBS was used as supernatant, and supernatant was changed every 3 days. ATIII-F in solution without hydrogel was used as control (red). Error bars indicate standard deviations (n ≥ 4). (b) Scheme of dynamics of affinity capture and release of ATIII peptide from hydrogel.

Figure 4. Release of ATIII-F from hydrogels using different ATIII-F concentrations or supernatant volumes. Hydrogels (10 μL) consisting of 2.5 mM KA7-starPEG and 14 kDa heparin were loaded with ATIII-F. Analysis of ATIII-F release from hydrogels using (a) different volumes of supernatant and 2 mM ATIII-F in hydrogel or (b) different starting concentrations of ATIII-F and 0.2 mL supernatant. Error bars indicate standard deviations (n ≥ 4).

profile shown in Figure 2. Because this system is based on a repertoire of affinity tags, the released compounds can be captured again by the matrix. The system can reach equilibrium if the volume of supernatant is small and not changed during the experiment. Obviously, this feedback process is dependent on the experimental design, which could be used to reflect different microenvironments in vivo. A small hydrogel in a large volume of solution would resemble an infinite sink, as a device placed in contact with the bloodstream. Incubation of a hydrogel in a relatively small volume of supernatant would mimic a closed environment with slow flow of body fluid. To demonstrate that the release and affinity capture of tagged compound is a dynamic process, hydrogels with 2 mM of nonfluorescent ATIII peptide were incubated with a solution of 14 μM ATIII-F (Figure 3). As shown in Figure 3a, a decrease of ATIII-F in the supernatant was observed, indicating the affinity capture of ATIII-F by hydrogel. After 6 days, the supernatant was replaced by PBS buffer, and the full volume of buffer was changed every 3 days. After two months, all affinity captured ATIII-F were released from the hydrogel. The exchange of ATIII tag between hydrogel and supernatant demonstrates that the affinity capture/ release is a dynamic process (Figure 3b). Given that the release of ATIII peptide from hydrogel is very slow, the concentration of ATIII-W in the hydrogel is very high and close to its initial concentration in the first 1−2 days. The ATIII-F can be quickly absorbed by the hydrogel, indicating that

natants, to gain insights into the release kinetics and to mimic various conditions for different potential applications. As the hydrogel system is also stable upon injection, we can also test its release profile as an injectable material. The drastic difference between ATIII-F and the other compounds indicates that their releases are governed by different mechanisms. Interestingly, loading ATIII-F turned the normally transparent KA7-starPEG/heparin hydrogels opaque. ATIII-F probably forms clusters with 14 kDa heparin in the hydrogel. Thus, instead of interior diffusion, the zero order dissociation of ATIII-F from clusters is the rate-limiting step for its release from hydrogel. This hypothesis explains why the release of the ATIII-F is very slow but constant, as the dissociation from clusters is not governed by Fickian diffusion. KA7-starPEG hydrogels with different concentrations of 14 kDa heparin were prepared and the release of ATIII-F was measured. As shown in Figure 2, the release of ATIII-F is independent of the 14 kDa heparin concentration. High heparin concentration would limit the diffusion of ATIII-F in the matrix; however, it has no effect on the release of ATIII-F. This result provides further evidence for the hypothesis of clusteringdissociation mediated release of ATIII-F. Whereas full replacement of supernatant was carried out in the release experiments shown in Figure 1c, half of the supernatant was changed every day in the experiments shown in Figure 2. This difference caused a remarkable extension of the release 2061

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Figure 5. Injection of peptide tag loaded hydrogel into agarose. Hydrogel with ATIII or ATIII alone was injected into a hole in agarose gel and further incubated overnight at 37 °C. (a) Agarose gel with two holes. (b,c) Rhodamine labeled hydrogel (2.5 mM rhodamine labeled KA7-starPEG and 14 kDa heparin) mixed with 2 mM ATIII-W. (d,e) Hydrogel (2.5 mM KA7-starPEG and 14 kDa heparin) mixed with 2 mM ATIII-R. (b+d) Dissection of agarose gel to examine the injected hydrogel. (c+e) Hydrogel extracted from agarose. (f) 2 mM ATIII-R was injected into a hole in agarose without hydrogel. Scale bar is 1 cm.

