Letter Cite This: Nano Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/NanoLett
Intracellularly Activatable Nanovasodilators To Enhance Passive Cancer Targeting Regime V. G. Deepagan,†,# Hyewon Ko,‡,# Seunglee Kwon,† N. Vijayakameswara Rao,† Sang Kyoon Kim,§ Wooram Um,‡ Sohee Lee,∥ Jiwoong Min,∥,⊥ Jeongjin Lee,‡ Ki Young Choi,† Sol Shin,‡ Minah Suh,‡,∥,⊥ and Jae Hyung Park*,†,‡,⊥ †
School of Chemical Engineering, College of Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Suwon 16419, Republic of Korea § Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea ∥ Center for Neuroscience Imaging Research, Institute for Basic Science, Suwon 16419, Republic of Korea ⊥ Department of Biomedical Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea ‡
S Supporting Information *
ABSTRACT: Conventional cancer targeting with nanoparticles has been based on the assumed enhanced permeability and retention (EPR) effect. The data obtained in clinical trials to date, however, have rarely supported the presence of such an effect. To address this challenge, we formulated intracellular nitric oxide-generating nanoparticles (NO-NPs) for the tumor site-specific delivery of NO, a well-known vasodilator, with the intention of boosting EPR. These nanoparticles are selfassembled under aqueous conditions from amphiphilic copolymers of poly(ethylene glycol) and nitrated dextran, which possesses inherent NO release properties in the reductive environment of cancer cells. After systemic administration of the NO-NPs, we quantitatively assessed and visualized increased tumor blood flow as well as enhanced vascular permeability than could be achieved without NO. Additionally, we prepared doxorubicin (DOX)-encapsulated NO-NPs and demonstrated consequential improvement in therapeutic efficacy over the control groups with considerably improved DOX intratumoral accumulation. Overall, this proof of concept study implies a high potency of the NO-NPs as an EPR enhancer to achieve better clinical outcomes. KEYWORDS: Nitric oxide, nitrated polysaccharide, vasodilation, enhanced permeability and retention effect, drug delivery
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combination therapy of NO and a chemotherapeutic drug has gained significant attention because NO not only improves the EPR by relaxing the smooth muscle layer to dilate blood vessels via the cyclic GMP signaling pathway,12,13 but also acts synergistically with the anticancer drug by upregulating p53 genes and blocking p-glycoproteins.14−16 NO is a gaseous free radical with an extremely short half-life (1 s) and is rapidly converted into nitrate by oxyhemoglobin in blood.17,18 Because of its reactive nature, NO is active only for a short distance, around 100 μm, from the site of production.19,20 Therefore, an ideal NO delivery platform for cancer therapy should be stable during blood circulation and should generate a biologically relevant concentration of NO at the tumor site. Unfortunately, the commonly used NO donors such as S-
n recent years, cancer therapeutics based on nanosized particles have been considered as potential alternatives to conventional chemotherapy because they can selectively deliver their payloads to tumor sites via the enhanced permeation and retention (EPR) effect.1−4 Despite the solid reputation of the EPR effect based on the huge success of cancer nanomedicines in preclinical studies, these nanomedicines have shown little clinical significance. For instance, the commercially available versions such as Doxil and Myocet have substantially reduced side effects but have failed to enhance the therapeutic outcomes of the diseases.5,6 This is primarily attributed to the finding that for conventional nanoparticles less than 10% of the original dose reached tumor sites after systemic administration.7,8 To improve the therapeutic efficacy of the nanomedicines, innovative approaches are needed to enhance the efficiency of passive targeting. One of the recent strategies has been focused on dilation of the tumor blood vessels to increase their permeability by providing vascular mediators including bradykinin9 and nitric oxide (NO).10,11 In particular, the © XXXX American Chemical Society
Received: February 4, 2018 Revised: February 27, 2018
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DOI: 10.1021/acs.nanolett.8b00495 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 1. Schematic representation of EPR boosted by DOX-NO-NPs. (a) NO-NPs can generate NO via reduction of hydrophobic nitrates back to hydroxyl groups in the presence of glutathione (GSH). (b) When the DOX-NO-NPs are administered intravenously, they extravasate into tumors through leaky tumor vasculature. Once taken up by tumor cells, they generate NO, which acts as a local vasodilator and releases DOX. The dilated blood vessels increase the blood flow in the tumor, thereby boosting EPR and facilitating the accumulation of the NO-NPs in the tumor tissue. This process repeats itself until an equilibrium is reached between the rates of NO generation and vasodilation.
