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. 2019 Mar 15;14:27–35. doi: 10.1016/j.isci.2019.03.008

Injectable Peptide Hydrogel Enables Integrated Tandem Enzymes' Superactivity for Cancer Therapy

Qingcong Wei 1,2, Shan Jiang 1, Rongrong Zhu 1, Xia Wang 1, Shilong Wang 1,∗, Qigang Wang 1,3,∗∗
PMCID: PMC6438909  PMID: 30921734

Summary

Elevation of the levels of reactive oxygen species and other toxic radicals is an emerging strategy to treat certain cancers by modulating the redox status of cancer cells. The biocatalytic upregulation of singlet oxygen by neutrophilic leukocytes should utilize robust enzymes and design carriers with protective microenvironment. Here, we utilize GOx-CPO as integrated tandem enzymes to in situ generate singlet oxygen, which could be not only for oxidative cross-linking of injectable hydrogel carriers but also for continuous tumor treatment by adjustable bioconversion of blood oxygen, glucose, and chloride ion. The tandem enzymes self-restrained within peptide hydrogel exhibited superactivity for upregulating singlet oxygen relative to free enzymes, which also avoids the diffusion of enzymes from tumor. This work will not only deepen the study of enzymes in biocatalysis but also offer an enzyme therapeutic modality for treating cancers.

Subject Areas: Biological Sciences, Medical Biochemistry, Cancer

Graphical Abstract

graphic file with name fx1.jpg

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Highlights

  • •

    Injectable peptide-based hydrogel was formed by tandem enzymatic 1O2 cross-linking

  • •

    Enzymes restrained in gel matrix exhibited superactivity for upregulating singlet oxygen

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    The hydrogel restricted enzymes within tumor rather than diffusing into normal tissues

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    The continuous upregulated 1O2 gave the hydrogel significant antitumor efficacy

Biological Sciences; Medical Biochemistry; Cancer

Introduction

Reactive oxygen species (ROS) including singlet oxygen (1O2), superoxide radicals, hydroxyl radicals, and H2O2 are formed as a natural by-product of the normal metabolism of oxygen, which have important roles in cell signaling and metabolic stability maintenance. Although the increase of ROS under environmental stimulus, called oxidative stress, is harmful to cells and organisms (Au and Madison, 2000, Balasubramanian et al., 1990, Lambrechts et al., 2005, Schrag et al., 2013, Shen et al., 1996), a high level of ROS can suppress tumor growth and resist bacterial infection. Therefore, ROS-elevating and ROS-eliminating strategies have been developed with the former being predominantly used for cancer therapy. As a successful exploration, photodynamic therapy utilizes in situ photosensitizers and light activation to elevate toxic ROS, especially singlet oxygen, for treating certain cancers (Celli et al., 2010). Obviously, in cells, the production of singlet oxygen rarely depends on light. In fact, the destruction of microorganism after phagocytosis by neutrophilic polymorphonuclear leukocytes relies on tandem enzymatic upregulation of ROS, which utilizes nicontinamide adenine dinucleotide phosphate oxidase to produce endogenous H2O2 or superoxide radical and further upregulates the singlet oxygen or hypochlorous acid level by myeloperoxidase (Klebanoff, 2005, Ying, 2008). Therefore, the bioinspired upregulation of singlet oxygen by integrating tandem enzymes could be an alternative pathway, which involves glucose oxidase (GOx) for the generation of H2O2 and chloroperoxidase (CPO) from Caldariomyces fumago to upregulate singlet oxygen.

