Melittin-Containing Hybrid Peptide Hydrogels for Enhanced

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Melittin-Containing Hybrid Peptide Hydrogels for Enhanced Photothermal Therapy of Glioblastoma Honglin Jin, Guifang Zhao, Jianli Hu, Quanguang Ren, Kui Yang, Chao Wan, Ai Huang, Pindong Li, Jue-Ping Feng, Jing Chen, and Zhenwei Zou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06431 • Publication Date (Web): 17 Jul 2017 Downloaded from http://pubs.acs.org on July 18, 2017

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ACS Applied Materials & Interfaces

Melittin-Containing Hybrid Peptide Hydrogels for Enhanced Photothermal Therapy of Glioblastoma †

Honglin Jin,1 Guifang Zhao,1† Jianli Hu,1† Quanguang Ren,† Kui Yang,† Chao Wan,† Ai Huang,† ‡

Pindong Li,† Jue-Ping Feng, Jing Chen,†,* and Zhenwei Zou†,* †

Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and

Technology, 1277 JieFang Avenue, Wuhan 430022, China. ‡

Department of Oncology, PuAi Hospital, Tongji Medical College, Huazhong University of

Science and Technology, Wuhan 430034, China

Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 JieFang Avenue, Wuhan 430022, Hubei Province, China. Fax: +86-2765650733; Tel: +86-27-85873100;

1

These authors contributed equally to this work.

ACS Paragon Plus Environment

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ABSTRACT. The design of biocompatible and efficacious anticancer biomaterials to achieve relatively low tumor recurrence rates is the main pursuit of cancer photothermal therapy (PTT). RADA 16-I is a synthetic amphiphilic peptide with the sequence RADARADARADARADA that can self-assemble into a peptide nanofiber hydrogel. In this study, we synthesized a novel Melittin-RADA32-Indocyanine green (ICG) hydrogel (“MRI hydrogel”), which contains melittin in the peptide hydrogel backbone and ICG in the hydrogel matrix, for enhanced PTT of glioblastomas. The MRI hydrogel exhibited physiologic characteristics similar to those of RADA16 hydrogel, while displaying concentration-dependent cytotoxicity to C6 glioma cells and photothermal effects. The in vivo biodistribution of the MRI hydrogel was visualized by NIR fluorescence and photoacoustic imaging. More importantly, in vivo PTT provided by the MRI hydrogel significantly reduced the tumor size and the tumor recurrence rate compared with the RADR16-ICG hydrogel and other controls, suggesting a synergistic effect of MRI hydrogelcarried melittin and ICG-based PTT treatment. Thus, MRI provide an alternative tool for the safe and efficient PTT treatment of tumors.

KEYWORDS. Peptide hydrogel, combination therapy, photothermal therapy, anticancer drug, melittin delivery


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INTRODUCTION Photothermal therapy (PTT), a hyperthermia-based therapeutic approach that employs photothermal conversion agents (PTCAs), is a powerful alternative over traditional cancer therapies for treating diverse tumors, mainly due to its spatiotemporal control and its precise and effective regulation of lesion destruction without the need of surgical intervention.1-3 The implementation of PTT for treating lesions often involves the use of PTCA-containing biomaterials, including inorganic, organic, and inorganic-organic hybrid materials.4 Among these, inorganic materials, such as gold nanoparticles,5-6 carbon nanotubes,7-8 graphene-based composites,9-10 and rare-earth nano-oxides,11 possess excellent photothermal conversion efficiencies but often face potential biodegradability concerns when applied for clinical translation.12-14 Pure organic photothermal materials, such as porphysomes,15 polypyrrole organic nanoparticles,16 and materials loaded with near-infrared (NIR) dyes (e.g., indocyanine green, ICG),17-18 have therefore raised significant attention recently. On the other hand, several studies have demonstrated that the combination of chemotherapy or other treatment modalities with PTT often results in synergistic antitumor effects.19-21 These may further decrease the rate of tumor recurrence, which is typically high in cancer PTT. One potential option to circumvent these issues is the use of peptide hydrogels, which are highly hydrated, porous, and biocompatible nanofibers formed by the crosslinking of short peptide molecules induced by stimuli such as temperature, pH, and salt.22-23 For example, the typical 16-residue peptide, RADARADARADARADA (RADA16-I), which possesses four repeats of RADA motifs with alternating hydrophobic and hydrophilic amino acids, can selfassemble into interwoven nanofibers under physiological conditions or in the presence of monovalent cations.24 Previously, these peptide hydrogels have been widely applied for 3D cell


