In Situ Nanoreactor for Photosynthesizing H2 Gas To Mitigate

Sep 5, 2017 - Department of Orthopaedics, National Taiwan University Hospital, Hsinchu Branch, Hsinchu 30059, Taiwan, ROC. •S Supporting Information...
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In Situ Nanoreactor for Photosynthesizing H2 Gas To Mitigate Oxidative Stress in Tissue Inflammation Wei-Lin Wan,† Yu-Jung Lin,† Hsin-Lung Chen,† Chieh-Cheng Huang,‡ Po-Chien Shih,† Yu-Ru Bow,† Wei-Tso Chia,*,§ and Hsing-Wen Sung*,†,‡ †

Department of Chemical Engineering and ‡Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC § Department of Orthopaedics, National Taiwan University Hospital, Hsinchu Branch, Hsinchu 30059, Taiwan, ROC S Supporting Information *

that is overproduced in inflamed tissues, because the solubility of H2 in biological settings is typically low. This difficulty may be overcome by the local delivery of H2 gas with high therapeutic concentration at inflammatory sites. H2 gas therapy is regarded to be safe and free of side effects.6 Inspired by natural photosynthesis, a multicomponent system that comprises a photosensitizer (chlorophyll a; Chla), an electron donor (L-ascorbic acid; AA), and a catalyst of H2 production (gold nanoparticles; AuNPs) that are encapsulated in a liposomal (Lip) system is proposed herein as a photodriven nanoreactor (NR) to generate H2 gas locally. The photosynthesis reaction between AA and AuNPs depends on Chla, which contains a hydrophilic porphyrin ring of greenish pigments and a lipophilic tail of phytol chain (Figure 1), absorbing light with wavelengths of 420−465 and 650−680 nm. Upon the absorption of photons by its porphyrin ring, Chla is excited (Chla*), and an electron−hole pair is thus generated.7 The hole in the excited Chla* can accept a new

ABSTRACT: Hydrogen gas can reduce cytotoxic reactive oxygen species (ROS) that are produced in inflamed tissues. Inspired by natural photosynthesis, this work proposes a multicomponent nanoreactor (NR) that comprises chlorophyll a, l-ascorbic acid, and gold nanoparticles that are encapsulated in a liposomal (Lip) system that can produce H2 gas in situ upon photon absorption to mitigate inflammatory responses. Unlike a bulk system that contains free reacting molecules, this Lip NR system provides an optimal reaction environment, facilitating rapid activation of the photosynthesis of H2 gas, locally providing a high therapeutic concentration thereof. The photodriven NR system reduces the degrees of overproduction of ROS and pro-inflammatory cytokines both in vitro in RAW264.7 cells and in vivo in mice with paw inflammation that is induced by lipopolysaccharide (LPS). Histological examinations of tissue sections confirm the ability of the NR system to reduce LPS-induced inflammation. Experimental results indicate that the Lip NR system that can photosynthesize H2 gas has great potential for mitigating oxidative stress in tissue inflammation.

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nflammation is associated with many diseases, including bacterial infection, sepsis, osteoarthritis, and asthma, all of which involve the overproduction of reactive oxygen species (ROS).1 ROS has a key role in immune responses to exogenous pathogens; for example, macrophages produce ROS that kill bacteria. However, the overproduction of ROS may amplify pro-inflammatory pathways;2 furthermore, the hydroxyl radical (•OH), which is the strongest ROS, is extremely reactive with nucleic acids, lipids, and proteins, causing significant destructions of host cells.3 Therefore, an antioxidant that can effectively scavenge cytotoxic •OH with no side effects is urgently sought. Hydrogen (H2) can reduce •OH to H2O, selectively scavenging the free radical while preserving other essential ROS for normal signaling regulation.4 The administration of H2-water (orally), H2-saline (via intravenous drip infusion), or H2-gas (via inhalation with air) has been shown to be promising in treating several inflammatory diseases in animal models.5 However, the amount of H2 that is absorbed by the body through these approaches may not suffice to scavenge the ROS © 2017 American Chemical Society

Figure 1. Composition/structure of photodriven NR and mechanisms of its photosynthesis of H2 gas in situ to reduce overproduction of oxidative stress in LPS-induced inflamed paw created in a mouse model. Received: July 18, 2017 Published: September 5, 2017 12923

