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Nanoclustered Cascaded Enzymes for Targeted Tumor Starvation and Deoxygenation-Activated Chemotherapy without Systemic Toxicity Yinchu Ma,†,‡,# Yangyang Zhao,‡,# Naveen Kumar Bejjanki,‡,# Xinfeng Tang,‡ Wei Jiang,‡ Jiaxiang Dou,‡ Malik Ihsanullah Khan,‡ Qin Wang,‡ Jinxing Xia,∥ Hang Liu,§ Ye-Zi You,§ Guoqing Zhang,‡ Yucai Wang,*,†,‡ and Jun Wang⊥ †
Intelligent Nanomedicine Institute, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, China ‡ Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences, University of Science and Technology of China, Hefei 230027, China § Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230027, China ∥ The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China ⊥ School of Biomedical Science and Engineering, South China University of Technology, Guangzhou International Campus, Guangzhou 510006, China S Supporting Information *
ABSTRACT: Intratumoral glucose depletion-induced cancer starvation represents an important strategy for anticancer therapy, but it is often limited by systemic toxicity, nonspecificity, and adaptive development of parallel energy supplies. Herein, we introduce a concept of cascaded catalytic nanomedicine by combining targeted tumor starvation and deoxygenation-activated chemotherapy for an efficient cancer treatment with reduced systemic toxicity. Briefly, nanoclustered cascaded enzymes were synthesized by covalently cross-linking glucose oxidase (GOx) and catalase (CAT) via a pH-responsive polymer. The release of the enzymes can be first triggered by the mildly acidic tumor microenvironment and then be self-accelerated by the subsequent generation of gluconic acid. Once released, GOx can rapidly deplete glucose and molecular oxygen in tumor cells while the toxic side product, i.e., H2O2, can be readily decomposed by CAT for site-specific and low-toxicity tumor starvation. Furthermore, the enzymatic cascades also created a local hypoxia with the oxygen consumption and reductase-activated prodrugs for an additional chemotherapy. The current report represents a promising combinatorial approach using cascaded catalytic nanomedicine to reach concurrent selectivity and efficiency of cancer therapeutics. KEYWORDS: catalytic nanomedicines, cascaded reactions, clustered enzymes, deoxygenation-activated prodrug, tumor starvation therefore, be used as a target for anticancer therapy.7,10 Over the past years, drugs that inhibit glycolytic enzymes and transporters, such as glucose transporter 1 (GLUT1),11,12 hexokinase (HK),13,14 6-phosphofructo 2-kinase-fructose-2,6biphosphatase 3 (PFKFB3),15 pyruvate kinase isozyme M2
G
lucose is the most abundant nutrient in blood and is a metabolic substrate commonly consumed by cells through glycolysis. Glycolytic intermediates of cancer cells have been demonstrated to fuel several important biosynthetic pathways, including those for lipids, nucleotides, and amino acids, which are essential for cancer progression and metastasis.1−5 Since cancer cells proliferate at a much higher rate than normal ones, glucose metabolism through glycolysis is also significantly faster in cancer cells.6−9 The metabolic difference of glucose between cancer and normal cells can, © XXXX American Chemical Society
Received: March 29, 2019 Accepted: July 10, 2019 Published: July 10, 2019 A
DOI: 10.1021/acsnano.9b02466 ACS Nano XXXX, XXX, XXX−XXX
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Cite This: ACS Nano XXXX, XXX, XXX−XXX
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Scheme 1. Schematic Showing the Nanoclustered Cascaded Enzymes for Targeted Tumor Starvation and DeoxygenationActivated Chemotherapy without Causing Systemic Toxicitya
a (A) Schematic showing the preparation of nanoclustered enzymes of glucose oxidase (GOx) and catalase (CAT) crosslinked via a pH-responsive linker, which was further coated with a serum albumin shell. (B) Schematic showing the working principles of BCETPZ@(GOx+CAT): (1) the albumin shell kinetically prevents the enzymes from rapid, premature exposure to blood glucose; (2) the selective release of cascaded enzymes in the tumoral acidic microenvironment; (3) GOx consumes tumoral oxygen (O2) and creates a hypoxia environment, producing H2O2 and gluconic acid; the CAT converts generated H2O2 into H2O without causing systemic toxicity; (4) the generated gluconic acid in step 3 further accelerates the degradation of nanoclustered enzymes; (5) the albumin-TPZ (BSATPZ) prodrugs conjugated on the clusters are activated by reductase in the hypoxia environment, which synergizes the gluocose-depletion therapy.
