Ultrasound-Triggered Nitric Oxide Release Platform Based on Energy

Nov 22, 2016 - Department of Medical Ultrasound, Shanghai Tenth people's ...... on portable focused ultrasound therapeutic apparatus (Chattanooga, USA...
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Ultrasound-Triggered Nitric Oxide Release Platform Based on Energy Transformation for Targeted Inhibition of Pancreatic Tumor Kun Zhang,†,‡,§ Huixiong Xu,‡,§ Xiaoqing Jia,† Yu Chen,† Ming Ma,† Liping Sun,‡ and Hangrong Chen*,† †

State Key Laboratory of High performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, P. R. China ‡ Department of Medical Ultrasound, Shanghai Tenth people’s Hospital, Tongji University School of Medicine, 301 Yan-chang-zhong Road, Shanghai 200072, P. R. China § Ultrasound Research and Education Institute, Tongji University School of Medicine, 301 Yan-chang-zhong Road, Shanghai 200072, P. R. China S Supporting Information *

ABSTRACT: Inspired by considerable application potential in various diseases, nitric oxide (NO) has gained increasing attention. Nevertheless, current NO release scaffolds suffer from some inevitable drawbacks, for example, high toxicity for NO donor byproducts, poor specificity, shallow penetration depth, and strong ionizing irradiation for triggers, all of which remain obstacles to clinical application. Herein, an ultrasound-triggered NO on-demand release system is constructed using natural L-arginine as NO donor and local ultrasound as trigger. The focused ultrasound can activate H2O2 to generate more oxygen-contained species (ROS) of stronger oxidation ability than H2O2 for oxidizing LA via the energy transformation from ultrasound mechanical energy to chemical energy, and thus produce more NO for ultimately suppressing the highly aggressive and lethal Panc-1 tumor. Moreover, a blood vessel−intercellular matrix−cell “relay” targeting strategy has been established and relying on it, over 7-fold higher retention of such NO release system in a subcutaneous xenograft mouse model of Panc-1 is obtained, which consequently results in a more evident inhibitory effect and a prolonged survival rate (80% ± 5% improvement in 60-day survival). KEYWORDS: Nitric oxide, ultrasound trigger, L-arginine donor, oxygen-contained species, Panc-1 tumor, “relay” targeting

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Up to now, N-diazeniumdiolate (NONOate)-based and Snitrosothiols (RSNO)-based NO scaffolds have been universally accepted as the dominant NO donors.5 Nevertheless, the toxic polyamine and carcinogenic nitrite amine after Ndiazeniumdiolates decomposition and the rapid clearance of RSNOs by the immune system remain obstacles to their clinical application.4,5 As for triggers, some endogenous triggers,6 for example, enzyme,7 thiyl radical, and copper ion,8,9 were initially explored. However, these endogenous triggers were not specific in tumors but universal in diverse organs, resulting in failure of on-demand release. Furthermore, some exogenous local triggers, for example, light,10 heat, and X-ray,11,12 were employed, but these exogenous triggers also encounter some

s a star molecule, nitric oxide (NO) molecules have gained increasing interest due to their extensive applications including cardiovascular homeostasis, platelet aggregation and adhesion, immune response to infection, bone metabolism and neurotransmission, and antibacterial/antibiofilm activity.1,2 Recently, it has been validated that NO molecules hold great potential in tumor therapy.1 However, low NO content not only failed to work but probably promoted further progression of disease, especially for cancers.3,4 Thus, maximizing NO in lesions is an indispensible prerequisite for acquiring excellent treatment outcomes but remains a challenge. In an attempt to deliver NO, stimulus-responsive NO release systems have attracted considerable attention, because they can realize smart release typically featuring spatial, temporal, or dose controlled delivery and enabling NO-mediated treatment.5 In designing stimulus-responsive NO release systems, the two components, NO donor and trigger, are the primary concerns.5 © 2016 American Chemical Society

Received: July 23, 2016 Accepted: November 22, 2016 Published: November 22, 2016 10816

