Organosilica-Based Hollow Mesoporous Bilirubin Nanoparticles for

Aug 2, 2019 - Antibodies were used according to the manufacturer's instructions, including antibody dilution. Synthesis of MSN. CTAC (2 g) and TEA (0...
0 downloads 0 Views 1MB Size
Subscriber access provided by Mount Allison University | Libraries and Archives

Article

Organosilica-Based Hollow Mesoporous Bilirubin Nanoparticles for Antioxidation-Activated Self-Protection and TumorSpecific Deoxygenation-Driven Synergistic Therapy Lingling Shan, Wenpei Fan, Weiwei Wang, Wei Tang, Zhen Yang, Zhantong Wang, Yijing Liu, Zheyu Shen, Yunlu Dai, Siyuan Cheng, Orit Jacobson, Kefeng Zhai, Junkai Hu, Ying Ma, Dale O Kiesewetter, Guizhen Gao, and Xiaoyuan Chen ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b02477 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 3, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Organosilica-Based Hollow Mesoporous Bilirubin Nanoparticles for Antioxidation-Activated

Self-Protection

and

Tumor-Specific

Deoxygenation-Driven Synergistic Therapy Lingling Shan,†,‡ Wenpei Fan,*,‡ Weiwei Wang,† Wei Tang,‡ Zhen Yang,‡ Zhantong Wang,‡ Yijing Liu,‡ Zheyu Shen,‡ Yunlu Dai,‡ Siyuan Cheng,‡ Orit Jacobson,‡ Kefeng Zhai,† Junkai Hu,§ Ying Ma,‡ Dale O. Kiesewetter,‡ Guizhen Gao,*,† Xiaoyuan Chen*,‡ †Institute

of Pharmaceutical Biotechnology, School of Biology and Food Engineering, Suzhou

University, Suzhou 234000, China ‡Laboratory

of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical

Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland 20892, United States §Department

of Chemistry & Biochemistry, University of Maryland, College Park, Maryland

20742, United States

ABSTRACT A major concern about glucose oxidase (GOx)-mediated cancer starvation therapy is its ability to induce serious oxidative damage to normal tissues through the massive production of H2O2 byproducts in oxygen-involved glucose decomposition reaction, which may be well addressed by 1 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 43

using a H2O2 scavenger, known as an antioxidation agent. Surprisingly, H2O2 removal accelerates the aerobic glycometabolism of tumors by activating the H2O2-dependent “redox signaling” pathway of cancer cells. Simultaneous oxygen depletion further aggravates tumor hypoxia to increase the toxicity of a bioreductive prodrug, such as tirapazamine (TPZ), thereby improving the effectiveness of cancer starvation therapy and bioreductive chemotherapy. Herein, a “nitrogenprotected silica template” method is proposed to design a nano-antioxidant called organosilicabased hollow mesoporous bilirubin nanoparticle (HMBRN), which can act as an excellent nanocarrier to co-deliver GOx and TPZ. In addition to efficient removal of H2O2 for self-protection of normal tissues via antioxidation, GOx/TPZ co-loaded HMBRN can also rapidly deplete intratumoral

glucose/oxygen

to

promote

synergistic

starvation-enhanced

bioreductive

chemotherapeutic effect for the substantial suppression of solid tumor growth. Distinct from the simple combination of two treatments, this study introduces antioxidation-activated self-protection nanotechnology for the significant improvement of tumor-specific deoxygenation-driven synergistic treatment efficacy without additional external energy input, thus realizing the renaissance of precise endogenous cancer therapy with negligible side effects.

KEYWORDS. mesoporous nanomaterials, organosilica, antioxidation agent, self-protection, synergistic therapy.