DOI: 10.1021/jacs.7b07492 J. Am. Chem. Soc. 2017, 139, 12923−12926

Communication

Journal of the American Chemical Society electron from AA, returning to its ground state.8 Colloidal AuNPs are used as a catalyst that collects the electrons that are released from the excited Chla* and the protons from the oxidized AA, promoting their conversion to H2 gas.7 AuNPs with a diameter of ≤5 nm reportedly have unique catalytic properties.9 In a bulk solution (BS), the reacting molecules (Chla, AA, and AuNPs) may not be located close enough to each other at all times, so they do not sufficiently react. Lips provide a design of a reactor that can uniquely confine reacting molecules within nanoscale dimensions by keeping the hydrophilic reactants (AA and AuNPs) in their aqueous cores and the amphiphilic reactant (Chla) in the lipid bilayers simultaneously. Unlike BS, this Lip NR enables the establishment of an optimal reaction environment for the reacting molecules under study. Figure 1 schematically depicts the composition/structure of the photodriven Lip NR and the mechanisms by which it produces H2 gas locally and scavenges the excess ROS in a mouse model of paw inflammation that is induced by lipopolysaccharide (LPS). The Lip NRs are suspended in phosphate-buffered saline (PBS), which is injected directly into the site of inflammation. Treatment with a laser (660 nm) activates the in situ Lip NRs to photosynthesize H2 gas locally, reducing the oxidative stress in inflamed tissues. The test Lips [1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(amino(polyethylene glycol)-2000) (DSPEPEG2000), and cholesterol] were prepared using a thin-film hydration technique in the presence of Chla, AA, and AuNPs. First, the Lip formulation was optimized, based on the highest concentration of each component that could be encapsulated. The encapsulated concentrations of Chla, AA, and AuNPs in the as-optimized Lips were determined to be 39.8 ± 9.7 μM, 1.8 ± 0.2 mM, and 5.9 ± 1.3 nM, respectively (n = 4 batches). Dynamic light scattering (DLS) measurements revealed that the size and zeta potential of the as-prepared Lips were 106.0 ± 37.9 nm (Figure S1) and −0.7 ± 2.3 mV, respectively (n = 10 batches). As confirmed by confocal laser scanning microscopy (CLSM), Chla (green color) was successfully embedded in the Lip membrane (Figure 2a); notably, to overcome the optical resolution limit of CLSM, the Lips that were used in this specific study had diameters of 1−3 μm. The embedment of Chla in the Lip membrane of the NR system was further elucidated by small-angle X-ray scattering (SAXS), using the plain Lips as a control. According to Figure S2, the SAXS profile of the plain Lips had three broad peaks, typical of unilamellar vesicles; the intensities of these peaks increased slightly when Chla was incorporated into the Lips. The measured SAXS profiles were further used to determine the electron density distributions in the normal direction, ρe(z), of the Lip membranes. The region with the lower electron density in ρe(z) (the valley) corresponds to the nonpolar layers of the lipid tails, while the two peaks of higher electron density represent polar lipid heads. The increase in electron density in the polar regions indicates that the hydrophilic porphyrin rings of Chla were located near the polar lipid heads (Figure 1). The lipophilic phytol chains of Chla were thus situated in the nonpolar layers of the Lips. Transmission electron microscopy (TEM) revealed that AuNPs were encapsulated in the aqueous compartments, mostly near the water/lipid interfaces, of the Lip NRs (Figure 2b). Thermodynamically, colloidal particles preferentially

Figure 2. (a) Fluorescence images of Chla embedded in Lip membrane of NR. (b) TEM images of AuNPs encapsulated in NR. (c) Spectral changes of AA encapsulated in NR following various periods of laser irradiation. (d) Bright field images of H2 bubble generation in an NR following laser irradiation. (e) Ultrasound images of H2 bubble generation in BS and NR following laser irradiation. (f) Cumulative H2 concentrations generated in BS and NR following laser irradiation. (g) Hydroxyl radical scavenging activities of BS and NR without/with laser irradiation. *P < 0.05.