(PKM2),16−18 lactate dehydrogenase A (LDHA),19 and monocarboxylate transporter 1 (MCT1),20 overexpressed in certain cancer types have been proven effective in either preclinical or clinical studies. Moreover, several groups have demonstrated the use of antiangiogenic therapy21,22 or blood vessel destruction to stop the blood supply that delivers glucose and other nutrients to cancers.23−25 However, previous strategies of glucose starvation usually yielded concurrent toxicities to normal cells since other types of cells, such as immune cells and stem cells, can also undergo aerobic glycolysis.26 Moreover, cancer cells could develop resistance to the inhibition of a particular metabolic pathway through the expression of alternate isoforms or the development of alternate pathways, a phenomenon known as metabolic plasticity.27
Apart from methods indirectly targeting glucose pathways, alternative strategies that can directly deplete intratumoral glucose are receiving increasing attention in anticancer therapy in that cancer cells are more sensitive to glucose concentration changes due to the cancer-specific up-regulation of intracellular aerobic glycolysis.16 Glucose oxidase (GOx) is potent for the oxidization of glucose to gluconic acid and hydrogen peroxide (H2O2), and it has been demonstrated to be effective in cancer inhibition via direct glucose depletion.28−37 Nevertheless, there are still a number of issues associated with this method. First of all, the byproduct H2O2 of glucose oxidation can initialize systemic toxicity and lethal chain reactions due to DNA damage and gene mutations.38−46 Second, the similar metabolic requirements of glucose in normal cells can induce serious off-target effects.47 Additionally, the adaptive upB
DOI: 10.1021/acsnano.9b02466 ACS Nano XXXX, XXX, XXX−XXX
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ACS Nano
Figure 1. Cascaded enzymes of glucose oxidase (GOx) and catalase (CAT) depleted oxygen (O2) and glucose. (A) Time-dependent changes in dissolved O2 in buffered saline containing varied concentrations of GOx and CAT in the presence of 5 mM glucose. (B) Corresponding O2 depletion rates. (C) Time-dependent changes in pH values of buffered saline containing GOx and CAT (GOx: 2 U/mL, CAT: 240 U/mL) with different concentrations of glucose (0, 2.5, 5, and 10 mM). GOx + CAT overall reaction: glucose + 1 2 O2 ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ gluconic acid
regulation of cancer cells by parallel energy supplies can lead to the failure of starvation therapy. As such, it is imperative to incorporate an intelligent material design concept in the therapeutic mode that can effectively deplete intratumoral glucose with high specificity and low toxicity for the cancer starvation treatment.48,49 Herein, we propose a cascaded catalytic nanomedicine by combining targeted tumor starvation and deoxygenationactivated chemotherapy for an efficient cancer treatment with reduced systemic toxicity (Scheme 1). Specifically, cascaded enzymes of GOx and catalase (CAT) oxidize glucose in a H2O2-free fashion throughout the whole process. To improve the targeting specificity for minimized side effects, the cascaded enzymes were cross-linked via a pH-responsive linker and further coated with a serum albumin shell (Scheme 1A). Despite the chronic thermodynamic instability of the nanoclusters in the physiological conditions, the albumin shell could kinetically prevent the enzymes from rapid, premature exposure to off-target blood glucose, which ensures the selective release of nanoclustered enzymes under the tumoral acidic conditions. In addition, the cascaded enzymes consume tumoral oxygen (O2) and create a hypoxic environment. To supplement the gluocose-depletion therapy, prodrugs that can be activated by reductase in the hypoxic environment were also incorporated in the system (Scheme 1B). The combination of cancer starvation and the subsequent hypoxia-activated chemotherapy provides a promising method for an effective synergistic cancer therapy.
We first optimized the ratios of GOx and CAT best for consuming glucose and O2. The dissolved O2 level in the phosphate-buffered saline (PBS) containing 5 mM glucose was real-time measured by a Foxy Fospor-R O2 probe after the addition of different unit ratios of GOx and CAT. In the presence of 2 U/mL of GOx alone, the O2 level was rapidly decreased from 14.3 mg/L to an equilibrium concentration of 0.62 mg/L within 2 min (Figure 1A). The consumption of O2 gradually became slower with the addition of CAT in a concentration-dependent manner, and CAT alone did not react with O2. As expected, the increase of GOx concentration in the enzyme cascade accelerated the O2 consumption rate (Figure S1A,B). The O2 consumption rate curves were a good fit with the exponential function of Y = a + b × e−kX, where X and Y are the incubation time and O2 concentration, respectively (Figure 1A). The exponent k, defined as the O2 depletion rate and obtained at different ratios of GOx and CAT, was calculated and shown in Figure 1B. The optimized ratio of GOx-CAT (2−240 U/mL) was determined by the k value, at which the O2 and glucose were rapidly consumed without producing H2O2, thereby retaining