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cultures, regenerative tissue engineering, and drug delivery.25-30 Therefore, various active agents have been incorporated, mainly by applying one of the following two approaches: (1) Gel matrix loading. Bioactive agents (such as enzymes and chemotherapeutic drugs) are encapsulated into the porous gel matrix to create reservoirs for sustained localized payload delivery. For example, Liu et al. have reported a RADA16-paclitaxel hydrogel for the controlled release of paclitaxel and inhibition of tumor cell growth in vitro.31 Therefore, peptide hydrogels might be excellent vesicles for PTCA delivery by gel matrix loading; (2) Peptide scaffold loading. Active agents (e.g., functional peptides) are chemically conjugated or directly coupled to designer peptide scaffolds. However, these fusion peptide systems comprising designer and functional peptide motifs are not ready to gelate. For example, Wang et al. have reported a hybrid peptide of RADA16-FGL

(containing

the

motif

of

the

neural

cell

adhesion

molecule,

EVYVVAENQQGKSKA) for spinal cord injury repair.32 However, this fusion peptide only formed a hydrogel after mixing with RADA16, limiting its scaffold cargo loading capacity. Additionally, synthesis and application of hybrid peptide hydrogels containing therapeutic peptides for in vivo cancer therapy have never been reported. Therefore, it would be desirable to develop a multifunctional peptide hydrogel platform based on a tailor-made gel for accommodating both therapeutic peptides and PTCAs. To construct a therapeutic hybrid peptide hydrogel scaffold, the selection of a suitable cytotoxic peptide (CP) is a prerequisite. Up to date, a variety of therapeutic peptides, mainly antimicrobial peptides, has been widely used in preclinical and clinical studies.33-34 Among these antimicrobial peptides, melittin, a cationic polypeptide composed of 26 amino acids derived from bee venom, is a potent anticancer agent mainly attributed to its strong membrane-disrupting ability.35 However, when used alone, the in vivo application of melittin is hampered by


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hemolysis as its main side effect.36 Recent studies suggest that the encapsulation of melittin into nanoscale materials can reduce hemolysis, possibly by shielding the direct interaction with red blood cells and normal tissue cells.37-38 Therefore, we hypothesize that scaffold loading of melittin onto peptide hydrogel nanofibers would likely not only attenuate hemolysis but also augment the antitumor efficacy of peptide hydrogel-based PTT. Here, we report the novel self-assembling peptide hydrogel melittin-RADA32-ICG (MRI), in which melittin was fused with RADA32 to produce a hydrogel scaffold into which ICG molecules were encapsulated. Taking advantage of its excellent biocompatibility and versatile cargo loading ability, this study aims to establish a multifunctional hybrid peptide hydrogel platform for the simultaneous melittin and ICG delivery to tumors, high antitumor efficacy, as well as NIR fluorescence and photoacoustic imaging, thus providing an alternative tool for the safe and efficient in vivo PTT treatment of tumors.

RESULTS Synthesis and Characterization of MRI Hydrogel. To develop a melittin-containing peptide hydrogel, we first designed an RADA16-melittin fusion peptide (RADARADARADARADA– GG–GIGAVLKVLTTGLPALISWIKRKRQQ–NH2) as a building block for the therapeutic peptide hydrogel. As expected, RADA16 gelates at a concentration of 0.5–2% in the presence of 0.9% NaCl (w/w). However, RADA16-melittin fusion peptide did not form any hydrogel even at higher concentrations than 2% (data not shown), suggesting that the incorporation of melittin into the peptide backbone affects the gelation of RADA16. To address this issue, we increased the weight ratio of the RADA motif by synthesizing a RADA32-melittin fusion peptide (Figure 1A). In the presence of 0.9% NaCl, ICG-loaded RADA32-melittin (1%, w/w) successfully formed