Enzymes in organisms are spatially restricted in tiny sophisticated subcellular organelles, and tandem enzymatic reactions could have higher overall reaction efficiency, even superactivity. This could be ascribed to the fact that intermediates generated in the previous reaction could be used promptly by the next enzymatic reaction before they diffuse into cytosols. It is evident that biomimetic enzyme nanocomposites, especially enzyme cascade system, could exhibit excellent catalytic efficiency and enhanced stability via the smart scaffold design (Liu et al., 2013, Wilner et al., 2009). Inspired by this, some vehicles could be designed and used to imitate tandem enzymatic reactions. Therefore the indispensable property of the vehicle is to entrap enzymes so that it could not only effectively retain enzymes around tumor cells and improve the bioavailability but also protect enzymes to maintain their activity, thereby allowing for continuous treatment (Hah et al., 2011, Parsons et al., 2009). Injectable and self-healing supramolecular hydrogels, including peptide-based ones are a particular promising platform due to their high structural similarity with extracellular matrices (ECMs), which could offer an ideal microenvironment for enzymatic catalysis (Medina et al., 2017, Tian et al., 2014, Wang and Yang, 2012, Wang et al., 2007, Zhou et al., 2014). Previous work from our laboratory has shown that enzymes integrated in optimized gel microenvironment maintain their high catalytic activity, even superactivity (Mao et al., 2014, Su et al., 2013, Wei et al., 2016a, Wei et al., 2016b). To enhance the antidegradable properties as biomedical vehicles (Jin et al., 2013, Puppi et al., 2010, Wang et al., 2010), peptide hydrogels could be covalently cross-linked with polysaccharide and synthetic polymers (Dai et al., 2015, Dong et al., 2017, Jeon et al., 2016, Sun et al., 2012, Yang et al., 2012). We hereby fabricated a mechanically enhanced peptide hydrogel by using in situ singlet oxygen cross-linking reaction (Cornwell and Smith, 2015, Schmidt and Summerer, 2013).

A novel strategy was developed by combining a GOx-mediated redox (Shenoy and Bowman, 2012, Wei et al., 2016a, Wei et al., 2016b, Wu et al., 2015) and a CPO-mediated redox (Kanofsky, 1984) into one cascade reaction system (Scheme 1) for controlled generation of singlet oxygen (Adam et al., 2005). Specifically, GOx oxidizes glucose to result in an elevated H2O2 level, which could be further used by CPO-H2O2-chloride system to upregulate the singlet oxygen level. Here, we prepared a furoyl-functionalized oligopeptide (NapFFK-furoyl, Figure S1A) and a furoyl derivative of glycol chitosan (GCF, Figure S1B). The peptide hydrogel with enhanced mechanical properties was then fabricated via the in situ singlet oxygen cross-linking catalyzed by GOx-CPO integrated tandem enzymes system, which could not only allow for rapid encapsulation of enzymes during gelation rather than diffusing into normal tissues but also enable syringe delivery of the gel into tumors. Moreover, the precursor solution containing CPO, GOx, NapFFK-furoyl, and GCF is able to form in situ hydrogels in tumors so that the encapsulated GOx and CPO utilize blood oxygen, glucose, and chloride to continuously generate singlet oxygen, which results in effective cancer cell apoptosis. Furthermore, the consumption of intratumoral oxygen and glucose by GOx likely starves cancer cells to death thus enhancing the anti-cancer efficacy (Fan et al., 2017, Huo et al., 2017, Li et al., 2017, Narayanan et al., 2016, Zhang et al., 2017, Zhang et al., 2018).

Scheme 1.

Scheme 1

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Schematic Illustration of the Hybrid Hydrogel Preparation via Dual-Enzyme (GOx and CPO)-Initiated Singlet Oxygen Cross-linking of NapFFK-furoyl (1.0 wt %) and GCF (1.0 wt %)

See also Figure S1.