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Figure 1. Characterizations of peptide−HMSN−LA synthesis process: (a,b) TEM images of the ultimate product, that is, peptide−HMSN−LA particles; (c) synthesis flowchart of peptide−HMSN−LA; (d,e) ζ-potential variation (d) and particle size (e) of modified HMSNs with different functional groups after each modification; (f) FTIR spectra of modified HMSNs with different functional groups after each modification.

ultrasound-responsive NO release in tumor, since the concentration of H2O2 in tumor is much higher than that in other normal organs in most tumor models.23−25 Instead of conventional triggers such as light, heat, and X-ray, the local ultrasound at a frequency of 1 MHz performed as the trigger to accelerate reaction between LA molecules in such a NO release system and H2O2 in Panc-1 tumor and release more NO when taking into account the compromise between high NO release and biosafety.16,17 In detail, local ultrasound is employed to activate the H2O2 in Panc-1 tumor to generate more highly reactive oxygen-containing species (ROS, termed as O*) via the energy transformation from ultrasound mechanical energy to chemical energy. Once this NO delivery system entered the tumor region, these ROS could effectively oxidize the LA molecules in this system to generate more NO molecules available for killing Panc-1 cells. Despite attracting tremendous effort,26 the most prevalent dual targeting strategy is always confronted with some inevitable limitations,27,28 for example, complex chelation process, poor universality and applicability, and the enhanced occurrence probability of protein corona.29−32 More significantly, it fails to successively deliver nanoparticles because of neglecting the transfer station, that is, intercellular matrix. Herein, after modifying the NO release system with cyclic decapeptide CGLIIQKNEC [CLT1-G-propargyl] capable of targeting fibrinogen,33,34 a “relay” targeting strategy was established. Such a targeting strategy, as a proof of concept, realized two relay deliveries, that is, blood vessel to intercellular

inevitable drawbacks, for example, light and heat feature shallow penetration and poor controllability, respectively, and X-ray is confronted with strong ionizing radiation. All of these limitations severely impair their potential applicability toward clinical translation. Ultrasound has recently aroused increasing interest as a preferable trigger for diagnostics of cancer,13−15 since it is noninvasive, nonionizing, and easily controllable and has high penetration depth, etc.16,17 Therapeutic ultrasound usually has a frequency range of 0.75−3 MHz, wherein 1 or 3 MHz is the most used frequency.18,19 Despite primary adsorption at a depth of 3−5 cm,19 the penetration depth of 1 MHz ultrasound could reach approximately 10 cm, and even deeper when using focused beams.20,21 For instance, Zaccagna and co-workers employed MR-guided focused ultrasound with a frequency of 0.95−1.35 MHz and a penetration depth range of 6−20 cm to treat patients suffering from pancreatic cancer.21 To overcome the issues of the aforementioned NO donors and triggers, an ultrasound-responsive NO release system has been constructed in this report, wherein hollow mesoporous silica nanoparticles (HMSNs) whose surfaces were modified by poly(ethylene glycol) (PEG) molecules and targeting peptides performed as carriers and L-arginine (LA) as NO donor was loaded in the mesopores and internal cavities of the modified HMSNs. As the natural NO donor, LA molecules that usually generate NO by nitric oxide synthase exhibit excellent biocompability7 and can spontaneously react with H2O2 to generate NO.22 A highly aggressive and lethal Panc-1 pancreatic cancer is employed as a general model for demonstrating the 10817

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Figure 2. In vivo evaluations of the blood vessel−intercellular matrix−cell “relay” targeting. (A−C) Schematic illustrations of this blood vessel−intercellular matrix−cell “relay” targeting strategy. Zone A represents no ultrasound irradiation, zone B represents tumor site treated with ultrasound irradiation, and zone C indicates the involved objects in zone A and zone B. In zone B, 1, 2, and 3 represent blood vessel wall, intercellular matrix, and tumor cell membrane, respectively. (D) Quantitative distributions of Si atom in normal organs and tumor after different treatments via ICP-OES method, and the significant differences of Si distribution in tumor between HMSN and any one of US + HMSN, peptide−HMSN, or US + peptide−HMSN were analyzed using the Student’s two-tailed t test (*p < 0.05 and **p < 0.01). (E) Macroscopic distributions of FITC-labeled different nanoparticles in tumor tissues after different treatments via confocal observations; scale bar is 100 μm. (F) Distributions of Dil-labeled different nanoparticles in related components of tumor tissues (i.e., blood vessels, tumor cells, and intercellular matrix) after different treatments via confocal microscopic observations, wherein blue color, green color, and red color represent Panc-1 cell nuclei, blood vessels, and Dil-labeled HMSN or peptide−HMSN, respectively; the scale bar is100 μm.