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melittin-RADA32-ICG (MRI) hydrogel, while direct mixing of melittin and ICG solution produced no hydrogel (Figure 1B). We next investigated size and shape, secondary structure, and melittin/ICG release properties of the MRI hydrogel by transmission electron microscopy (TEM), circular dichroism (CD), and membrane dialysis, respectively. As revealed by TEM, RADA16, melittin-RADA32 (MR), and the MRI hydrogel self-assembled into networks of interwoven nanofibers with diameters of 8.1 ± 2.5, 11.0 ± 2.5, and 16.2 ± 2.8 nm, respectively (Figure 1C). CD measurements exhibited negative maximum molar residue ellipticities at 216 nm for both RADA16 and MRI hydrogel, which is characteristic for a β-sheet structure, clearly indicating that the fusion of melittin and RADA32 peptide does not hinder β-hairpin folding and β-sheet formation. Moreover, the negative maximum molar residue ellipticities at 208 and 222 nm (Figure 1D), a typical spectrum for an α-helix structure, further supports the successful encapsulation of melittin into the MRI hydrogel. As the ICG molecules loaded in the MRI hydrogel interfere with an accurate measurement of the peptide concentration, we examined the release of the MR hydrogel-loaded peptide under physiologic conditions instead of the MRI hydrogel itself. According to Figure 1E, the MR hydrogel exhibited a remarkably slower peptide release rate compared with free melittin (Figure 1E). At 24 h, melittin was completely released from the dialysis membrane, while the MR hydrogel only exhibited a release rate of 42%. Compared with the release profile of the MR hydrogel backbone peptide, MRI hydrogel-loaded ICG was quite stable and showed only marginal content release (17% at 24 h; Figure 1F). In contrast, free ICG exhibited a very quick release profile with 51% of the ICG released from the dialysis membrane at 24 h and 62% was released at 48 h. Taken together, these results show that we successfully synthesized a hybrid, ICG-loaded melittin-RADA32 peptide hydrogel.


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Cytotoxic Properties. Hemolysis presents the main side effect of melittin, which limits its in vivo application. As MR hydrogel exhibited a much slower peptide release rate than melittin, we hypothesized that the incorporation of melittin into MR hydrogel might reduce its cytotoxicity. To test this hypothesis, we accessed the hemolysis effect of MR hydrogel using red blood cells (RBC). As shown in Figure 2A and B, only minimal hemoglobin release (11.5%, n = 3) was observed for MR hydrogel with a peptide concentration of 50 µM. However, very low concentrations of melittin (3.2 µM) and Triton (1%; positive control) resulted in the complete lysis of RBC. To explore whether the melittin encapsulation into the MRI hydrogel via backbone loading would impart the cytotoxic properties of the MRI hydrogel, we evaluated its antitumor efficiency against murine glioma C6 cells by calcein-AM and PI staining after 24 h incubation. In this experiment, different volumes of the MRI hydrogel were pre-added into 96-well plates prior to incubation with C6 cells. Viable cells and dead or late apoptotic cells were stained green with calcein-AM and red with PI, respectively, which revealed only green signals for the RADA16 hydrogel group, indicating little or no adverse effect on C6 cells (Figure 3A). In comparison, treatment with a relatively smaller concentration of MRI hydrogel (200 µM) resulted in significant cell death or late apoptosis, as indicated by a large number of red signals, while a relatively larger MRI hydrogel concentration (400 µM) led to almost complete cell death or late apoptosis. A quantitative experiment was also performed using the MTT assay. According to MTT, RADA16 hydrogel slightly increased the cell viability under the tested conditions (100– 400 µM). As for MRI hydrogel, the addition of 100, 200, and 400 µM of MRI hydrogel resulted in cell death rates of 36%, 77%, and 98% (Figure 3B), respectively, indicating concentrationdependent toxicity to C6 cells.