nitrosothiols and diazeniumdiolates start generating NO by hydrolysis during blood circulation before they reach the tumor site.21 To date, no efforts have been made to develop a polymeric nanoparticle system for enhanced vasodilation via tumor-targeted NO delivery. Herein, we report a novel class of tumor-targeted NO donor, simply composed of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic nitrated dextran (NO-Dex), which inherently releases NO in the presence of glutathione (GSH), a compound abundant at the intracellular level of tumors. This amphiphilic PEG-b-NO-Dex spontaneously self-assembled into stable nanoparticles in aqueous solution. The NO-generating nanoparticles (NO-NPs) also underwent a hydrophobic to hydrophilic transition, leading to the disruption of their micellar structure in the presence of GSH (Figure 1a). This unique feature was advantageous not only in supplying NO at the tumor site but also in delivering a payload that was encapsulated in the hydrophobic cores of the NPs. Our main hypothesis was that once they are taken up by cancer cells, the doxorubicin (DOX)-encapsulated NO-NPs (DOX-NO-NPs) start releasing NO and DOX simultaneously (Figure 1b). Consequently, the released NO would dilate tumor blood vessels, increase the vessel permeability, and boost EPR to initiate a positive feedback of inviting more DOX-NO-NPs into the tumor interstitium, by which their therapeutic efficacy is determined.
We synthesized PEG-b-NO-Dex to prepare GSH-sensitive NO-NPs by simply modifying the hydroxyl groups of PEG-bDex into nitro esters. The control NPs, which are insensitive to GSH, were prepared by conjugating lithocholic acid (LA) to PEG-b-Dex (Figure S1, Supporting Information). The chemical structures of the polymers including PEG-b-NO−Dex and PEG-b-LA-Dex were confirmed by 1H nuclear magnetic resonance (1H NMR), and Fourier transform infrared (FTIR) spectroscopy (Figure S2 and S3a,b). The degree of nitration of the synthesized PEG-b-NO-Dex was estimated to be 2.03 nitrates per unit sugar molecule of dextran based on the elemental analysis. First, to prove that the nitration did help the block copolymer to self-assemble in aqueous solution, 1H NMR spectra of the PEG-b-Dex were recorded before and after nitration in D2O. The data clearly showed the characteristic peaks of PEG and dextran before nitration (Figure 2a) but only the PEG peak was present after nitration (Figure 2b). This suggests that after nitration the protons in the dextran block were not interacting with water (D2O) as it self-assembled into a compact core.22 In addition, red fluorescence of Nile red was observed only in the aqueous solution of PEG-b-NO-Dex which could encapsulate the hydrophobic dye in the nanoparticular core (Figure 2c). These data suggest that the PEG-b-NO-Dex self-assembled to form the PEG corona and nitrated dextran core. The critical micelle concentration of PEG-b-NO-Dex was 16.92 μg/mL (Figure S3c), which is similar to those of other amphiphilic B
DOI: 10.1021/acs.nanolett.8b00495 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 2. Physicochemical characterization of NO-NPs. (a,b) 1H NMR of the PEG-b-Dex in D2O, before nitration (a) and after nitration (b). (c) Photoluminescence spectrum of Nile red. The insert images were taken under UV excitation (i) before nitration and (ii) after nitration. (d,e) The size distribution of NO-NPs at 24 h without (d) and with 10 mM GSH (e). The insert is a TEM image of the NPs. Scale bar: 100 nm. (f) Photoluminescence spectra of Nile red-loaded NO-NPs in the presence of 10 mM GSH. The black arrow indicates the reduction in fluorescence intensity.
