Biodegradable Hollow Mesoporous Organosilica Nanotheranostics for

Subscriber access provided by Gothenburg University Library

Article

Biodegradable Hollow Mesoporous Organosilica Nanotheranostics for Mild Hyperthermia-Induced Bubble-Enhanced Oxygen-Sensitized Radiotherapy Nan Lu, Wenpei Fan, Xuan Yi, Sheng Wang, Zhantong Wang, Rui Tian, Orit Jacobson, Yijing Liu, Bryant C. Yung, Guofeng Zhang, Zhaogang Teng, Kai Yang, Minming Zhang, Gang Niu, Guangming Lu, and Xiaoyuan Chen ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b08103 • Publication Date (Web): 31 Jan 2018 Downloaded from http://pubs.acs.org on February 1, 2018

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 free 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 accessible to all readers and 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.

ACS Nano 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 36 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

Biodegradable Hollow Mesoporous Organosilica Nanotheranostics for Mild Hyperthermia-Induced Bubble-Enhanced Oxygen-Sensitized Radiotherapy Nan Lu,†,

‡, §

Wenpei Fan,*,§ Xuan Yi,⊥ Sheng Wang,§ Zhantong Wang,§ Rui Tian,§ Orit

Jacobson,§ Yijing Liu,§ Bryant C. Yung,§ Guofeng Zhang,ǂ Zhaogang Teng,† Kai Yang,⊥ Minming Zhang,‡ Gang Niu,§ Guangming Lu,*,† and Xiaoyuan Chen*,§ †

Department of Medical Imaging, Jinling Hospital, Medical School of Nanjing University,

Nanjing, Jiangsu 210002, China ‡

Department of Radiology, the Second Affiliated Hospital, Zhejiang University School of

Medicine, Hangzhou, Zhejiang 310009, 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 ⊥

School of Radiation Medicine and Protection & School for Radiological and

Interdisciplinary Sciences (RAD-X), Medical College of Soochow University, Suzhou, Jiangsu 215123, China

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

ǂ

Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of

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

ABSTRACT: Alleviation of tumor hypoxia has been the premise for improving the effectiveness of radiotherapy, which hinges upon the advanced delivery and rapid release of oxygen within the tumor region. Herein, we propose a “bubble-enhanced oxygen diffusion” strategy to achieve whole tumor oxygenation for significant radiation enhancement based on the “bystander effect”. Towards this end, sub-50 nm CuS-modified and 64Cu labeled hollow mesoporous organosilica nanoparticles were constructed for tumor-specific delivery of O2saturated perfluoropentane (PFP). Through the aid of PFP gasification arising from NIR laser-triggered mild hyperthermia, simultaneous PET/PA/US multi-modality imaging and rapid oxygen diffusion across the tumor can be achieved for remarkable hypoxic radiosensitization. Furthermore, the multi-functional oxygen-carrying nanotheranostics also allow for other oxygen-dependent treatments, thus greatly advancing the development of “bubble-enhanced synergistic therapy” platforms.

KEYWORDS: mesoporous organosilica, bubble, tumor oxygenation, radiosensitization, multi-modal imaging

INTRODUCTION Featuring high body penetration length and precise positioning, radiotherapy (RT) has been widely used for noninvasive treatment of deep-seated tumors inside the body with minimal side effects.1-2 However, owing to the insufficient oxygen supply in blood vessels during rapid tumor growth, and the increasd oxygen diffusion distance as well as decreased oxygen