between COOH grafted on HMSNs and NH2 tethered to one tail end of PEG molecules whose other tail end was functionalized by N3 was carried out to modify HMSNCOOH with PEG and N3 and yield HMSN-N3, as confirmed by further decrease of ζ potential and emergence of N3 characteristic peak at 2111 cm−1. Afterward, alkynyl-ended CLT1-G-propargyl peptides were further chelated through click chemistry between CCH on one tail end of CLT1-Gpropargyl peptides and NN+N− (N3) tethered to HMSNN3). A significant increase in ζ potential from −45 ± 2.1 to −31 ± 1.8 mV indicates the successful chelation of CLT1-Gpropargyl peptides on HMSNs, yielding peptide−HMSN. The chelating amount of CLT1-G-propargyl peptides is ca. 3.2% ± 0.18% after quantifying sulfur atoms in peptide−HMSN nanoparticles via inductively coupled plasma-atomic emission spectrometry (ICP-AES) method.34 Ultimately, LA molecules were loaded in peptide−HMSN via electrostatic affinity to harvest peptide−HMSN−LA. The loading amount of LA in peptide−HMSN−LA is ca. 17% ± 0.8% via thermogravimetry (TG) analysis (Figure S2), since LA molecules can completely decompose at above 680 °C (Figure S3). After loading LA molecules, the ζ potential increases from −31 ± 1.8 to −14 ± 0.7 mV. Due to the strong electrostatic affinity between peptide−HMSN and LA molecules and peptide shielding effect, less than 30% of LA molecules leak from peptide−HMSN−LA within 27 h via the UV−vis method (Figure S4) even under

matrix and intercellular matrix to tumor cell. Depending on the successive “relay” deliveries, more ultrasound-responsive NO release systems entered the Panc-1 tumor, resulting in more evident inhibitory effect against the Panc-1 tumor.

RESULTS AND DISCUSSION Synthesis of Peptide-Chelated NO Release System (Peptide−HMSN−LA). HMSNs can be regarded as an ideal platform because of their unique characteristics, large surface area, tunable pore volume, and facile surface modification.35,36 Herein, HMSNs with a diameter of 410 nm as carrier were synthesized via a well-established method (Figure 1a,b),16,37 and the surface area and pore size are 1007 ± 40 m2/g and 3.3 ± 0.1 nm (Figure S1), respectively. CLT1-G-propargyl peptides can be facilely conjugated via the following successive reactions (Figure 1c). In detail, NH2 was first grafted onto HMSNs via the reaction between 3-aminopropyltriethoxysilane (APTES) and OH in HMSNs, resulting in a significant increase in ζ potential (Figure 1d) from −33 ± 1.2 to 27 ± 1.0 mV, and an emerging characteristic peak of NH2 at 1529 cm−1 in the Fourier transform infrared (FTIR) spectra (Figure 1f). These results indicate the successful modification of NH2 on HMSNs to yield HMSN-NH2. Subsequently, succinic anhydride was added to react with NH2 and produce COOH, obtaining HMSN-COOH, as demonstrated by the decrease of ζ potential from 27 ± 1.0 to −45 ± 2.1 mV and an emerging characteristic peak of COOH at 1721 cm−1. Furthermore, amidation reaction 10818

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Figure 3. In vitro exploration of the 2nd relay from intercellular matrix to Panc-1 cells through ultrasound-enhanced permeability of Panc-1 cell membrane: (a) Schematic image of permeability variation of Panc-1 cell membrane between no US group and US group; (b) confocal images of Panc-1 cells treated with and without ultrasound irradiation, followed by incubation with peptide−HMSN−FITC for 8 h, wherein FITC intensity represents the total integrated intensity of green fluorescence obtained via calculating the integral optical density (IOD), and the scale bar is 80 μm.