block copolymers.23 As demonstrated in the dynamic light scattering and transmission electron microscopy (TEM) images, the NO-NPs and the control NPs were spherical and monodispersed with average hydrodynamic radii of 134 ± 3.2 nm and 113 ± 3.1 nm, respectively (Figure S4a,b). Moreover, both types of nanoparticles exhibited excellent stability for at least 5 days in phosphate buffered saline (PBS; pH 7.4) (Figure S4c). These characteristics of NO-NPs such as the small size and high stability are considered to be beneficial to design the cancer nanomedicines.24 The NO generation from organic nitrates is associated with various factors such as GSH, organic nitrate reductase, p450, and hypoxia.25,26 However, in living cells GSH is the primary and most abundant source, which can reduce the organic nitrates to produce NO. To demonstrate the responsiveness of the NO-NPs toward the reductive environment of the cells, we incubated the particles in 0 and 10 mM of GSH solution. As shown in Figure 2d, the NO-NPs retained their structural integrity only in the absence of GSH. Conversely, the NO-NPs were destabilized in the presence of 10 mM GSH (Figure 2d,e). Furthermore, a time-dependent gradual decrease in fluorescence intensity was observed only when the Nile red-loaded NO-NPs were in the presence of 10 mM GSH (Figure 2f and Figure S5). That is probably due to the reduction of hydrophobic nitrates back to hydrophilic hydroxyl groups, which results in NO generation and destabilized their structural integrity. To validate this claim further, the level of GSHdependent NO generation from NO-NPs was assessed using a NO-sensitive fluorescent dye, 4-amino-5-methylamino-2,7difluorofluorescein (DAF-FM) (Figure S6). When incubated
the NO-NPs with 10 mM GSH solution, the intensity of DAFFM increased as a function of time, suggesting that the NO was generated from the NO-NPs in the presence of GSH. On the other hand, no significant increase in the intensity of DAF-FM was observed in the absence of GSH. These results imply that the NO generation is mediated by the reduction of the NONPs considering the fact that GSH is the major cellular reductant and NO is the product of nitrate reduction reaction. Griess reagent was used to indirectly quantify the amount of NO released from the nanoparticles by measuring the total nitrate and nitrite concentration. We observed that 0.918 μM of the NO, corresponding to a 1.97% conversion rate of the NOreleasing moiety to NO, was released within 48 h in a sustained manner (Figure 3a). No such release was observed with the Control NPs. Subsequently, DOX, a hydrophobic anticancer drug, was encapsulated into the nanoparticles by the solvent exchange method. We measured the DOX release profile of the DOXloaded samples (DOX-Control NPs and DOX-NO-NPs) in the presence and absence of 10 mM GSH for 72 h (Figure 3b). As expected, we observed enhanced release behavior for the DOXNO-NPs in the presence of GSH, compared to that in the absence of GSH. The difference in the release profile of the DOX-NO-NPs might be attributed to the conversion of hydrophobic nitrate ester to hydrophilic dextran. In contrast, no significant difference was observed for the DOX-Control NPs due to the absence of the GSH-sensitive functional group. Thereafter, the intracellular uptake and NO delivery behavior of the NPs were evaluated by visualizing NP-treated HT29 cells under confocal microscopy. The DAF-FM was used to visualize C
DOI: 10.1021/acs.nanolett.8b00495 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 3. In vitro release profiles and fluorescence imaging. (a) In vitro NO release profile of nanoparticles in the presence of 10 mM GSH. (b) In vitro DOX release profiles of DOX-control NPs and DOX-NO-NPs in PBS (pH 7.4) as a function of time. The error bar represents standard deviation (n = 3). * p < 0.05 calculated by one-way ANOVA test. (c) Confocal microscopic images showing the intracellular NO (green), DOX (red), and cell nuclei (blue). Scale bar: 20 μm. (d) Total nitrate and nitrite concentrations in spent media after treatment of NO-NPs.