and Figure S3a). Without changing the supernatant during the experiments, larger supernatant volumes led to higher release profile. In small volume (0.2 mL) equilibrium of the affinity capture system was reached, and the concentration of the released compound plateaued (capping concentration). To further analyze how the initial concentrations of ATIII-F in hydrogel placed in a small volume system influenced the release profiles and final concentrations at equilibrium, hydrogels were formed with different amounts of ATIII-F and incubated with 0.2 mL PBS buffer. The supernatants were not changed during the experiment. As the initial concentration of ATIII-F in hydrogel increased (0.5 mM, 1 mM and 2 mM), the percentage of released compound decreased, although the absolute amount was higher (Figure 4b and Figure S3b). All three curves reached a plateau after 8 days. It is important to note that the amounts of released ATIII-F during the first 2 days were independent from the initial concentrations in the hydrogel, because the two phase system was still far from equilibrium, and the release profile was determined by the clustering/dissociation process. The tagged compounds can be both affinity-captured into and released from the heparin hydrogel. As we changed the supernatant regularly in the early experiments (Figures 1−3), the systems could not reach equilibrium. Without changing the supernatant (Figure 4), the equilibrium between affinity-capture and release could be reached over more than 1 week and is dependent on the initial concentrations of tagged compound in hydrogel as well as the volume of supernatant. As expected, larger supernatant volume led to more released compound. Interestingly, higher initial concentration of ATIII-F in the hydrogel resulted in higher absolute release but lower percentage of release. Clustering/aggregation of ATIII-F at high concentration could provide extra energy to stabilize the tagged compound in

Figure 6. Release of ATIII-F at 37 °C from control hydrogel or injectable hydrogel passed through the needle of a μL-syringe. Hydrogel consisted of 2.5 mM KA7-sterPEG, 2.5 mM 14 kDa heparin, and 2 mM ATIII-F. The supernatant was 1 mL cell culture medium with 10% FBS. Release at 24 °C (room temperature) is shown as a control. Measurements were stopped when no change in fluorescent intensity could be measured. Error bars indicate standard deviation (n ≥ 4).

there are enough binding sites for the ATIII-F in the heparin hydrogel. Different from the experiments described in Figure 1 and 2, ATIII-F was loaded to the preformed hydrogel. Therefore, its distribution in hydrogel is mediated by the binding to and the diffusion in the hydrogel. In contract, the hydrogel formed in the presence of ATIII-F (Figure 1c) led to even distribution in the matrix. The difference between these two loading methods has led to different release profiles. Hydrogels with ATIII-F were incubated with different volumes of PBS buffer. Whereas 10 μL hydrogels in 5 mL solution could resemble the situation of an infinite sink, 10 μL hydrogels in 0.2 mL solution is closer to an isolated microenvironment (Figure 4a 2062

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Figure 7. Release of fluorescent peptide tags using hydrogel as a control barrier. A 5 μL hydrogel consisting of 2.5 mM KA7-starPEG and 14 kDa heparin was placed at the end of a capillary. 0.1 mL of 2 mM fluorescently labeled peptide in PBS were filled in the capillary and the capillary was sealed. Capillary was placed in 50 mL PBS. (a) Scheme of hydrogel as a control barrier for peptide tag release. (b) Release of peptide tag from capillary with hydrogel as the control barrier. Supernatant was not changed throughout the experiment. The first 14 weeks concentration was measured regularly and then after 31 weeks. Error bars indicate standard deviation (n = 4).