in intercellular matrix are further facilitated to enter tumor cells through ultrasound-enhanced permeability, realizing the second “relay” delivery, as depicted in Figure 2B. Though smaller nanoparticles are, in principle, more easily able to cross the blood vessels via the enhanced permeability and retention (EPR) and induced more retention,40 the overhigh retention of small nanoparticles via EPR effect probably covers up the case of ultrasound-enhanced retention, which is disadvantageous for demonstrating this relay targeting. To address it, the relatively large model particles, that is, HMSNs with 400 nm diameter were used herein. To demonstrate the successive two “relay” targeting strategy, in vivo experiments were conducted on nude mice bearing Panc-1 solid tumor. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethy-lindocarbocyanine perchlorate (Dil)-labeled peptide−HMSN (peptide−DLM/Dil@HMSN) particles were prepared, wherein solid DL-menthol (DLM) was loaded to prevent Dil from leaking out of HMSNs’ cavity even after ultrasound irradiation (Figure S6). Optical microscopic observation (Figure S7) shows numerous peptide−DLM/Dil@HMSN diffused out of blood vessels and entered the extravascular (intercellular) matrix after ultrasound irradiation, demonstrating the significantly enhanced permeability of blood vessels of Panc-1 solid

ultrasound irradiation. Noticeably, after each modification, the particle diameter gradually increases, as shown in Figure 1e. In an attempt to inhibit protein corona and improve dispersity, poly(ethylene glycol) (PEG) modification has become one of the most prevalent approaches, because it can be used to improve the stability of nanoparticles and simultaneously mitigate the negative effect of protein corona on targeting,29 consequently prolonging the blood half-life of nanoparticles. Therefore, after chelating PEG and CLT1-Gpropargyl peptides, the blood half-life of peptide−HMSN−LA is considerably improved (Figure S5), and typically, the blood half-life is prolonged from 32 ± 3 to 69 ± 5 min. Evaluations on This Two “Relay” Targeting. Differing from previously reported dual-targeting methods with an emphasis on dual active ligands, the “relay” targeting consists of ultrasound-enhanced permeability and peptide-mediated active targeting. The principle of “relay” targeting is shown in Figure 2A−C. In detail, ultrasound-enhanced permeability first promotes more nanoparticles to cross blood vessels in tumor tissue and then actively target fibronectin−fibrin complexes rich in intercellular matrix,33 realizing the first “relay” delivery, since ultrasound can enhance the permeability of blood vessels and cell membrane via sonoporation.38,39 Afterward, these particles 10819

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Figure 4. Ultrasound-responsive NO release and in vitro cytotoxicity evaluation. (a) NO concentration after reaction between different samples with excessive H2O2 of a fixed concentration of 5 μM, and the significant differences between peptide−HMSN−LA or US + peptide− HMSN−LA and peptide−HMSN were analyzed using the Student’s two-tailed t test (**p < 0.01). (b) NO concentration after different treatments, that is, control 1 (peptide−HMSN−LA + US-20%), experiment 1 (Panc-1 cells + peptide−HMSN−LA + US-20%), control 2 (peptide−HMSN−LA + US-50%) and experiment (Panc-1 cells + peptide−HMSN−LA + US-50%); the significant differences between cellcontaining groups (experiments 1 and 2) and cell-free groups (controls 1 and 2) were analyzed using the Student’s two-tailed t test (*p < 0.05, **p < 0.01). (c) Panc-1 cell viability after different treatments, that is, control group (peptide−HMSN), peptide−HMSN−LA, peptide− HMSN−LA + US (0.2 W, 20% and 50% duty cycles), peptide−HMSN−LA + US (0.6 W, 20% and 50% duty cycles), and peptide−HMSN−LA + US (1.0 W, 20% duty cycle). (d) Systematic schematic of ultrasound-responsive NO release from peptide−HMSN−LA. (e) Confocal images of Panc-1 cells stained by calcein and propidium iodide (PI) after different treatments. (f) Flow cytometry data of Panc-1 cells stained by annexin V−FITC and PI after aforementioned different treatments; the ultrasound power is 1.0 W−20% duty cycle. Note, the used mass concentration of peptide−HMSN−LA is 0.8 mg/mL, and the frequency is 1 MHz; the total duration is 6 pulses, and the duration of each pulse is 15 s with a 30 s interval between two adjacent pulses.