the intracellular NO content. The images (Figure 3c) confirmed that the DOX-NO-NPs were efficiently taken up by the cancer cells, resulting in intracellular delivery of NO and drug. Conversely, very little NO signal was observed from the cells treated with DOX-Control NPs and free DOX. After analyzing the effectiveness of the DOX-NO-NPs as delivery vehicles for DOX and NO into the cells, we evaluated their in vitrocytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HT29 human colon carcinoma cells were treated with the bare and DOX-loaded nanoparticles. When the cells were treated with PEG-b-Dex and the bare NPs, we did not observe any remarkable toxicity at their concentrations of up to 500 μg/mL (Figure S7a). However, for the DOX-loaded nanoparticles, we observed a higher cell death for DOX-NO-NPs than for the DOX-Control NPs (Figure S7b). The accelerated drug release behavior of the DOX-NO-NPs inside the cancer cells might be the reason for the discrepancy in the observed cytotoxicity. The Griess reagent was used to further analyze the total nitrate and nitrite content in the medium in which the cells were incubated for 24 h. We observed a dose-dependent increase in the nitrate and nitrite concentrations (Figure 3d), suggesting that the intracellular NO produced from NO-NPs can diffuse to the extracellular space where NO would act as vasodilator in vivo. Encouraged by the NO generation results in vitro, we performed in vivoquantitative analysis of the changes in tumor blood flow after systemic administration of the NONPs to the HT29 tumor-bearing mice using three-dimensional ultrasound power Doppler (Figure 4a,b). The results clearly indicate that for the first 6 h the blood flow in the tumor tissue rapidly increased to 30% of the base level and then gradually
returned to the normal level as time progressed, whereas the saline-treated groups did not show any significant increase in vascularity for the period of time tested. To visualize the early events upon administration of NO-NPs, the mice were treated with saline and NO-NPs, followed by laser speckle contrast imaging of the tumor vasculature (Figure 4c). Before treatments of saline or NO-NPs, only the prominent blood vessels were seen in the tumors. However, the microvasculature became clear in the mice that received the NO-NPs at 6 h postinjection, implying enhanced blood flow of the tumor vasculature. We did not observe such behavior in the salinetreated tumors. Additionally, we observed tumor blood vessels using twophoton microscopy through the intravital imaging window on a subcutaneous HT29 tumor xenograft mouse model (Figure 4d,e). Before treating the mice with saline, Control NPs, or NO-NPs, the tumor blood vessels (red color, baseline) were brightened by Texas Red-labeled dextran (TR-Dex, 70 kDa). Compared with the 12 h post saline or Control NPs treatment, NO-NPs treatment induced much more extravasation of the TR-Dex into the tumor tissue from the blood vessels, implying their higher permeability. Compared with the 12 h post saline treatment, NO-NPs treatment induced much more extravasation of the TR-Dex into the tumor tissue from the blood vessels implying their higher permeability. It should be noted that the fluorescein-labeled dextran (FITC-Dex, 150 kDa) was used to visualize the blood vessels (green color, after treatment) differentiating them from the extravasated TR-Dex molecules. Much of the careful observation also revealed that the tumor blood vessels were dilating in several regions (Figure S8), which might explain the observed increase in the EPR. A cellD
DOI: 10.1021/acs.nanolett.8b00495 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 4. In vivo vasodilation effect after systemic administration of NO-NPs. (a) Representative power Doppler images depicting tumor vascularity. The colors indicate the flow directions toward (red) or away from (blue) the transducer. (b) Sonographic measurement of changes in vascularity upon treatment with saline and NO-NPs. The red arrow indicates the time point of intravenous administration. (c) Representative laser speckle images of changes in tumor vascularity. (d) Experimental illustrations for dual-color imaging of tumor vasculature using two-photon microscopy. (e) Representative two-photon microscopic images of tumor vasculature, labeled using TR-Dex (70 kDa) (red) or FITC-Dex (150 kDa) (green). Scale bar, 50 μm.
fluorescence (NIRF) imaging technique. NPs were fluorescently tagged via chemical conjugation of Cy5.5. The Cy5.5labeled nanoparticles were injected into the tail vein of HT29 tumor-bearing mice, and NIRF images were recorded at predetermined intervals (Figure 5a). The mice were divided into two groups, the Control NPs and NO-NPs group. Significant NIRF signals were found in the whole body in the initial hours, suggesting prolonged circulation of nanoparticles in the blood. The mice that were treated with NO-NPs showed a significant increase in the accumulation of nanoparticles, compared to the control group. At 24 h, the mice were sacrificed, and the total intensities of fluorescence were calculated for the excised tumor. The results suggested that the NO-NPs had 1.75-fold higher accumulation in the tumor tissues than that of the control group (Figure 5b). Such results together with the aforementioned two-photon data imply that by dilating the blood vessels the NO-NPs are capable of
impermeable NO-sensitive fluorescent dye (4,5-Diaminofluorescein, DAF-2) was used to confirm the extracellular presence of vasodilatory NO in tumor tissue. After treating the mice with saline or NO-NPs, the tumoral region was observed using an in vivofluorescence molecular imaging system. Interestingly, extracellular NO (green color) was apparently found near the blood vessels (red color) of NO-NP-treated mice, but no significant NO signal was detected in saline-treated mice (Figure S9). Although NO would be initially generated at the intracellular level from NO-NPs, the results imply that NO diffused through the cell membrane into the extracellular matrix and distributed nearby the blood vessels as is the case with NO produced by the intracellular enzymes.27 Such results partially confirm that the NO released from the NO-NPs in the tumor tissue might have dilated the blood vessels to facilitate the EPR effect. The in vivo tumor homing abilities of the NO-NPs and Control NPs were monitored by a real-time near-infrared E
DOI: 10.1021/acs.nanolett.8b00495 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 5. In vivo biodistribution and antitumor efficacy of DOX-loaded NPs in HT29 tumor bearing mice. (a) Time-dependent biodistribution of Cy5.5-labeled NO-NPs. (b) Ex vivo fluorescence intensities of tumors at 24 h postinjection. The error bars represent the standard deviations (n = 3; *p < 0.05 calculated by one-way ANOVA). (c) DOX content in major organs and tumor lysate. (d) Changes in tumor volume for each treatment group (*p < 0.01 calculated by one-way ANOVA). Error bars in the graph represent standard deviations (n = 5). (e) Changes in tumor weights after treatment with the samples. Scale bar, 1 cm.