Page 2 of 36

Page 3 of 36 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

transport capacity with tumor propagation/expansion, there exists severe hypoxia within most solid tumors, which causes strong resistance to RT.3-5 In view of the indispensable role of oxygen in increasing the sensitivity of hypoxic cancer cells to X-ray radiation and fixing the RT-induced DNA damage,6 multiple strategies have been proposed to enhance the RT efficacy via radiosensitization.7-10 However, Compared with the recently reported method of in situ oxygen production, which relies heavily on the tumor microenvironment,11-12 the tumor-specific oxygen delivery may serve as a more effective and versatile strategy for oxygenation of various hypoxic tumors, which thus underscores the importance of the pursuit of high-performance oxygen-carrying compounds. Owing to the high affinity towards oxygen molecules, perfluorocarbons (PFCs) have been used as preferential oxygen-carrying candidates.13-14 The particular interest in PFCs stems from their ability to provide a strong mechanical driving force for the fast release and distribution of oxygen throughout the whole tumor via hyperthermia-induced liquid-gas transformation,15 which contributes greatly to the improved tumor oxygenation. Considering the hydrophobicity of PFC, a biocompatible nanocarrier with a hollow cavity is necesary to encapsulate the PFC liquid.16 Importantly, the size of the hollow-structured nanocarrier should be restricted below 50 nm to avoid rapid uptake by the reticuloendothelial system (RES) and simultaneously achieve rich tumor accumulation through the enhanced permeability and retention (EPR) effect.17-18 For clinical purposes, the nanocarrier must be biodegradable to avoid long-term toxicity.19 Besides, facile surface modification is an additional requisite, allowing for structural multi-functionalization of the nanocarrier. To satisfy the above multi-faceted criteria, herein, biocompatible and biodegradable sub-50 nm hollow mesoporous organosilica nanoparticles (HMONs) have been successfully constructed for efficient storage of hydrophobic perfluoropentane (PFP, one type of PFC with low boiling point of 29 °C)20 liquid. By taking advantage of thiol group modification, ultra-


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

small CuS nanoparticles and 64Cu can be attached firmly and chelated stably on the surface of HMONs to realize photoacoustic (PA) imaging and positron emission tomography (PET) imaging, respectively. Benefiting from the nonhazardous mild-hyperthermia upon low power near-infrared (NIR) laser irradiationof CuS, the liquid PFP can be gasified into bubbles for not only enhancing the tumor cell uptake of HMONs, but also intensifying the ultrasound (US) imaging signal. It is hypothesized that the generated bubbles can greatly promote the free diffusion of oxygen from cell to cell inside tumors. The “bystander effect” results in homogeneous tumor oxygenation for remarkably enhanced radiosensitization. Prospectively, the developed bubble-enhanced oxygen-sensitized RT along with judiciously designed oxygen-carrying nanotheranostics in this study will open up new dimensions for multi-modal PET/PA/US image guided radiosensitization of various tumors (Scheme 1) without endogenous oxygen or depth limitations.

Scheme1. Schematic of the construction of O2-PFP@HMCP for PET/PA/US imaging, mildhyperthermia-induced PFP bubble release, and oxygen-sensitized radiotherapy. 4 ACS Paragon Plus Environment

Page 4 of 36

Page 5 of 36 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

RESULTS AND DISCUSSION Distinguished from the preparation of large HMONs by using strong acid and alkali as etching agents,21 herein, a mild “ammonia-assisted selective etching” method was developed to make sub-50 nm HMONs. In brief, according to the “chemical homology” principle, the core/shell structured mesoporous SiO2/organosilica nanoparticles (denoted as MSN@MON, Figure

1a)

were

fabricated

through

the

co-hydrolysis

and

co-condensation

of

tetraethoxysilane (TEOS) and bis[3-(triethoxysilyl)propyl]tetrasulfide (BTES) by using cetyltrimethylammonium chloride (CTAC) as the pore forming agent. It should be emphasized that the addition of 0.1g triethanolamine (TEA)22 as the catalyst is the key to restrict the size of MSN@MON below 50 nm. Considering that the Si-C bonds within the MON shell are more stable and stronger than the Si-O bonds within the MSN core, we strategically used ammonia to selectively etch away the inner MSN core, thus yielding disulfide-bridged HMON (Figure 1b, S1a and S1b). By using mild ammonia instead of strong HF/NaOH as the etching agent, the obtained HMON demonstrated a uniform hollow spherical morphology with an average diameter of around 40 nm. Ultra-small CuS nanoparticles are well known for strong absorbance in near-infrared (NIR) region and high photothermal conversion efficiency,23-24 so the direct conjugation of CuS onto the surface of HMON (HMON@CuS) through thiol groups may endow excellent PA and photothermal performance. The white to dark green color change (Figure 1c) and the energy dispersed X-ray (EDX) spectrum (Figure S2) confirm the successful formation of HMON@CuS with 40 wt.% Cu, as measured by inductively coupled plasma (ICP) analysis. However, CuS decoration did not have notable effect on the hollow structure and particle size of HMON (Figure 1c and S1c). Finally, PEG 2000 was modified onto the surface of HMON@CuS (HMON@CuS-PEG, denoted as HMCP) to further improve the dispersity and