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Next, we evaluated the mechanisms responsible for MRI hydrogel-directed cell death based on flow cytometry and TEM analysis. In this experiment, cells were pre-seeded in 6-well plates and then treated with different concentrations of MRI hydrogel. As shown in Figure 3C, RADA16 hydrogel had no additional adverse effect on C6 cells compared to the PBS control. However, the percentages of early apoptosis and late apoptosis or necrosis induced by MRI hydrogel were 19.5% and 50.7%, respectively, suggesting that late apoptosis or necrosis was the main cause of cell death. We further detected the ultrastructural changes of MRI hydrogel-treated C6 cells. In contrast to control cells, we clearly observed typical morphological features of apoptosis, including chromatin condensation, cell shrinkage, vacuole formation, and mitochondrial swelling (Figure 3D and S1), indicating that MRI hydrogel is capable of inducing cellular structure changes and cell apoptosis. Photothermal and Photoacoustic Properties. After confirming the direct cell-killing capability of the MRI hydrogel, we next examined its photothermal and photoacoustic properties. Temperature changes and photoacoustic signals were recorded by a hand-held thermal infrared camera and a multispectral photoacoustic tomography imaging system, respectively. When subjected to continuous irradiation with an 808-nm NIR laser, PBS solution and RADA16 hydrogel exhibited no temperature increase (Figure 4A,B). Under the same condition, dramatic temperature changes correlated to temperature enhancement of 30–50 °C were detected for MRI hydrogel and RADA16-ICG (RI) hydrogel, suggesting strong photothermal conductivity. Encouraged by this photothermal effect of the MRI hydrogel, we investigated whether it could be applied as a photoacoustic tomography (PAT) contrast agent. As expected, the MRI hydrogel exhibited intensive photoacoustic signals, which were of comparable intensity to those of an ICG solution with the same ICG concentration (Figure 4C). Furthermore, we studied the in vivo PAT


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imaging capability of the MRI hydrogel in C6 tumor-bearing mice. At 3 h post injection, the MRI hydrogel showed significant PAT signals in tumors (red signal), which were rich in blood vessels (green signal; Figure 4D). However, the injection of ICG solution only resulted in very weak PAT signals, indicating the superiority of the MRI hydrogel over free ICG for an application as PAT contrast agent (Figure S2). Taken together, these results indicate that the loading of the MR hydrogel with ICG conferred photothermal and photoacoustic properties to the resulting MRI hydrogel. In Vivo Biodistribution of MRI Hydrogel. To further study the NIR imaging capability of the MRI hydrogel and its in vivo biodistribution patterns, whole-body NIR fluorescence imaging was performed in C6 tumor-bearing mice after intratumoral injection of the MRI hydrogel or ICG solution at various time points. For ICG-treated mice, strong fluorescence signals were not only confined to the tumor area, but also spread to tumor-surrounding tissues at 5 min post injection and continued to spread until 3 h post injection (Figure 5A). However, these signals were dramatically decreased at 24 h post injection, indicating quick clearance or rapid degradation of ICG. In contrast, no fluorescence signal could be observed at the tumor area at 5 min post injection for MRI hydrogel-treated mice, whereas the signal became detectable at 3 h and was increased at 24 h post injection (Figure 5A). It is noticeable that the signal was mainly distributed at tumor areas, demonstrating the maintenance of the MRI hydrogel’s gel shape after intratumoral administration. This suggests that the fluorescence of the MRI hydrogel maintained highly quenched at earlier time points and protected ICG from rapid clearance and degradation, which may facilitate efficient PTT treatment. Moreover, the main tissues were dissected at 24 h post injection and subjected to NIR fluorescence imaging. This clearly exhibited significantly enhanced fluorescence signals for tumor tissues collected from the MRI hydrogel-treated group


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compared with the ICG-treated group (Figure 5B,C), which is consistent with in vivo imaging results. Collectively, these results demonstrate the feasibility of using NIR imaging to track the MRI hydrogel distribution in vivo. In Vivo Antitumor Properties.To further investigate the in vivo PTT effect of the MRI hydrogel, we then tested its antitumor effectiveness in a C6 tumor-bearing mouse model. When the tumor size reached 75 mm3, the mice were injected with MRI hydrogel, RI hydrogel, or PBS. These mice were divided into the following five groups, and some of these groups were subjected to 808-nm NIR laser irradiation at 24 h post injection at 2 W cm−2 for 8 min: (a) PBS control, (b) PBS + laser, (c) MRI gel, (d) RI gel + laser, (e) MRI solution, and (f) MRI gel + laser. The injection of MRI hydrogel or RI hydrogel led to an increase of the tumor temperature of 22 °C, which increased up to 53 °C upon NIR laser irradiation (Figure 6A). In comparison, only a slight temperature change (∆T = 8 °C) was observed for the control group (PBS + laser), suggesting that the injected MRI or RI hydrogel provided sufficient hyperthermia to fight cancer cells upon NIR laser irradiation. Following the laser treatment, mice in the MRI hydrogel/laseror RI hydrogel/laser-treated group developed eschars on the tumors, in contrast to the laser or MRI hydrogel alone groups (Figure 6B). During the first several days, the eschars healed and the tumors in both the MRI hydrogel/laser- and RI hydrogel/laser-treated groups have been destroyed (Figure 6C), indicating that the PTT treatment with ICG-loaded peptide hydrogel was effective. However, all tumors in the hydrogel/laser-treated group recurred with a significantly larger tumor size compared with the tumors of the MRI hydrogel/laser group at twelve days post hydrogel injection (Figure 6D), suggesting a critical role of melittin in preventing tumor recurrence. Unlike the MRI hydrogel/laser group, tumors in mice that received PBS or PBS/laser