rhodamine labeled hydrogel (2.5 mM rhodamine labeled KA7starPEG with 2.5 mM 14 kDa heparin) containing 2 mM ATIIIW was injected into an agarose gel.22 The holes were introduced into agarose gel using a syringe during the agarose gel preparation (Figure 5a). The agarose gel was incubated at 37 °C in PBS, and the rhodamine labeled hydrogel was injected. After injection, the agarose gel was further incubated overnight at 37 °C. The sample was dissected to examine hydrogel formation. As shown in Figure 5b,c, hydrogel formation is not disturbed by ATIII-W peptide and the injection procedure. Almost no rhodamine-labeled material could be detected outside the injected bulky hydrogel. This result demonstrated that a coherent bulky matrix can be formed in the confined area, while no small polymer fragment was generated, which could cause drug releasing behavior different from a bulky system. To demonstrate that the tagged compound was not leaked during the injection procedure, hydrogel was formed with nonlabeled KA7-starPEG and heparin in the presence of ATIIItagged rhodamine (ATIII-R). The resulting hydrogel was injected into the agarose gel holes (Figure 5d), while only a minor release of the labeled ATIII conjugate could be observed. Dissection of the agarose gel led to the recovery of the intact heparin hydrogel (Figure 5e). In contrast, direct injection of ATIII-R led to an instant spreading of the dye throughout the agarose gel (Figure 5f). In order to prove that the injectable system is also compatible with cell culture conditions and the injection does not disturb the release profile, the ATIII-F containing hydrogel was formed and incubated in cell culture medium with 10% fetal bovine serum (FBS) at 37 °C, mimicking the physiological environment. As shown in Figure 6, the release of the fluorescent compound was constant, while the release profile was slightly faster than at room temperature. The preformed hydrogel was pushed through a syringe. The injected hydrogel exhibited the same release profile in cell culture medium with 10% FBS as the control material. Similar to the KA7-starPEG/heparin hydrogel, the hydrogels loaded with ATIII-F did not swell when the formed hydrogels were incubated in PBS (Supporting Information Figure S8). Swelling of biomaterials is often dependent on their environment such as supernatant composition. The nonswelling behavior shows that the resulting systems can possess constant volume and be engineered for many other applications in the future, such as 3D bioprinting or in vivo injection.

the hydrogel, in addition to their binding to heparin. This can explain the shift of equilibrium to hydrogel and low percentage of release at high ATIII-F concentration. Given that the peptide tags can compete with KA7-starPEG for heparin binding, we investigated their effects on hydrogel formation, network mesh size, as well as the stiffness. Given that ATIII is the strongest heparin-interacting peptide in this study, we studied the hydrogels in the presence or absence of ATIII conjugates. Even 20 mM ATIII-rhodamine conjugate (ATIII-R) did not interfere with the hydrogel formation. We studied the influence of ATIII-R on hydrogel mesh size using electron microscopy. Interestingly, 2 mM ATIII-R did not cause significant changes in the pore sizes, while the heparin concentrations have shown a more remarkable effect on network structure (Supporting Information Figure S4). The pore size of hydrogel containing 2.5 mM heparin is larger than those with 1.25 mM or 5 mM heparin, while the hydrogel containing 1.25 mM heparin is stiffer than those containing 2.5 mM and 5 mM heparin. The presence of 2 mM ATIII-F has caused a slight increase of stiffness for the hydrogel containing 1.25 mM heparin (Supporting Information Figure S5). The structures of peptides and heparin as well as their interaction in the physical hydrogel represent a very important subject for our future research to understand the matrix network. It seems that there are sufficient binding sites for the peptide tags in the noncovalently assembled matrix, as the presence of peptide tags do not cause remarkable effects on the hydrogel. While the hydrogel pore sizes are in the micromolar range, the release of peptide tags could be mainly mediated by the heparin−peptide interaction, and thus relatively independent from the hydrogel formulation and pore size. Confocal fluorescence microscope images have indicated hydrogel pore sizes in a similar range. (Supporting Information Figure S6). We investigated the stability of ATIII peptide against proteolysis, as well as the possibility to improve the stability by using its all-D analogue, as heparin interacts with many peptides in both all-L or all-D configurations.20 As expected, while ATIIIR can be degraded by trypsin, whereas its all-D counterpart did not show any detectable proteolysis (Supporting Information Figure S7). While different drug delivery systems would require different stabilities of the tags in vivo, this method provides us the opportunity to tune their stability. 3.3. Injection of Hydrogel. To demonstrate that the releasing system is compatible with medical applications, 2063