Results and Discussion

Preparation and Characterization of Hydrogels and Test of Singlet Oxygen Quantum Yield

We prepared the mechanically enhanced supramolecular hydrogel by simply mixing several of the following components to allow for enzyme-induced singlet oxygen cross-linking. Briefly, 5.0 mg NapFFK-furoyl was dissolved in 250 μL alkaline solution (pH = 10), then 0.5 M HCl solution was added to adjust pH value to 7–8, and this was followed by the addition of 167 μL GCF solution (3 wt %) and the dual-enzyme-initiated system (5 mM NaCl, 15 mM glucose, 75U GOx, and 75U CPO). The total volume was adjusted to 500 μL by adding water, and the resulting solution was mixed thoroughly and kept at 37°C for 120 min to obtain hybrid hydrogels. The final concentrations of dual enzyme are both 0.15 U per 1 mg hybrid hydrogel for GOx and CPO. As shown in Scheme 1, glucose was oxidized by GOx-mediated redox reactions, and the H2O2 product immediately participated in the oxidation of chloride via CPO-catalyzed reactions. The generated hypochlorite subsequently reacted with H2O2, and singlet oxygen was produced to oxidize the furoyl groups. The resulting endoperoxides underwent cross-linking to construct the final gel network.

Next, the morphological images obtained from scanning electron microscopy (Figures 1A and 1B) and transmission electron microscopy (Figures S2A and S2B) showed that the cross-linking process facilitated the formation of denser and more entangled irregular fibers in the hybrid gel network. These results are consistent with the frequency sweep test results (Figure 1C) that the storage modulus (G′) of the hybrid hydrogel (average 534 Pa) dominated over that of the supramolecular hydrogel (average 47 Pa). This indicated that the supramolecular hydrogel's network was effectively enhanced by the polysaccharides. Moreover, the effect of enzymes on peptide self-assembling was investigated by adding enzymes into peptide pre-gel solution. Rheological results (Figure S3) showed that the gelation was not affected and the G′ value (average 45 Pa) of supramolecular gel with enzymes was almost the same as that of supramolecular gel without enzymes. Therefore the effect of adding enzymes on peptide self-assembling is negligible. The gelation point was reached at approximately 180 s (Figure S4A), whereas the complete gelation process took approximately 2 h. During the gelation process, the viscosity increased gradually owing to the increasing degree of cross-linking as well as the self-assembly of peptide residues (Figure 1D). This gradual change in viscosity is a critical characteristic of enzymatic gelation system that guarantees the injectable property as well as the shape recovery after injection. Finally, the self-healing property (Figure S4B) of hybrid gels could be beneficial to its biomedical applications.

Figure 1.

Figure 1

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Morphological and Rheological Characterization, Singlet Oxygen Assay

(A and B) Scanning electron microscopic images (scale bar, 2 μm) of (A) cryo-dried supramolecular hydrogel (2 wt % NapFFK-furoyl) and (B) hybrid hydrogel (1 wt % hydrogelator and 1 wt % GC-furoyl). The inner images in (A) and (B) are supramolecular hydrogel and hybrid hydrogel, respectively. See also Figure S2.

(C) Frequency sweep tests of hybrid gel (1 wt % hydrogelator and 1 wt % GC-furoyl) and supramolecular gel (2 wt % NapFFK-furoyl) at a fixed strain of 0.2%. See also Figure S3.

(D) The viscosity test profile during the gelation process of hybrid gel. See also Figure S4.

(E) EPR spectra of dual enzymatic system in the presence of TEMP and DMPO.

(F) Singlet oxygen assay by the absorbance attenuation of 9,10-anthracenediyl-bis(methylene)dimalonic acid in various aqueous systems: Rose Bengal as positive control, immobilized enzymes of hybrid hydrogel, free enzymes, and blank hydrogel (without loading enzymes) as negative control. Error bars represent mean ± SD (n = 3).

See also Figure S5.