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

stability, as shown by the narrow dynamic light scattering (DLS) size distribution (Figure S3). The Fourier transform infrared (FT-IR) spectra and zeta potential change also confirm the successful conjugation of CuS and PEG (Figure 1d and 1e). Moreover, HMON@CuS exhibits a large surface area of 282.4 m2/g (Figure S4a) and mesopore size of around 3.88 nm (Figure S4b), which allows for sufficient encapsulation of a large variety of payloads, including hydrophobic cargoes. As the disulfide bonds in the framework of HMON are inclined to be cleaved in the reductive tumor microenvironment,25-26 HMON was found to be gradually degraded in simulated glutathione (GSH) solutions (Figure S5). Accordingly, HMCP exhibits a similar time-dependent biodegradable behavior (Figure 1f-h and S6). Meanwhile, the dissociated ultra-small CuS nanoparticles arising from the biodegradation of HMCP can be eliminated through feces and urine,27 which faclitates the in vivo excretion of CuS and further improves the biosafety of HMCP, thus promising for biological applications.


Page 6 of 36

Page 7 of 36 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-c) TEM images of (a) MSN@MON, (b) HMON, and (c) HMON@CuS. Inset of (b): Photograph of HMON aqueous solution. Inset of (c): Photographs of HMON@CuS aqueous solutions. (d) FT-IR spectra of HMON and HMCP. The new bands at 2878 cm-1 and 1464 cm-1 in HMCP indicate C-H stretching in PEG. (e) Zeta potentials of HMON, HMON@CuS, and HMCP. The changes in zeta potentials demonstrate successful conjugation of CuS and PEG. (f-h) TEM images of bio-degradable HMCP immersed in 10 mM GSH aqueous solution for (f) 3 days, (g) 2 weeks, and (h) 3 weeks.

Owing to the hollow cavity, HMON is an ideal nanocarrier for encapsulating PFP via vacuum impregnation.15,28 In contrast to the apparent phase separation between free PFP and PBS (Figure S7a), PFP loaded HMON (PFP@HMON) can be well dispersed in PBS(Figure S7b), indicating the efficient encapsulation of PFP into HMON without leakage. Moreover, by loading PFP into the cavity of HMCP (PFP@HMCP), the strong NIR absorption of HMCP (Figure 2a) makes the liquid-gas phase transformation of PFP possible, as HMCP demonstrates excellent yet stable photothermal performance within at least five cycles of NIR (808 nm) laser irradiation (Figure 2b). According to the linear regression curve between cooling stage and negative natural logarithm of driving force temperature of HMCP (Figure S8), the photothermal conversion efficiency of HMCP was calculated to be 73.9%. Meanwhile, the photothermal effect of HMCP is dependent on its concentration as well as NIR laser power density (Figure 2c-e), which allows for temperature-responsive gasification of PFP, as shown by the gradually emerging PFP bubbles with prolonged NIR laser irradiation (Figure 3a-c). Furthermore, many more bubbles were observed for oxygensaturated PFP@HMCP (O2-PFP@HMCP) upon NIR irradiation (Figure 3d-f), which should be attributed to the release of both PFP and O2.


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

Figure 2. (a) UV-vis spectra of HMON and HMCP. (b) Photothermal stability of HMCP within 5 cycles of NIR laser irradiation. (c) Temperature rise profiles of HMCP aqueous solution with different concentrations upon NIR laser irradiation (power density: 0.5 W/cm2) for 3 min. (d) Temperature rise profiles of HMCP aqueous solution upon NIR laser irradiation with different power densities (concentration of HMCP: 100 µg/mL) for 3 min.(e) Photothermal images of HMCP aqueous solution with different concentrations upon NIR laser irradiation (laser power density: 0.5 W/cm2, upper row) for 3 min, and 100 µg/mL of HMCP aqueous solution upon NIR laser irradiation with various power densities (bottom row) for 3 min.