Originating from an old notion of anti-angiogenesis,1 cancer starvation therapy likely provides a direct and effective treatment protocol to suppress tumor growth via blood vessel occlusion,2,3 which remains desirable and sophisticated. In parallel to conventional cancer-starvation methods 2 ACS Paragon Plus Environment

Page 3 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

based on vascular occlusion to deprive tumors of nutrient supply,4-7 deoxygenation nanotechnology has emerged and been used for intratumoral starvation therapy.8,9 For example, magnesium silicide nanoparticle is a representative deoxygenation agent that can rapidly remove intratumoral oxygen to starve cancer cells.10 By comparison, an unconventional enzyme-catalyzed starvation strategy that involves the glucose oxidase (GOx)-catalyzed depletion of glucose and oxygen,11 has been extensively investigated to produce a much stronger starvation effect.12-15 Interestingly, considerable deoxygenation creates a tumor microenvironment with decreased oxygen to further increase the toxicity of hypoxia-sensitive prodrugs,16-19 bridging the gap between starvation therapy and bioreductive chemotherapy toward highly efficient deoxygenation-driven synergistic therapy. However, glucose decomposition inevitably yields excess amount of H2O2 byproducts,20 which are highly toxic because of their irreversible oxidative damage to the surrounding normal tissues.21,22 Thus, off-target oxidative toxicity should be minimized during tumor-specific deoxygenation-driven synergistic therapy through the co-administration of GOx and bioreductive prodrugs. To remove excess H2O2 for detoxification, we aim to introduce a natural biocompatible antioxidant called bilirubin (BR) which has wide anti-inflammatory applications in multiple diseases.23,24 In contrast to other antioxidants absorbing diverse types of reactive oxygen species (ROS),25-27 BR preferentially scavenges H2O2 to eliminate oxidative damage.28-30 Of special note, tumors need to take up a much larger amount of glucose than normal tissues for growth and angiogenesis.31 As such, cancer cells become more sensitive to GOx-catalyzed glucose depletion than normal cells. Besides, another difference between cancer and normal cells is the much larger amount of reactive oxygen species (ROS, including H2O2) in cancer cells due to their stronger metabolic activity.32 The H2O2 level in cancer cells is elevated by an elevated H2O2 production

3 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 43

rate.33 H2O2 plays an important role in regulating tumor signal transduction pathways.34,35 Compared to normal cells, tumor cells are featured with their increased metabolic activity,36 which results in changes of the cellular redox state in response to the production of large amounts of H2O2, which is called the H2O2-dependent “redox signaling” pathway.37-39 Accordingly, a right shift in the reaction equilibrium of glucose decomposition occurs to increase the GOx activity to enhance the efficacy of starvation therapy. Meanwhile, the resulting deoxygenation-aggravated tumor hypoxia further improves the cytotoxicity of bioreductive prodrugs.40 Consequently, the antioxidant BR plays two indispensible roles in scavenging excess H2O2 for self-protection of normal tissues and inducing intratumoral glucose/oxygen depletion for synergistic starvationenhanced bioreductive chemotherapy against cancer. Towards this end, intrinsically hydrophobic BR molecules should be engineered into hydrophilic BR vehicles for the co-delivery of GOx and bioreductive prodrugs under the assistance of nanotechnology. In this current study, we identified a typical paradigm of BR vehicles, namely, organosilicabased hollow mesoporous bilirubin nanoparticle (HMBRN), synthesized through a previously unreported “nitrogen-protected silica template” method (Figure 1a). This well-designed watersoluble HMBRN not only retained the antioxidant activity of BR, but also served as an excellent nanocarrier for the co-encapsulation of GOx and tirapazamine (TPZ, a bioreductive prodrug) because of its large surface area, uniform mesoporous channels, and hollow cavity. In contrast to the oxygen richness and low glucose dependency of normal tissues, high glucose demand and hypoxia microenvironment cause the tumors to become more susceptible to GOx-catalyzed starvation therapy and TPZ-induced bioreductive chemotherapy. The GOx/TPZ co-loaded HMBRN could specifically activate the H2O2-dependent “redox signaling” pathway to regulate the expression of related proteins (e.g., Nrf2, GLUT1, HIF-1α, p53, etc.),41-43 and simultaneously