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treatment retained a rapid growth rate. Compared with the PBS control group, a tumor inhibition rate of 99% was obtained, as calculated by both tumor volume and tumor weight (Figure 6E). Moreover, we observed that MRI hydrogel injection remarkably shrank the tumor size by 69% (calculated based on tumor weight) compared with the PBS control group, indicating high antitumor efficacy of the MRI hydrogel. Interestingly, MRI solution exhibited a similar tumor inhibitory effect (77%, calculated based on tumor weight) compared with MRI hydrogel , possibly due to the rapid gelation of the MRI solution after intratumor injection (data not shown). We further recorded the mice’s body weight during the experiments to evaluate the efficacy of the MRI hydrogel against C6 tumors. As shown in Figure 6F, no obvious body losses in groups of mice that received MRI hydrogel/laser or MRI hydrogel alone treatment were observed. Additionally, histological examination of the tissues taken from the MRI hydrogel-treated mice showed no obvious histological changes compared to those of control mice (Figure S3). Altogether, these results indicate that our hydrogel system has good biocompatibility when locally administered. We further examined the dissected tumors histologically to evaluate changes of the tumor cells. In contrast to the control groups, the MRI hydrogel alone group showed obvious drug responses, as revealed by a significant amount of apoptotic cells, vacuole degeneration, as well as cheese necrosis areas (Figure 7). Strikingly, a large number of relatively small nucleus cells were detected in the periphery of vacuole areas, which appeared to be infiltrated lymphocytes, indicating inflammatory reactions induced by tumor apoptosis or necrosis. This suggests that the MRI hydrogel can induce tumor cell apoptosis or necrosis in vivo.

DISCUSSION


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Peptide hydrogels represent attractive molecular building blocks due to their ease of synthesis, versatile gelation approaches, and excellent biocompatibility.23 For the delivery of anticancer drugs, especially in PTT, it would be desirable to load PTCAs together with therapeutic agents to produce synergistic antitumor effects for reducing the tumor recurrence rate.20-21 We successfully incorporated melittin into the backbone of RADA hydrogel and encapsulated ICG into the gel matrix to construct a multifunctional hydrogel. This was achieved by increasing the RADA motif to construct a RADA32-melittin peptide, as the RADA16-melittin peptide was not able to form a hydrogel. In fact, our group, as well as other groups, failed to synthesize active peptide (AP)containing hydrogels from a single RADA16-AP fusion peptide, possibly due to the limited number of RADA motif repeats of RADA16, which is insufficient to carry the relatively larger AP cargo.32, 39 Previously, Standley et al. reported a method to incorporate CPs to form peptide nanofibers by making a peptide-amphiphile, which comprises a hydrophobic alkyl tail, a β-sheet promoting sequence, and a CP.40-41 Although this peptide-amphiphile efficiently delivered CPs, the amphiphile itself, without conjugated CPs, was toxic to cells, and the synthesis involved complicated chemical reaction steps. In this context, we have developed a facile synthesis approach to prepare hybrid functional hydrogels self-assembled from a single fusion peptide. A variety of inorganic nanomaterials has been applied for cancer PTT due to their excellent photothermal effects resulting from strong NIR absorbance.15-16, 18 To the best of our knowledge, this is for the first time that hybrid peptide hydrogels were used for the photothermal treatment of tumors. In our study, the MRI hydrogel did not only show effective photothermal effects, but also revealed the following unique properties: (1) Unique anti-tumor effect. In this study, we found that the viability of RADA16 hydrogel-treated cells marginally increased under our tested conditions (Figure 3B). In fact, Tang et al. observed a similar phenomenon that at a relatively


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lower concentration (

Melittin-Containing Hybrid Peptide Hydrogels for Enhanced

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