environment.28 This enormous concentration difference is likely to contribute tumor site-specific release of NO from NONPs. Moreover, the released NO in normal tissue is quickly converted to inactive nitrate and nitrite ions. However, due to the low pH and hypoxic conditions in the tumor tissue the nitrate and nitrite ions generated from the released NO are converted back to biologically active NO molecules.29 These actions enhanced the EPR in the tumor region but not in the normal tissue. This selective enhancement in EPR is a highly desirable quality for any nanomedicines in order to improve the accumulation at the target site. The antitumor efficacies of the DOX-loaded nanoparticles and the free DOX were evaluated by measuring the tumor volumes and weights post-treatment (Figure 5d,e). The mice treated with DOX-NO-NPs had the lowest tumor volumes and tumor weights, compared with the mice treated with the DOXControl NPs, free DOX, or saline. On day 14, the tumor
enhancing the natural tendency of the nanoparticles to accumulate passively in tumor tissue. The drug distributions and antitumor efficacies of free DOX, DOX-Control NPs, and DOX-NO-NPs were assessed in HT29 tumor-bearing mice. The amount of DOX in each organ was quantified at 12 h after the systemic administration of DOXloaded nanoparticles (Figure 5c). The results show that the DOX content in the tumors of the mice treated with the DOXNO-NPs (14.50 μg/g tissue) was significantly higher than in those treated with the DOX-Control NPs (9.33 μg/g tissue) or the free DOX (2.16 μg/g tissue). Interestingly, the DOX contents of other major organs showed little difference between the groups. This preferential accumulation of DOX in the tumors might be attributed to the increases in blood flow and vasodilatory effect of the NO that were generated in situ by the DOX-NO-NPs. In particular, the GSH concentration in cancer cells is 100−1000-fold higher than that in blood or extracellular F
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Nano Letters volumes were 544.5, 393.8, 308.6, and 149.9 cm3 for the saline, free DOX, DOX-Control NP, and DOX-NO-NP groups, respectively. These significant reductions in tumor volume strongly indicated that the enhanced EPR of the DOX-NO-NPs delivered DOX more effectively over the control groups. Furthermore, the body weights of the animals treated with DOX-NO-NPs showed no drastic changes compared with those of the animals treated with free DOX (Figure S10). Hematoxylin and eosin (H&E) staining also confirmed that the DOX-NO-NPs showed maximal cell death in the tumor and minimal damage to other major organs (Figure S11), compared to the other groups. It should be noted that NO is a potent secondary messenger, which has both beneficial and harmful effects on tumor progression. The concentration of NO is a major factor that determines the activity of NO. For instance, a lower concentration of NO has been shown to trigger an antiapoptotic response by up-regulating Bcl-2 genes and increasing metastasis in many cancer types.29,30 Conversely, a higher concentration of NO has been reported to be proapoptotic by causing DNA damage and by upregulating the p53 gene, transforming growth factor-β, and poly(ADPribose) polymerase.31−33 The nanocarrier should deliver a high concentration of NO to achieve a desirable therapeutic outcome in cancer treatment. Fortunately, the high degree of nitration (46.59 μM of NO equivalent/mg nanoparticles) implies that the NO-NPs can generate significant amounts of NO, which swings the balance toward the beneficial effects of NO. Nitrated polysaccharide has tremendous advantages over low-molecular weight NO donors such as large NO carrying