4 ACS Paragon Plus Environment

Page 5 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

protect normal tissues through BR-mediated H2O2 scavenging, which contributed greatly to promoting intratumoral GOx-catalyzed glucose decomposition and subsequent deoxygenationactivated TPZ toxicity. By harnessing the discrepancy between normal tissues and tumors, this study offers a concept of applying antioxidation-activated self-protection nanotechnology for healthier yet more effective intratumoral deoxygenation-driven synergistic starvation-enhanced bioreductive chemotherapy without the need for external energy input. Besides, this study may also shed light on the exploration of other biocompatible, endogenous, and tumor-specific treatment technologies. RESULTS Silica nanoparticles are well known for their ability to protect functional biomolecules from harsh environments. In this study, BR was silanized by using 3-(aminopropyl)triethoxysilane (APTES) through amidation to form silanized BR (BR-silane, Figure S1a). Traditionally, BRsilane was directly conjugated onto the surface of silica nanoparticles through dehydration between Si-OH bonds. However, such silica-BR nanoparticles are not soluble in water and the surfaceconjugated BR was easily attacked by the complicated physiological environment. To hybridize BR-silane within the organosilica framework, we developed a “nitrogen-protected silica template” method to construct organosilica-based HMBRN, in which BR could be protected/shielded by the organosilica shell to ensure the high dispersity of HMBRN. Featuring high stability, high loading capacity, and stimuli-responsive biodegradability, smallsized HMON may preferentially serve as an advanced drug vehicle because of its long half-life in blood circulation (Figure S2). Herein, sub-50 nm HMON with an average size of 41.5 nm (Figure S3a) was synthesized through an “ammonia-assisted hot water etching” method (Figure 1a). First, mesoporous silica nanoparticles (MSNs, Figure S4) were synthesized via the hydrolysis of

5 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 43

tetraethyl orthosilicate (TEOS). Then a mesoporous organosilica shell was deposited on the MSN core to obtain MSN@MON (Figure 1b) via the co-hydrolysis of bis[3-(triethoxysilyl)propyl] tetrasulfide (BTES) and TEOS. Finally, hollow-structured HMON (Figure 1c) was obtained by selectively etching away the inner MSN core because the Si-C bonds within the MON shell exhibited much stronger resistance against ammonia etching than the Si-O bonds within the MSN core. Particularly, thioether hybridization (Figure 1d, e) allowed GSH-induced biodegradation.44,45 The sub-50 nm HMON with thioether hybridization was used as a template for the in situ “growth” of BR within the framework of organosilica during synthesis so that the mesoporous channels were not blocked. In accordance with the “chemical homology” principle, BR-hybridized MON shell was deposited on the MSN core (Figure 1f) through the co-hydrolysis of BTES/TEOS and BRsilane in a N2 gas flow to prevent the oxidation of BR. Owing to the higher stability of the BRhybridized MON shell than the MSN core, ammonia triggered outward etching, thereby producing HMBRN with a hollow cavity (Figure 1g, h). In comparison with HMON, HMBRN showed the spectra with a typical Raman shift at 620 cm−1 (Figure 1i) and UV/Vis absorption at 425 nm (Figure 1j), thereby confirming the successful hybridization of BR within the framework of organosilica. All of the major elements (Si/O/S) were homogeneously distributed within the framework of HMBRN (Figure 1k, l). Moreover, the as-prepared sub-50 nm HMBRN with high water dispersity exhibited a narrow hydrodynamic size distribution (78.2 ± 5 nm, Figure S5). Such a “nitrogenprotected in situ growth” strategy involving thioether-hybridized HMON as a template enabled the controllable synthesis of sub-50 nm HMBRN (Figure S3b) with uniform spherical morphology, high BR hybridization percentage (20 wt.%, Figure S6), and GSH-responsive biodegradability (Figure S7), in addition to BR-mediated antioxidation.

6 ACS Paragon Plus Environment

Page 7 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 1. (a) Scheme of the synthetic procedures of HMON and HMBRN for co-delivery of GOx/TPZ. HMON and HMBRN were synthesized by an “ammonia-assisted hot water etching” method and “nitrogen-protected silica template” method, respectively. The addition of bissilylated organosilica precursor with thioether moiety, bis[3-(triethoxysilyl)propyl] tetrasulfide (BTES), produced core/shell-structured MSN@MON (or MSN@MON(BR)) and hollow-structured 7 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 43

HMON (or HMBRN) with thioether hybridization. The thoiether hybridization allowed for GSHresponsive biodegradation of HMON and HMBRN. (b, c) TEM images of (b) MSN@MON and (c) HMON. Scale bar, 50 nm. (d) Schematic of the thioether-hybridized framework of HMON. (e) Raman spectrum of HMON. (f, g) TEM images of (f) MSN@MON(BR) and (g) HMBRN. Scale bar, 50 nm. (h) Schematic of the thioether/BR dual-hybridized framework of HMBRN. (i) Raman spectrum of HMBRN. The Raman shift of 620 cm-1 represents the specific stretching vibration of BR within HMBRN. (j) UV/Vis absorption spectra of HMON, BR, HMBRN. (k) Energydispersive X-ray spectroscopy (EDS) spectrum of HMBRN. (l) Elemental mapping images of HMBRN.