tumor by ultrasound irradiation. This result enables the first “relay” delivery from intravascular matrix to tumor intercellular matrix. In in vitro experiments, stronger fluorescence intensity after ultrasound (US) irradiation represents more retention of FITC-labeled peptide−HMSN in Panc-1 cells, as demonstrated in Figure 3. This result suggests that ultrasound irradiation also enhanced the permeability of Panc-1 cell membrane and allowed nanoparticles to enter Panc-1 cells through sonoporation as well as endocytosis, benefiting from the second relay delivery from intercellular (extravascular) matrix to cell. As a result, depending on the enhanced permeability of blood vessels and cell membrane by ultrasound, 2-fold accumulation of model particles in Panc-1 tumor is obtained via the comparison between HMSN and US + HMSN (Figure 2D,E). It is well established that fibronectin−fibrin complexes primarily distribute in intercellular matrix of tumor tissue and CLT1-G-propargyl peptides could target fibronectin−fibrin complexes in the intercellular matrix of different tumors with

little binding to normal tissues.33,34 Noticeably, there are rich fibronectin−fibrin in Panc-1 solid tumors, as demonstrated in Figure S8. Hence, the chelated CLT1-G-propargyl peptides can perform as an intermediate to link the two relays, which will benefit the retention of nanoparticles. More experimental results indeed demonstrate that more CLT1-G-propargyl peptide-chelated particles (peptide−HMSN) accumulate in tumor than peptide-free HMSNs, as can be found in Figure 2D,E. Noticeably, compared to the significantly enhanced distribution in tumor region, the distribution of HMSNs in other organs also shows a little increase, which is attributed to the increased permeability by scattered and transmitted waves of the local ultrasound,41 since the employed ultrasound is a type of plane wave rather than focused wave and the employed animal model (nude mice) is so small with their organs closely distributed. In vivo distributions of different Dil-labeled nanoparticles in tumor tissues were explored to further test the successive two 10820

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Figure 5. Principle investigation of ultrasound-responsive NO release. (a) Detailed principle schematic of ultrasound-responsive NO release from peptide−HMSN−LA. (b) Standard curve of H2O2 concentration (X axis) and UV−vis absorbance intensity (Y) obtained by hydrogen peroxide assay kit. (c) Variations of H2O2 concentration in Panc-1 cells treated with different groups, that is, control, US alone, peptide− HMSN−LA and US + peptide−HMSN−LA; the significant differences either between US + peptide−HMSN−LA and US alone or between US alone and control were analyzed using the Student’s two-tailed t test (*p < 0.05, **p < 0.01). (d) Laser confocal images (Ex 488 nm and Em 535 nm) of Panc-1 cells stained by pH indicator, BCECF AM, probe after treatment with two different groups, that is, peptide−HMSN−LA and US + peptide−HMSN−LA; channel 1 represents BCECF AM, and channel 2 represents bright-field image. Note, BCECF AM is a pHsensitive fluorescence probe, and as the pH decreases, the fluorescence intensity will gradually drop. (e) Laser confocal images (Ex 488 nm and Em 525 nm) of Panc-1 cells stained by DCFH-DA after treatments with above different groups. Note, DCFH-DA was employed to detect reactive oxygen and stronger green fluorescence intensity means more reactive oxygen. In all experiments, the mass concentration of peptide− HMSN−LA is 0.8 mg/mL, and the ultrasound power is 1.0 W−20% duty cycle; the total duration is 6 pulses, and each pulse duration is 15 s with a 30 s interval between two adjacent pulses.