capacity, improved tumor site-specific delivery, and increased retention time. Nevertheless, no efforts have been made to develop a biocompatible polymeric NO donor to boost EPR. This is probably because of the insoluble nature of nitrated polysaccharide in aqueous media. In this study, we clearly showed that PEGylated nitrated polysaccharide can be used as an EPR-enhancing drug delivery platform for the stimuliresponsive dual delivery of NO and DOX. The NO-NPs have the ideal characteristics of nanocarriers such as easy scale up and the simultaneous triggered release of NO and drug in the presence of GSH. This type of accelerated drug release is highly beneficial, as it can also help to overcome drug resistance. Most nanoformulations that have been shown to increase the median lifespan in mouse models have failed to perform consistently in cancer patients. This is mainly due to the anatomical and physiological differences between the mouse models and human tumors.34 The blood vessels of the human tumors are better organized and less leaky than the tumors in mouse models, leading to a call for reliable means to boost EPR in tumors for improving overall therapeutic outcomes.35 We consider the developed NO-NPs as a universal EPR enhancer, which may contribute to advancing the clinical implementation of translating nanomedicines.
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cytotoxicity; two-photon microscopic images, fluorescence images for extracellular NO and in vivo toxicity (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: +82-31-290-7288. Fax: +82-31-299-6857. E-mail:
[email protected]. ORCID
Jae Hyung Park: 0000-0002-5043-9455 Author Contributions #
V.G.D. and H.K. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the Basic Science Research Programs (20100027955 and 2018R1A2B3006080) of the National Research Foundation (NRF), Republic of Korea.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.8b00495. Materials, experimental details, the synthetic scheme, 1H NMR spectra, and additional characterization of NONPs and Control NPs; in vitro NO release behavior and G
DOI: 10.1021/acs.nanolett.8b00495 Nano Lett. XXXX, XXX, XXX−XXX
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Nano Letters (22) Nguyen, T.−K.; Selvanayagam, R.; Ho, K. K. K.; Chen, R.; Kutty, S. K.; Rice, S. A.; Kumar, N.; Barraud, N.; Duong, H. T. T.; Boyer, C. Chem. Sci. 2016, 7, 1016−1027. (23) Oerlemans, C.; Bult, W.; Bos, M.; Storm, G.; Nijsen, J. F.; Hennink, W. E. Pharm. Res. 2010, 27, 2569−2589. (24) Cuong, N. V.; Li, Y. L.; Hsieh, M. F. J. Mater. Chem. 2012, 22, 1006−1020. (25) Blanco, E.; Shen, H.; Ferrari, M. Nat. Biotechnol. 2015, 33, 941− 951. (26) Agvald, P.; Adding, L. C.; Artlich, A.; Persson, M. G.; Gustafsson, L. E. Br. J. Pharmacol. 2002, 135, 373−382. (27) Denicola, A.; Souza, J. M.; Radi, R.; Lissi, E. Arch. Biochem. Biophys. 1996, 328, 208−212. (28) Torchilin, V. P. Nat. Rev. Drug Discovery 2014, 13, 813−827. (29) Fukumura, D.; Kashiwagi, S.; Jain, R. K. Nat. Rev. Cancer 2006, 6, 521−534. (30) Maeda, H.; Nakamura, H.; Fang, J. Adv. Drug Delivery Rev. 2013, 65, 71−79. (31) Xu, W.; Liu, L. Z.; Loizidou, M.; Ahmed, M.; Charles, I. G. Cell Res. 2002, 12, 311−320. (32) Wang, X.; Zalcenstein, A.; Oren, M. Cell Death Differ. 2003, 10, 468−476. (33) Blecher, K.; Martinez, L. R.; Tuchman-Vernon, C.; Nacharaju, P.; Schairer, D.; Chouake, J.; Friedman, J. M.; Alfieri, A.; Guha, C.; Nosanchuk, J. D.; Friedman, A. J. Nanomedicine 2012, 8, 1364−1371. (34) Lammers, T. J. Controlled Release 2015, 91, 3−6. (35) Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Nat. Rev. Cancer 2017, 17, 20−37.
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