With a large surface area (348.8 m2/g, Figure S8a), uniform interpenetrating mesopores (average size of 3.5 nm, Figure S8b), and a hollow cavity, HMBRN could be applied to co-deliver GOx and TPZ. Herein, GOx was covalently anchored onto the surface of HMBRN via dehydration condensation between the Si-OH groups of organosilica and GOx-silane (Figure S1b). The internal cavity endowed HMBRN with a large TPZ loading capacity (45 wt.%, Figure S9). The positively charged TPZ was loaded into the cavity of negatively charged HMBRN through electrostatic adsorption. Similar to the acidity-driven release of positively charged DOX from silica nanoparticles,46,47 the TPZ-loaded HMBRN also showed a pH-responsive controlled release profile. In the acidic solution, the excess amount of H+ competed with the positively charged TPZ molecules in the negatively charged HMBRN to weaken the electrostatic interaction between TPZ and HMBRN, which could accelerate the TPZ release from HMBRN at pH < 7 (Figure S10). Therefore, the TPZ-loaded HMBRN demonstrated faster release of TPZ molecules in the acidic environment than that under neutral condition. Besides, HMBRN-GOx/TPZ exhibited a faster TPZ

8 ACS Paragon Plus Environment

Page 9 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

release profile in the glucose (1 mg/mL)-containing buffer solution (pH = 7.4) than that in the acid buffer solution (pH = 3.6), which should be attributed to the H2O2 generation from GOx-catalyzed glucose decomposition. The generated H2O2 could oxidize the framework-incorporated disulfide bonds into sulfoxides and sulfones to decompose the organosilica framework, which would induce the degradation of HMBRN and speed up the release of the loaded TPZ. In the presence of glucose and oxygen, the GOx/TPZ co-loaded HMBRN (denoted as HMBRN-GOx/TPZ) underwent a typical three-step reaction (Figure 2a): GOx-catalyzed decomposition of glucose into gluconic acid and H2O2 and depletion of oxygen, BR-mediated removal of H2O2, and hypoxia-triggered transformation of TPZ into benzotriazinyl (BTZ). This “trilogy” reaction provided the basis for specific deoxygenation-driven synergistic starvation-enhanced bioreductive chemotherapy against cancer and selective antioxidation-activated self-protection for normal tissues.

9 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 43

Figure 2. (a) Schematic of three-step chain reactions: GOx-catalyzed glucose decomposition and oxygen depletion, BR-induced H2O2 scavenging, and hypoxia-triggered transformation of nontoxic TPZ into toxic BTZ. (b) Evaluation of the H2O2-scavenging effect of HMBRN. After 1 h of incubation of 200 µM H2O2 and varied concentrations (7.8–500 µg/mL) of HMBRN, the remaining concentrations of H2O2 were measured by using a H2O2 assay kit. (c) The concentrations of H2O2 arising from 1 h of reaction between varied concentrations (0.125–4 mg/mL) of HMONGOx and 1 mg/mL glucose. (d) The concentrations of H2O2 arising from 1 h of reaction between varied concentrations (0.125–4 mg/mL) of HMBRN-GOx and 1 mg/mL glucose. (e-g) After 1 h of incubation of varied concentrations (0.125–4 mg/mL) of HMON-GOx (and HMBRN-GOx) and 10 ACS Paragon Plus Environment

Page 11 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

1 mg/mL glucose, (e) GOx enzymatic activity, (f) dissolved O2 concentration, and (g) pH value were measured by a typical GOx activity assay kit, oxygen sensor, and pH sensor, respectively. n = 4, mean ± s.d., *P < 0.05, ***P < 0.001.