accumulate at the interfaces between two immiscible phases.10 Aqueous AA has an ultraviolet absorption band at ca. 250 nm.11 Figure 2c displays spectral changes of AA that was encapsulated in the aqueous core of the NR system during laser irradiation. Under laser irradiation, the absorption band at 250 nm gradually weakened, disappearing in ca. 10 min, reflecting the consumption of AA in the photosynthesis of H2 gas. The H2 gas that was generated in the NR system then diffused out through its Lip membrane in the form of a gas bubble. The process in which the H2 gas bubble was generated by a laser-irradiated NR (3 μm in diameter) was observed using CLSM. According to Figure 2d, a gas bubble began to appear at the outer membrane of the Lip NR after exposure with a laser for 10 s, suggesting that laser irradiation rapidly activated photosynthesis in the NR system to generate H2 gas. The ability of the NR system in PBS to form H2 gas bubbles upon laser treatment was further elucidated using an ultrasound imaging system with BS that contained free Chla, AA, and AuNPs as a control. Before laser treatment (t = 0 s in Figure 2e), both BS and NR systems (BS−Laser and NR−Laser) yielded no gas bubbles. During laser irradiation, only a few gas bubbles were detected in the BS system (BS+Laser), whereas significantly more H2 bubbles were observed in the sample that contained the NR system (NR+Laser). The formation of H2 bubbles in the NR+Laser system continued for ca. 10 min, with the most produced in the first 5 min. Ten minutes of laser irradiation was therefore used to study the anti-inflammatory capacities of BS and the NR in the following in vitro and in vivo experiments. The cumulated concentrations of H2 gas that were generated from BS and the NR locally during laser exposure were individually measured using gas chromatography. According to Figure 2f, the cumulated H2 concentrations from the NR consistently exceeded those from BS. The above analytical results indicate that the reacting molecules that were encapsulated in the NR reacted more effectively than those free reacting molecules that were suspended in BS. The •OH scavenging activity of the laser-irradiated NR was studied using a hydroxyl radical antioxidant capacity (HORAC) activity assay; laser-irradiated BS was the control. As presented 12924

DOI: 10.1021/jacs.7b07492 J. Am. Chem. Soc. 2017, 139, 12923−12926

Communication

Journal of the American Chemical Society in Figure 2g, following exposure to the laser, the NR exhibited a significantly higher level of HORAC activity than the BS, verifying that more H2 gas was photosynthesized in the former system than in the latter (Figure 2e,f). The cytotoxicity of the Lip NRs was evaluated by incubating them with a mouse macrophage cell line (RAW264.7) using a WST-1 assay, without/with the production of H2 gas by laser irradiation; macrophages that received no Lip NRs were used as a control. According to Figure S3, the viability of the macrophages was not significantly affected by co-culturing with the test NRs, without/with the production of H2, up to a concentration of 5 mg/mL. H2 is known to be nontoxic even at high concentration.6 These findings indicate that the asprepared Lips can serve as an excellent NR for the photosynthesis of H2 gas, as they do not influence cell viability, enabling them to be used to mitigate cellular inflammatory responses. The anti-inflammatory effect of NR+Laser on the LPSinduced inflammatory responses of RAW264.7 cells was examined. LPS, a main component of the outer membrane of Gram-negative bacteria, is a powerful activator of macrophages and has been used to create animal models of bacterial inflammation. LPS stimulates macrophages, resulting in the overproduction of ROS and causing the excess expression of pro-inflammatory cytokines such as interleukin (IL)-6 and IL1β.2 The mode of action of LPS as an activator of RAW264.7 cells was determined by a histogram analysis of the degree of its intracellular ROS production. The ROS level within the cells was quantified by using a commercially available kit to detect the conversion of 2′,7′-dichlorofluorescein diacetate (DCFDA) to 2′,7′-dichlorofluorescein (DCF), which is highly fluorescent at 530 nm.12 As shown in the histogram (Figure S4), the amount of ROS produced due to LPS stimulation was timedependent; the ROS level peaked at ca. 6 h. Therefore, the RAW264.7 cells that had been treated with LPS for 6 h were used to study the anti-inflammatory effects of BS and the NR without/with laser irradiation. As shown in Figure S5, when activated by the laser, both BS and the NR effectively reduced the excess production of ROS in a concentration-dependent manner (P < 0.05). The highest NR concentration that was used in this experiment was 5 mg/mL, but the reduction of ROS overproduction was maximized at an NR concentration of 2.5 mg/mL, which was therefore used in subsequent studies. The concentrations of Chla/AA/AuNPs that were used in BS were those encapsulated in NR. Notably, the reduction in the amount of ROS by the NR+Laser exceeded that by the BS+Laser (P < 0.05, Figure 3a). Chla, AA, and AuNPs are known individually to have antioxidant effects.13 However, neither BS−Laser nor NR−Laser [including Lips that contain Chla, AA, or AuNPs alone (Figure S6)] significantly reduced the levels of LPS-stimulated overproduced ROS (P > 0.05), suggesting that the reduction of ROS levels by BS and the NR probably involved their laser-irradiated photosynthesis of H2 gas. Reduction of the overproduction of ROS in LPS-stimulated RAW264.7 cells by BS or NR ± Laser was visualized using a cell-permeable fluorogenic probe of ROS (CellROX), which fluoresces in red upon oxidation by ROS.14 The CLSM results herein (Figure 3b) indicate that only NR+Laser effectively reduced the LPS-induced ROS overproduction in cells. Previous studies have shown that H2 gas can easily penetrate cellular membranes and be rapidly distributed into cytosol and