Page 8 of 36

Page 9 of 36 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-c) Optical microscopy images of PFP@HMCP aqueous solution (a) before and after NIR laser irradiation for (b) 10, and (c) 20 s. (d-f) Optical microscopy images of O2PFP@HMCP aqueous solution (d) before and after NIR laser irradiation for (e) 10 s, and (f) 20 s. (g) O2 concentration changes of water after adding PFP@HMCP with or without NIR laser irradiation under a N2 atmosphere. (h) O2 concentration changes of water after adding O2-PFP@HMCP with or without laser irradiation under a N2 atmosphere.

The above temperature-responsive O2 releaseis expected to increase the dissolved oxygen concentration in the hypoxic environment. As shown in Figure 3g and 3h, when adding O2PFP@HMCP instead of PFP@HMCP, the dissolved oxygen concentration was rapidly elevated and could persist for several minutes, suggesting the stable encapsulation of oxygen saturated PFP in HMCP. After exposure to NIR irradiation, a sharp rise of dissolved oxygen concentration was observed due to the burst release of O2 from PFP gasification. Moreover, 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

the dissolved oxygen concentration remained at a relatively high level within 15 min postirradiation, indicating that O2-PFP@HMCP serves as an excellent oxygen reservoir for alleviating hypoxia. It has been reported that bubbles can create permeable defects in lipid bilayers,29 which may contribute to enhancing the cellular uptake of nanoparticles. In order to verify this hypothesis, our designed HMCP was labeled with FITC to allow for quantitative evaluation and visual observation of intracellular transport via flow cytometry analysis and confocal luminescence imaging, respectively. The NIR laser power density was tuned to maintain the temperature around 42 °C in cells, so that the generated mild hyperthermia would not damage the cells. No matter whether exposed to NIR laser irradiation, both HMON and HMCP were biocompatible (Figure S9). As shown in Figure 4a, although HMCP-FITC combined with NIR laser irradiation enhanced the U87MG cell uptake to some extent owing to the mild temperature rise,30 the cellular uptake of PFP@HMCP-FITC could be further elevated upon NIR irradiation. This may be ascribed to the generated PFP bubbles driving more nanoparticles into the cytoplasm, as exhibited by the stronger green fluorescence intensity of PFP@HMCP-FITC around the blue nuclei than that of HMCP-FITC upon NIR laser irradiation (Figure 4b). Moreover, the bio-TEM images provided visual evidence of a greater number of PFP@HMCP nanoparticles in U87MG cells subjected to NIR laser irradiation (Figure 4c, the red arrows). By contrast, only few nanoparticles were endocytosed without any treatment, and the intracellular transport efficiency of HMCP was also suboptimal even with exposure to NIR laser irradiation (Figure 4c, the yellow arows), which further confirmed the bubble-enhanced tumor cell uptake of nanoparticles. Therefore, it can be concluded that the mild hyperthermia-induced liquid-gas transformation of PFP enhances the tumor celluptake of nanoparticles in addition to promoting the fast intracellular/intercellular delivery and release of oxygen, which may substantially alleviate the tumor cell hypoxia.


Page 10 of 36

Page 11 of 36 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 4. (a) Flow cytometry analyses of U87MG cells incubated with HMCP-FITC and PFP@HMCP-FITC for 15 min without or with NIR laser irradiation. Inset of (a): The corresponding relative mean fluorescence intensity. (b) Confocal luminescence imaging of U87MG cells after incubated with FITC conjugated HMCP and PFP@HMCP for 15 min without or with NIR laser irradiation. Scale bar: 50 µm. (c) Bio-TEM images of U87MG cells incubated with HMCP and PFP@HMCP for 15 min without or with NIR laser irradiation. The yellow arrows refer to internalized HMCP nanoparticles upon NIR laser irradiation. The red arrows refer to internalized PFP@HMCP nanoparticles upo nNIR laser irradiation.