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Figure 8. Bioactivity analysis of ATIII-CsA. (a) Bioactivity of CsA and ATIII-CsA. ATIII-CsA can selectively inhibit cyclophilin (Cyp), but not calcineurin (CaN). (b,c) CD4+ enriched eFluor670-labeled T cells were cultured in the presence or absence of 1 μM cyclosporin A (CsA) or its derivative ATIII-CsA for 3 days. (b) Representative histograms of eFluor670 dilution. Open histograms show cell proliferation of stimulated cells with numbers indicating the percentage of cells that have divided (i.e., inside the respective gate). Negative DMSO control (black), ATIII-CsA (red), and CsA (blue) as a control for inhibition of proliferation. Closed histograms show nonstimulated cells (DMSO control) (gray, 96.6 ±0.3% undivided cells). (c) Total number of viable cells after 3 days of culture. Dots and horizontal lines in graphs indicate individual replicates and mean values, respectively. (d) Dose−response curve for the inhibition of the PPIase activity of cyclophilin 18 (Cyp18) by ATIII-CsA. (e) Cumulative release of ATIII-CsA from hydrogel. The supernatant (culture medium with 10% FBS) was changed every week. Error bars indicate standard deviation (n = 3−5).

KA7-starPEG/heparin hydrogel is very stable.3 The affinity binding of tag peptides to the hydrogel suggests that it could be used as a reloadable biomaterial for drug release. Based on these features, we designed a sustainable releasing system (Figure 7a). A 5 μL hydrogel was formed at one end of a glass capillary, and the capillary was filled (0.1 mL) with a solution of ATIII-F, KA7F, or KA5-F at high concentration (2.0 mM). The other end of the capillary was sealed. Each capillary was placed in 50 mL of

3.4. Constant Release of Peptide Tags through Hydrogel as a Control Barrier. In a simple releasing system, the repertoire of (BA)n peptides cannot cause constant release. Unlike the ATIII, the (BA)n tags do not form cluster in the hydrogel, and their releases are governed by Fick’s second law of diffusion. Whereas ATIII represents an exception as an affinity tag, we asked whether there is a general method to engineer a system for a constant release of the repertoire of affinity tags. The 2064

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PBS buffer, and the release of the fluorescent compound was measured by fluorescent spectrometry (Figure 7b). Given that the volume of buffer was 500 times larger than the reservoir in the capillary and 10 000 times larger than the hydrogel, it could be considered as an infinite sink for the release experiment over a long period of time. In this design, the hydrogel functions as both an affinity column to capture tagged compound from the reservoir and a drug releasing system to the supernatant. Because the release of tagged compounds is driven by the gradient between reservoir and supernatant, the kinetics should be different from the release system described before. Remarkably, the sustained release of peptides over the period of 31 weeks (Figure 7b) was similar to their profiles of the first hours in the buffer/hydrogel system (Figure 1c). The hydrogel at the end of the capillary was still intact after more than 1 year. Such systems could lead to constant drug releasing devices, whose reservoirs do not need to be refilled regularly. In this experiment, the tagged compounds were affinitycaptured from one end of the hydrogel and released from the other end of the hydrogel. A weak heparin binder such as KA5 will cause a slower loading and faster release as compared to the relatively strong binders KA7 and ATIII. This could explain why the three compounds have shown similar release profiles from the reservoir. It is worthy to note that by changing the design of loading and releasing, such as the in situ loading (Figure 1), the loading to a preformed hydrogel (Figure 3), as well as this design of reservoir system (Figure 7), completely different release profiles of a tagged compound could be achieved. 3.5. Release of Tagged Bioactive Cyclosporin A. The immunosuppressive drug cyclosporin A (CsA) has a complex mechanism for its biological functions. It inhibits both the peptidyl prolyl cis/trans isomerase cyclophilin and the phosphatase calcineurin, but only the calcineurin inhibition is associated with its immunosuppressive activity.37 CsA is very hydrophobic and thus surfactants, such as cremophor EL, have been used in its formulation. In recent years, nonimmunosuppressive cyclophilin inhibitory CsA derivatives have been used in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) treatment,37,38 in studying distal renal tubular acidosis,39 as well as for neuro-protective therapy. We have reported a nonimmunosuppressive CsA derivative Cs9,40 which has been shown to protect brain mitochondria from permeability transition.41,42 These results suggested a sound basis for new therapies for cerebral ischemia and its devastating consequences. Most importantly, Cs9 can selectively inhibit cyclophilin, thus avoiding the adverse risks of massive immunosuppression. To pave the way for creating an injectable biomaterial for neuro-protective therapy and thus bypass the requirement in drug design of crossing blood-brain-barrier, a release system for highly soluble and nonimmunosuppressive cyclosporin derivatives would be of great interest. We aimed to design a tagged CsA derivative that could inhibit cyclophilin, but does not cause immunosuppression (Figure 8a). The residue 1 of CsA is important for its interaction with the immunological target calcineurin, but not the presenter protein cyclophilin. Therefore, modification at this position has been expected to impair the calcineurin inhibition and immunosuppression.43 An ATIII tag was conjugated to position 1 of CsA and the immunosuppressive effect of ATIII-CsA on murine T cells and T cell subsets was tested.44 As shown in Figure 8b, CsA inhibited the proliferation of stimulated T cells, while no inhibitory effect was observed in the ATIII-CsA treated samples. CsA treatment reduced the viability of T cells, whereas ATIII-CsA treatment had no effect