To confirm the generation of singlet oxygen from our dual-enzyme-initiated system, we used data from its electron para-magnetic resonance (EPR) spectrum as the direct evidence. 2,2,6,6-tetramethylpiperidine (TEMP) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were employed as the singlet oxygen trapping agent and other ROS trapping agent, respectively. As shown in Figure 1E, there were clear 1:1:1 triplet EPR signals for the free dual-enzyme system when adding TEMP, with no detectable signal upon addition of DMPO. Therefore the EPR results showed that the dual-enzyme-initiated system could effectively generate singlet oxygen without the simultaneous generation of other ROS such as superoxide and/or hydroxyl radicals. We next investigated the singlet oxygen quantum yield for free enzymes (Φfree), immobilized enzymes of hybrid hydrogel (Φimmob), and blank hydrogel (Φblank) in aqueous solution employing Rose Bengal (RB, ΦRB = 0.76) as a reference (Lin et al., 2013). Φfree, Φimmob, and Φblank were calculated according to the absorbance attenuation (ΔA) of 9,10-anthracenediyl-bis(methylene)dimalonic acid. The slope of a linear fitted curve corresponded to the absolute amount of singlet oxygen in which the Φimmob, Φfree, and Φblank were 0.71, 0.62, and 0.00 respectively (Figure 1F). The activity of the immobilized enzymes was slightly higher (1.14-fold) than that of the free enzymes, which might have resulted from the overall increased reaction efficiency promoted by the cascade reaction. It is possible that GOx and CPO might encapsulate themselves in close proximity during the gelation process. This would have allowed the product of H2O2 in GOx-mediated redox to be immediately utilized by CPO to generate hypochlorite and further singlet oxygen. However, GOx and CPO were separated in the free enzyme system, which resulted in a lower local concentration of generated H2O2 around CPO due to the partial diffusion of H2O2 into the bulk solution (Liu et al., 2013, Wilner et al., 2009). These results demonstrate that the dual enzyme successfully maintained their activity within the gel matrix. Moreover, the time-dependent enzymatic activity was evaluated by the same singlet oxygen quantum yield assay. Cubic pieces of 100 μL hybrid hydrogel were incubated at 37°C and tested the singlet oxygen generation at different time nodes. As shown in Figure S5, the singlet oxygen generation rate decreased gradually and reached ca. 28% after 48-h incubation. These results indicated that the dual enzyme would lose activity gradually, which would be a major parameter for the in vivo anti-tumor experiments.

Biocompatibility of Hydrogels and In Vitro Generation of Singlet Oxygen

Furthermore, we sought to determine the in vitro biocompatibility of both the gel matrix ingredients and the cell inactivation ability of the hybrid hydrogel via CCK8 assays. The glioblastoma cell line (U87 cells) was chosen as the model. Hydrogels were cut into fine fragments as standby. First, cells were cultured with blank hydrogel (without enzymes). No significant cytotoxic effects were observed (Figures 2A and S6) indicating that the gel matrix components had good biocompatibility under the treated concentrations (1 μg/mL to 100 μg/mL hydrogel). Then, the cytotoxicity of hybrid hydrogel was evaluated. Results shown in Figure S6 indicated a relative higher cell viability (>100%) at low concentrations (1 μg/mL to 5 μg/mL), whereas there was an increasing cytotoxicity with hybrid hydrogel in excess of 5 μg/mL (Figure 2A). Therefore the elevated therapeutic singlet oxygen gave the hybrid hydrogel significant cell proliferation inhibition, with an IC50 of approximately 31 μg/mL (ca. 4.65×10−3 U/mL for both GOx and CPO). The cytotoxicity of free enzymes by using the same amount of enzyme as hybrid hydrogel group was shown in Figure S7. The results exhibited similar cell viability as that of hybrid hydrogel, with an IC50 of approximately 6.20×10−3 U/mL for both GOx and CPO. Overall, the accumulative damage of fatty acids, proteins, and DNA by singlet oxygen might ultimately lead to cell apoptosis, giving it an anti-cancer effect (Kriska et al., 2005, Maes et al., 2011, Nam et al., 2016). In addition, the degradation behavior of the hybrid hydrogels was evaluated by incubating gels in PBS (pH = 7.4) for 120 h. Results in Figure S8 showed that the dry weight of hybrid hydrogels decreased gradually and eventually reached a plateau after 72 h incubation. The weight loss (ca. 32%) might be due to the loss of uncross-linked peptide molecules.

Figure 2.

Figure 2

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Cell Viability and In Vitro Singlet Oxygen Generation

(A) Cell viability of U87 with different concentrations of blank hydrogel and hybrid hydrogel. See also Figures S6 and S7.