Figure 3. (a) DCF intensities and (b) CLSM images of LPS-induced ROS production in RAW264.7 cells following various treatments. (c) Levels of inflammatory cytokines IL-6 and IL-1β and (d) corresponding fluorescence images in RAW264.7 cells following various treatments. *P < 0.05; n.s. = not significant.

organelles owing to its small molecular size, making it very effective in scavenging the intracellularly overproduced oxidants.6 To further evaluate the anti-inflammatory effect of NR+Laser on LPS-stimulated RAW264.7 cells, the expression of proinflammatory cytokines IL-6 and IL-1β in such cells was measured using commercial enzyme-linked immunosorbent assay (ELISA) kits. According to Figure 3c, BS+Laser treatment only minimally affected the expression levels of IL-6 and IL-1β in RAW264.7 cells, while the LPS-stimulated overproductions of IL-6 and IL-1β were dramatically reduced by treatment with NR+Laser (P < 0.05). Double immunocytochemistry staining versus of nuclei was performed to visualize the intracellular expression levels of IL-6 and IL-1β. NR+Laser treatment greatly reduced levels of IL-6 and IL-1β in LPS-stimulated cells (Figure 3d). The above results indicate that NR+Laser effectively reduces inflammatory responses of LPS-stimulated macrophages, and so may have the potential to mitigate oxidative stress in inflamed tissue. The feasibility of using NR+Laser to mitigate oxidative stress in inflamed tissues was investigated using a mouse model with LPS-induced paw inflammation. The dose-dependent effect of the NR (2.5 mg NR/mL) on the reduction of the amount of ROS in inflamed paws was first studied. Changes in the amount of ROS in inflamed tissues were measured using a luminescent probe, L-012, which could be detected noninvasively using an in vivo imaging system (IVIS). The detected luminescent signal (representative of ROS level) in the blank control (inflamed paw treated with PBS that contained no NR) was stronger than that in the healthy paw (Figure S7), confirming the LPSinduced overproduction of ROS in the inflamed paw. A dosedependent decrease in the strength of luminescent signal was observed in mice that were treated with NR+Laser, revealing the effectiveness of NR+Laser in reducing excess oxidative stress in tissue inflammation. Since paw edema was observed following treatment with 30 μL NR because the injection volume was excessive, 20 μL NR was used to mitigate tissue inflammation. 12925

DOI: 10.1021/jacs.7b07492 J. Am. Chem. Soc. 2017, 139, 12923−12926

Journal of the American Chemical Society

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The intensity of luminescence that was emitted from the inflamed tissue that was treated with NR+Laser was significantly less than that of the inflamed tissue that was treated with BS+Laser (Figures 4a and 4b), suggesting the

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Figure 4. (a) IVIS images and (b) corresponding L-012 luminescence intensities of ROS in LPS-induced inflamed paws following treatment with BS and NR without/with laser irradiation. (c) Levels of inflammatory cytokines IL-6 and IL-1β and (d) corresponding fluorescence images and (e) H&E staining images of inflamed paws following various treatments without/with laser irradiation. *P < 0.05.

decline of ROS level. Treatment with NR+Laser significantly reduced the LPS-induced IL-6 and IL-1β levels below those following treatment with BS+Laser (P < 0.05, Figures 4c and 4d). Histological examinations of tissue sections that were stained with hematoxylin−eosin (H&E) also established the superiority of NR+Laser over BS+Laser, by revealing less infiltration of the inflammatory cells (Figure 4e). In summary, a multicomponent NR system that can photosynthesize H2 gas in situ upon photon absorption was successfully developed. The molecules that were encapsulated in the NR system reacted more strongly than those in a BS system, yielding more H2 gas, and providing a high local therapeutic concentration. Analyses of levels of ROS and cytokines and histological examinations of inflamed tissues demonstrate that the developed photodriven NR system effectively reduced oxidative stress, revealing its great potential for mitigating tissue inflammation.

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Communication

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b07492. Experimental details and additional data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] (W.-T.C.) *[email protected] (H.-W.S.) ORCID

Hsin-Lung Chen: 0000-0002-3572-723X Hsing-Wen Sung: 0000-0002-0789-5236 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology (MOST 105-2119-M-007-008) of the Republic of China, Taiwan. 12926

DOI: 10.1021/jacs.7b07492 J. Am. Chem. Soc. 2017, 139, 12923−12926