Figure 2b showed the inverse relationship between the remaining H2O2 concentration and the original HMBRN concentration, indicating the ability of HMBRN to eliminate H2O2. Meanwhile, the antioxidant effect of BR on the aerobic glycometabolism was reflected by the reaction between HMON-GOx/HMBRN-GOx and glucose. Different from the slight dependence of H2O2 yield on the concentration of HMON-GOx (Figure 2c), the generated H2O2 concentrations showed an abnormally declining tendency as the HMBRN-GOx concentrations increased (Figure 2d). This finding further confirmed the excellent H2O2-scavenging effect of HMBRN. As compared to the inhibition of the GOx activity by the H2O2 production in the GOx-catalyzed glucose decomposition reaction,48,49 the selective removal of H2O2 via HMBRN-mediated antioxidation could cause a right shift in the reaction equilibrium of glucose decomposition to increase the GOx activity. Therefore, the activity of HMBRN-GOx was measured to be higher than that of HMON-GOx (Figure 2e). Besides, the rapid decrease in dissolved oxygen concentration and pH (Figure 2f, g) demonstrated the outstanding deoxygenation effect of HMBRN-GOx. Despite the lower GOx activity, HMON-GOx could still catalyze the oxidization of the whole glucose (1 mg/mL) into gluconic acid within 1 h reaction. There was no significant difference between the pH values of HMBRN-GOx + Glucose (1 mg/mL) and HMON-GOx + Glucose (1 mg/mL) because these two reactions produced comparable amount of gluconic acid at the same initial concentration (1 mg/mL) of glucose.

11 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 43

HMON and HMBRN conjugated with PEG-silane were found to be highly biocompatible (Figures S11, S12), thus all the HMON and HMBRN particles used for the following in vitro/in vivo experiments were modified with PEG. The antioxidation-activated self-protection ability of HMBRN was illustrated in two normal cell lines: human umbilical vein endothelial cells (HUVECs) and human embryonic kidney transformed cells (293T). High concentration (200– 1000 µM) of H2O2 caused normal cell death to varied extents, which was reversed by the addition of HMBRN (Figure S13). The decreased concentrations of HMBRN (7.8–250 µg/mL) also exhibited H2O2-scavenging activity to offset the H2O2-induced oxidative damage (Figure S14). Therefore, the combination of GOx and HMBRN likely eliminated the adverse effect of H2O2 byproducts from GOx-catalyzed glucose decomposition (Figure 3a). Considering that the H2O2 production in the GOx-catalyzed glucose decomposition reaction might in turn inhibit the GOx activity,48,49 such low GOx concentration of HMON-GOx with low enzymatic activity might be difficult to decompose a high concentration (4.5 mg/mL) of glucose in the DMEM medium during 24 h of incubation. Higher concentrations of HMON-GOx catalyzed the oxidization of more glucose into higher concentrations of H2O2 to kill more normal cells, so the viabilities of 293T cells and HUVEC cells were dependent on the concentrations of HMON-GOx (Figure 3b, c). In sharp contrast to the severe cytotoxicity of HMON-GOx, HMBRN-GOx demonstrated negligible destructive influence on the viabilities of 293T and HUVEC cells (Figure 3b, c), which indicated that the advantage of HMBRN in antioxidation-activated self-protection accounted for the much higher biocompatibility of HMBRN-GOx than that of HMON-GOx.

12 ACS Paragon Plus Environment

Page 13 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 3. (a) Schematic of the opposite effects of HMON-GOx and HMBRN-GOx on normal tissues. The substantial generation of H2O2 arising from the HMON-GOx-activated glucose decomposition can destroy normal tissues, whereas the antioxidant HMBRN can scavenge the H2O2 byproducts to protect normal tissues from oxidative damage. (b, c) Comparison of cytotoxicity of HMON-GOx and HMBRN-GOx to normal cells. Viabilities of (b) 293T cells and (c) HUVEC cells after 24 h of incubation with varied concentrations (25, 50, 100, 200 µg/mL) of HMON-GOx and HMBRN-GOx. (d-f) Concentrations of (d) glucose, (e) H2O2 and (f) ATP in 293T/HUVEC/U87MG cells after incubation with HMBRN-GOx and HMON-GOx for 2 h and 24 h, respectively. Red, green, and black bars refer to 293T, HUVEC, and U87MG cells, respectively. The glucose/H2O2/ATP concentrations significantly differ in between the normal and tumor cells. n = 4, mean ± s.d., *P