(B) Oxidized SOSG confocal fluorescence images of U87 cells with free enzymes (0.3×10−3 U/mL for both GOx and CPO) and hybrid hydrogel (2 μg/mL) after 12-h incubation. Scale bar, 50 μm.

(C) Oxidized SOSG confocal fluorescence images of U87 cells with free enzymes (0.015 U/mL for both GOx and CPO) and hybrid hydrogel (100 μg/mL) at different time intervals. Scale bar, 50 μm.

(D) Mean fluorescence intensity in (B) and (C) at 4 h. Statistical evaluation was conducted by using analysis of variance (Student's t test).

Error bars represent mean ± SD (n = 3). p value < 0.05 was considered statistically significant. **p < 0.01, *p < 0.05.

To better understand free enzymes and hybrid-hydrogel-mediated singlet oxygen generation in tumor cells, free enzyme and hybrid-hydrogel-loaded U87 cells were incubated with Singlet Oxygen Sensor Green (SOSG) (Flors et al., 2006)—which could be rapidly oxidized by singlet oxygen to a green fluorescent molecule (SOSG endoperoxides, SOSG-EPs). As shown in Figure 2B, strong green fluorescence was observed by confocal laser scanning microscopy (CLSM) in each group after 12-h incubation at a low gel concentration (2 μg/mL) with a well fusiform shape of cells. Furthermore, the fluorescence intensity (Figure 2D) of hybrid-hydrogel-treated cells was slightly higher than that of free-enzyme-treated cells, indicating a high level of singlet oxygen generation, which suggested the higher anticancer efficacy of hybrid hydrogel than free enzymes. The same results could also be obtained at a higher gel concentration (100 μg/mL) as shown in Figure 2C. CLSM images (Figure 2C) revealed stronger green fluorescence (Figure 2D) in hybrid hydrogel group indicating a higher concentration of SOSG-EPs accumulated in cells incubated with hybrid hydrogel as compared with free enzymes. Furthermore, as shown in Figure 2C, fluorescence intensity gradually increased over time and cell morphology became spherical from its previous fusiform shape. Collectively, these results are consistent with the singlet oxygen quantum yield of Φfree and Φimmob (Figure 1F). Moreover, in vitro experimental results indicate that an encapsulated dual enzyme in the hybrid hydrogel could effectively upregulate the level of singlet oxygen, resulting in effective inhibition of the proliferation of tumor cells. Therefore, these results laid a solid foundation for the following in vivo antitumor experiments.

In Vivo Antitumor Property

At last, tumor-bearing mice were weighed and randomly divided into different treatment groups. When the tumor volume reached 100 mm3, subjects were given a single intratumoral dose (10 μL) of PBS, blank hydrogel (oligopeptide + chitosan), or hybrid hydrogel (hydrogel 454.5 mg/kg, GOx 68.2 U/kg, and CPO 68.2 U/kg) every other day. On day 12, mice were euthanized and the tumors were collected, washed, weighed three times with saline, and fixed in 10% neutral-buffered formalin. As shown in Figure 3A, the hybrid hydrogel significantly inhibited tumor growth compared with the other two groups. There was a slight increase in tumor volume in the hybrid hydrogel group (104.06 mm3). However, a rapid growth in tumor volume for the physiological saline (707.88 mm3) and blank hydrogel (678.42 mm3) treatment groups was observed (Figure 3B). Furthermore, the tumor weight was also minimal for the hybrid hydrogel group (Figure 3C). These results demonstrated not only that the hybrid hydrogel could effectively immobilize the dual enzyme in its matrix but also that the immobilized dual enzyme could effectively generate singlet oxygen to kill tumor cells. Besides, the body weights of the mice injected with either blank hydrogel or hybrid hydrogel were like those of mice receiving PBS, indicating a negligible toxicity for mice in both these groups (Figure 3D). The further histological response of these external stimuli on major organs of the mice were evaluated by H&E staining after the treatments, and no noticeable tissue damages and side effects were found (Figure S9). Therefore integrated tandem enzymes can be used for the non-invasive cancer therapy.