a high cell viability (>95%, Figure S1C). The pH change of PBS containing GOxCAT (2−240 U/mL) and different glucose concentrations, as a result of the generation of gluconic acid, was monitored by a pH meter. For glucose concentrations at 2.5, 5, and 10 mM glucose, the pH decreased steadily over time and reached 6.1, 5.7, and 5.5 within 240 min, respectively, whereas the pH value remained unchanged for the control without glucose (0 mM) (Figure 1C). Preparation of Tumoral Acidic MicroenvironmentActivatable Nanoclustered Cascaded Enzymes. After investigating the O2 and glucose consumption behaviors of free GOx-CAT, we immobilized the enzymes by cross-linking GOx and CAT with a block copolymer of poly(ethylene glycol)block-poly(2-hydroxyethyl methacrylate) (PEG-b-PHEMA) bearing 2-(2-carboxyethyl)-3-methylmaleic anhydride (CMA) pendant groups (PEG-b-PHEMACMA, Figure S2). We selected this amide linker derived from the cis-aconityl bond as it has been demonstrated to undergo rapid hydrolysis under the acidic conditions.50−53 The degree of polymerization of PHEMA was calculated to be 30, and 43% of its hydroxyl groups were successfully conjugated with CMA (Figure S3 and S4). GOx-CAT were then cross-linked with PEG-b-PHEMACMA by forming an amide bond between the anhydride of the copolymer and amine groups of the GOx-CAT. The amide
RESULTS AND DISCUSSION GOx/CAT Cascade for Depletion of O2 and Glucose. In the GOx/CAT dual-enzyme cascade system, GOx oxidizes glucose to produce gluconic acid and toxic H2O2 (eq 1), which is immediately converted into H2O and O2 catalyzed by CAT (eq 2). The overall reaction (eq 3) results in the consumption of two glucose molecules per O2 molecule and leads to the rapid depletion of both nutrients, which serves as the catalytic basis for tumor starvation and deoxygenation induced by cascaded enzymes. glucose and O2 depletion: glucose + O2 + H 2O GOx ⎯⎯⎯⎯⎯→ gluconic acid + H 2O2
(1)
CAT detoxicity: H 2O2 ⎯⎯⎯⎯⎯⎯→ H 2O + 1 2 O2
(2)
(3)
C
DOI: 10.1021/acsnano.9b02466 ACS Nano XXXX, XXX, XXX−XXX
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ACS Nano
Figure 2. Preparation of nanoclustered cascaded enzymes and the acidity-triggered release of original enzymes. (A) Size and polydispersity index (PDI) of CE@(GOx+CAT) prepared by cross-linking GOx and CAT with PEG-b-PHEMACMA at different weight ratios. (B) Time course size and size distribution (PDI) of CE@(GOx+CAT) in saline before and after the addition of albumin. Time dependent changes in (C) size, (D) pH value, (E) dissolved O2 level, and (F) glucose concentration in buffered saline of BCE@(GOx+CAT) (GOx: 2 U/mL, CAT: 240 U/mL) at pH 6.5 and 7.4 with (solid lines) or without (dashed lines) of 5 mM glucose.
all the formulations was similarly determined as listed in Table S1. The acidity triggered disassembly of the nanoclusters was first assessed using cryo-transmission electron microscopy, which showed a uniform spherical shape for BCE@(GOx +CAT) with a diameter of ∼200 nm at pH 7.4 (Figure S9). At pH 6.5, small particles (∼10 nm) presumably from detached single enzymes were observed, indicating the cleavage of the CMA-based cross-linkage at slightly acidic pH. The dropping in particle size of BCE@(GOx+CAT) was further confirmed by time-course DLS measurements, which revealed the degradation was completed within 4 h in acidic media (i.e., pH = 6.5, Figure 2C). On the contrary, the diameter remained unchanged at pH 7.4 without glucose, which supported the higher stability of the cross-linkage under neutral conditions. However, the CMA-based bonds also underwent slow degradation upon long-term storage (Figure S10), indicating that BCE@(GOx+CAT) needed to be stored in the dried form, for example, after the freeze-drying process. The release of GOx and CAT from BCE@(GOxFITC+CATRhoB) was slowed at pH 7.4 and was accelerated at acidic pH without glucose (Figure S11). Furthermore, as opposite to BCE@(GOxFITC+CATRhoB) (Figure S12), the BSA shell of BCE@(GOx+CAT) could protect the enzyme release from nanoclusters in the presence of glucose which supported the stability the cross-linkage under neutral conditions (Figure S11). As controls, BCE@GOxFITC (Figure S13) and BCE@ CATRhoB (Figure S14) showed similar stability and enzyme release properties with BCE@(GOxFITC+CATRhoB), i.e., keeping intact structure at pH 7.4 and releasing enzymes at pH 6.5. Chemical modifications of enzymes have been commonly reported to reduce their catalytic activities.54 Thus, we further investigated the recovery of active enzymes by evaluating their
bond was ultrasensitive under acid conditions (pH < 6.5), recovering the original amine groups by hydrolysis. The extent of cross-linking could be readily adjusted by systemically varying the weight ratios of PEG-b-PHEMACMA/(GOx+CAT) from 1:1 to 20:1 (wt/wt, the concentrations of GOx and CAT were both fixed at 1.0 mg/mL). Both GOx and CAT had a size of ∼10 nm, whereas size of PEG-b-PHEMACMA was not detectable from dynamic light scattering (DLS) (Figure 2A and S5). Large aggregates with size of ∼600 nm were formed from DLS measurement when the weight ratio was set to 1:1. As the ratio increased to 5:1, nanoclustered enzymes of GOX and CAT (CE@(GOx+CAT)) with sizes