In contrast to normal U87MG cells, hypoxic U87MG cells were cultured in a low-oxygen atmosphere of 1% O2/5% CO2/94% N2. When exposed to X-rayradiation, hypoxic cells exhibited a much higher viability than normal cells, which confirmed the stronger resistance of hypoxiccells to RT (Figure S10). We then examined the potential of our designed O2PFP@HMCP in overcoming the hypoxic resistance and improving the RT effects. As the temperature of an incubator at 37 °C is higher than the boiling point of PFP, NIR laser 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

irradiation is not required for inducing PFP gasification. Figure S11 shows that O2PFP@HMCP exhibited low cytotoxicity at 37 °C, which indicates that the generated PFP and oxygen bubbles did not result inpermanent damage to the cells. However, O2-PFP@HMCP gave rise to a significant radiation enhancement effect, much better than HMCP and PFP@HMCP, which may be attributed to the rapid PFP bubble-induced O2 release for greatly increased sensitivity of hypoxic U87MG cells to X-ray radiation (Figure 5a and Figure S12). Moreover, the calcein AM/PI stained images also demonstrate a larger population of dead cells following treatment with O2-PFP@HMCP + RT (reflected by the stronger red fluorescence intensity in comparison to other treatments) as well as an elevated hypoxic cell death rate with increasing incubation time and X-ray doses (Figure S13 and S14). The corresponding mechanism of hypoxic radiosensitization by O2-PFP@HMCP was explored by probing the intracellular O2 level using a [Ru(dpp)3]Cl2 indicator. It can be observed that the red fluorescence of [Ru(dpp)3]Cl2 was quenched in the cells treated by O2PFP@HMCP and NIR laser irradiation for 15 min, which indicated the greatly diminished hypoxia arising from the rapid O2 release and diffusion through cells. Contrastingly, other nanoparticles without O2 did not improve the oxygenation, as seen by the unchanged red fluorescence of [Ru(dpp)3]Cl2 (Figure 5b and 5c). Furthermore, the intensified oxygenation was accompanied by the enhanced X-ray-induced reactive oxygen species (ROS) generation, as evidenced by the recurrent green fluorescence of DCFH-DA (a probe for detecting ROS) in the cells treated by O2-PFP@HMCP + RT in Figure 6a and Figure S15. DNA damage by ROS was assessed by single cell gel electrophoresis (comet) assay (Figure 6b). In general, the longer tail of fluorescent DNA stain signifies greater DNA damage.31-32 Although the DNA damage of hypoxic cells is not highly dependent on the X-ray dosage, the addition of O2PFP@HMCP caused much more significant DNA damage than that of PFP@HMCP (Figure 6c). Therefore, it is O2 rather than PFP that brings about significant DNA damage for X-ray-


Page 12 of 36

Page 13 of 36 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

triggered hypoxic cell destruction, while gasified PFP bubbles play an indispensable role in driving the fast intracellular O2 release and intercellular O2 diffusion, which further improves oxygenation and enhances hypoxic radiosensitization based on the so-called “bystander effect”.

Figure 5. (a) Relative viabilities of hypoxic U87MG cells incubated with HMCP, PFP@HMCP, and O2-PFP@HMCP for 24, 48, and 72 h after exposure to 6 Gy of X-ray irradiation. (b) Flow cytometry analysis of O2 in hypoxic U87MG cells incubated with HMCP, PFP@HMCP, and O2-PFP@HMCP for 15 min without or with NIR laser irradiation. Inset of (b): The corresponding relative mean fluorescence intensity. (c) Confocal luminescence images of hypoxic U87MG cells after incubated with HMCP, PFP@HMCP, and O2-PFP@HMCP without or with NIR laser irradiation. Scale bar: 50 µm. ***P < 0.001.


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

Figure 6. (a) Flow cytometry analysis of ROS in hypoxic U87MG cells incubated with fresh medium, HMCP, PFP@HMCP, and O2-PFP@HMCP without or with X-ray irradiation. Inset of (a): The corresponding relative mean fluorescence intensity. (b) Single cell gel electrophoresis (comet) assay of hypoxic U87MG cells incubated with fresh medium, PFP@HMCP, and O2-PFP@HMCP before and after exposure to X-ray irradiation. (c) Quantitative analysis of DNA damage of hypoxic U87MG cells incubated with fresh medium, PFP@HMCP, and O2-PFP@HMCP before and after exposure to X-ray irradiation. *P

Biodegradable Hollow Mesoporous Organosilica Nanotheranostics for

Recommend Documents