(Figure 8c). On the other hand, the ATIII-CsA is a potent inhibitor of cyclophilin isomerase activity (Figure 8d), with a Ki value of 58 ± 3 nM. Therefore, the ATIII-CsA is a monospecific cyclophilin inhibitor without immunosuppressive activity. The release of ATIII-CsA from hydrogel at 37 °C was monitored using the inhibition assay of cyclophilin (Figure 8d/ e). The supernatant was changed every week. The supernatant samples inhibited cyclophilin in the enzyme activity assay over the entire experimental period of 5 weeks (Figure 3e). These experiments have demonstrated that the hydrogel system can be used to release a drug conjugate for 5 weeks in a stable and constant manner.

4. CONCLUSION A biomaterial design with an integral system of drug delivery is reported, where both the assembly process of matrix formation and affinity capture/release of tagged compounds are based on the noncovalent interaction of heparin with one class of peptide. It was found that the release profiles of the (KA)n and (RA)n tags have a simple structure−function relationship. On the other hand, the ATIII tag behaves with a different mechanism and possesses release kinetics close to zero order. It was demonstrated that this system is compatible for many different applications, such as reloadable releasing systems and injectable material. Furthermore, we have shown the application as portable tag for designing conjugate of particular drug molecule, e.g., CsA. The ATIII peptide and the (BA)n peptides have exhibited completely different release profiles. It will be very interesting to investigate the release of other peptide motifs in the future. For example, a number of peptides containing repetitive lysine-rich motifs have anticancer and antimicrobial activity.45−47 The study of these peptides could result in releasable tags, which are themselves therapeutic reagents.

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ASSOCIATED CONTENT

S Supporting Information *

Complete experimental procedures for peptide and peptide− starPEG conjugate synthesis, hydrogel preparation, release experiment setup, ATIII-CsA activity assay, and immunosuppressive assay are given in the Supporting Information. This material is available free of charge via the Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (+) 49 351 463 43040. Fax: (+) 49 351 463 40322. Email: [email protected]. Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Ulrike Hofmann and Peggy Berg for technical support. We thank Michael Thompson for critical reading of the manuscript. R.W., W.L., A.B., and Y.Z. were supported by the Bundesministerium für Bildung und Forschung (BMBF) through Grant 03Z2EN12. 2065

dx.doi.org/10.1021/bm500186a | Biomacromolecules 2014, 15, 2058−2066

Biomacromolecules

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Article

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ABBREVIATIONS Cyp, cylophilin; CsA, cyclosporin A; CaN, calcineurin; ECM, extra cellular matrix; VEGF, vascular endothelial growth factor; FGF-2, fibroblast growth factor 2, BMP-2, bone morphogenetic protein 2; SDF-1α, stromal-cell derived factor-1α; ATIII, antithrombin III; PEG, poly(ethylene glycol); starPEG, fourarm PEG polymer; FBS, fetal bovine serum; HIV, human immunodeficiency virus and; HCV, hepatitis C virus

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dx.doi.org/10.1021/bm500186a | Biomacromolecules 2014, 15, 2058−2066