Figure 3.

Figure 3

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In Vivo Anti-tumor Experiments

(A) Photographs of the collected U87 tumor tissues after intra-tumoral injection with physiological saline, blank hydrogel, and hybrid hydrogel at day 12.

(B) Tumor volume changes of the three groups over the course of the treatments.

(C) Tumor weight changes of the three groups over the course of the treatments.

(D) Body weights of mice in different groups after treatment.

(E) TUNEL and Ki-67 immunostaining of xenografts. The positive expressions turn to brown color. Statistical evaluation was conducted by using analysis of variance (Student's t test).

Error bars represent mean ± SD (n = 3). p value < 0.05 was considered statistically significant. **p < 0.01, *p < 0.05. See also Figures S8 and S9.

In addition, the in vivo Ki-67 and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) detection in nude mice bearing U87 xenografts treated with PBS, blank hydrogel, and hybrid hydrogel was performed by immunohistochemistry. As shown in Figure 3E, TUNEL assay was used for detection of cell apoptosis. There were only a few positive cells in mice treated with PBS and blank hydrogel. However, a significantly increased ratio of TUNEL-positive cells was observed in hybrid-hydrogel-treated group, which proved the apoptotic induction effect of hybrid hydrogel on U87 xenografts. Furthermore, hybrid hydrogel inhibited cancer cell proliferation in a much stronger manner than PBS and blank hydrogel, as evidenced by Ki-67 staining.

Discussion

In summary, we have prepared an injectable, mechanically enhanced supramolecular hydrogel by using singlet oxygen oxidative cross-linking of furoyl-functionalized oligopeptide and glycol chitosan. The hydrogel could be intratumorally injected due to their shear-thinning property from supramolecular hydrogel networks and the increasing viscosity, which could effectively immobilize enzymes within tumor rather than diffusing into normal tissues. GOx and CPO are likely restricted in the ECM-like gel networks at a close distance, and H2O2 generated by GOx-mediated redox reaction could be used promptly by CPO-chloride catalytic system, resulting in the superactivity of immobilized dual enzyme when compared with that of the free dual enzyme. The continuous generation of upregulated singlet oxygen by integrated tandem enzyme reaction gave the hydrogel significant in vitro and in vivo antitumor efficacy. Therefore, this injectable peptide hydrogel provides a tool for localized delivery of enzymes for high-efficiency and ROS-responsive deep-seated tumor therapy.

Limitations of Study

The control of free enzymes has not been tested in vivo because free enzymes could cause discomfort (even death) of the mice after intratumoral injection.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 51473123, 31570849, 51402215, 81671105), the National Key Research and Development Program (No. 2016YFA0100800), the Scientific Research Foundation for Doctors (No. qd16112) and Youth Foundation (No. 2016QK11) of Henan Normal University.

Author Contributions

Conceptualization, S.W. and Q. Wang; Methodology, R.Z., X.W., S.W., and Q. Wang; Investigation, Q. Wei, S.J., S.W., and Q. Wang; Writing – Original Draft, Q. Wei and S.J.; Writing – Reviewing & Editing, S.W. and Q. Wang; Funding Acquisition, Q. Wei, X.W., S.W., and Q. Wang. Q. Wei, and S.J. contributed equally to this work.

Declaration of Interests

The authors declare no competing interests.

Published: April 26, 2019

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2019.03.008.

Contributor Information

Shilong Wang, Email: wsl@tongji.edu.cn.

Qigang Wang, Email: wangqg66@tongji.edu.cn.

Data and Software Availability

The authors provide detailed description of methods and original data upon request.

Supplemental Information

Document S1. Transparent Methods and Figures S1–S9
mmc1.pdf (1.7MB, pdf)

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Associated Data

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Supplementary Materials

Document S1. Transparent Methods and Figures S1–S9
mmc1.pdf (1.7MB, pdf)

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