“relay” targeting strategy. In comparison to Dil@HMSN alone, more Dil@HMSN nanoparticles leak out of intravascular matrix and enter intercellular matrix and Panc-1 cells after exposure to US irradiation, suggesting ultrasound can enhance the permeability of blood vessels and cell membrane. As well, CLT1-G-propargyl peptides promote more peptide−Dil@ HMSN to remain in tumor, as confirmed by the comparison between US + Dil@HMSN and US + peptide−Dil@HMSN (Figure 2F), demonstrating occurrence of actively targeting intercellular matrix. Therefore, after experiencing the successive two “relay” deliveries, peptide−HMSN particles after ultrasound irradiation show the highest accumulation (7.40% ± 0.25%) in tumor (∼2.5-fold higher than peptide−HMSN alone and 7-fold higher than HMSN alone), and this accumulation is higher than the sum of two groups, that is, US + HMSN (1.35% ± 0.12%) and peptide−HMSN alone (2.76% ± 0.18%),

exhibiting a synergistic retention effect, which will be beneficial for in vivo NO release and NO-mediated Panc-1 apoptosis. Evaluations on Ultrasound-Responsive NO Release Behavior. Besides participating in the construction of “relay” targeting, ultrasound can also perform as a trigger to accelerate the reaction between H2O2 and LA molecules loaded in peptide−HMSN−LA and produce more NO. To demonstrate it, in vitro ultrasound-responsive NO release behavior was evaluated in detail, and Griess assay method whose principle is shown in Figure S9 was employed to monitor NO release.5 In Figure 4a, more NO release from peptide−HMSN−LA (4.4 ± 0.4 μM) than peptide−HMSN (1.2 ± 0.2 μM) and US + peptide−HMSN (1.4 ± 0.2 μM) is observed after direct reaction with excessive H2O2 solution of a fixed molar concentration (5 μM), validating the reaction between LA in peptide−HMSN−LA and H2O2 indeed occurred. Especially upon exposure to US irradiation, the NO concentration further 10821

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ACS Nano increases from 4.4 ± 0.4 to 12.5 ± 1.1 μM on comparison between peptide−HMSN−LA and US + peptide−HMSN−LA, which convincingly demonstrates the occurrence of ultrasoundresponsive NO release behavior. The ranking of NO accumulative release amount of different groups is control < US + peptide−HMSN < peptide−HMSN−LA < US + peptide−HMSN−LA. It is worth noting that neglectable NO concentration is found in the absence of H2O2 (Figure S10), suggesting that H2O2 is important for generating NO via direct reaction with LA molecules entrapped in peptide−HMSN−LA. Since Panc-1 cells produce a much higher concentration of H2O2 than in normal organs,42 in vitro NO release in Panc-1 cells especially after exposure to US irradiation can be expected. Herein, we compare the NO release behavior of peptide− HMSN−LA between incubations with and without Panc-1 cells to investigate the in vitro ultrasound-responsive NO release behavior. It is found that NO release in the absence of Panc-1 cells can be almost neglected, as shown in Figure 4b. In contrast, the NO concentration substantially increases once Panc-1 cells are introduced, indicating more NO release from peptide−HMSN−LA through the reaction between entrapped LA and H2O2 in Panc-1 cells. This result also demonstrates rich H2O2 in Panc-1 cells. Noticeably, Figure 4b shows that higher ultrasound power and larger duty cycle promote more NO release, further confirming the ultrasound-responsive NO release behavior of peptide−HMSN−LA. Although the NO release amount is positively proportional to ultrasound intensity that is typically associated with ultrasound power and duty cycle, ultrasound with 1 W−20% duty cycle as the optimal parameter was employed in terms of safety because overlarge intensity could induce hyperpyrexia according to our previous report.16 Figure 4c is the schematic of ultrasound-responsive NO release behavior. It is believed that this ultrasoundtriggered NO release is predominantly attributed to the ultrasound-accelerated reaction between LA molecules and H2O2, and this detailed mechanism will be progressively elucidated and confirmed in the following parts. In Vitro Treatment Evaluations Using This UltrasoundResponsive NO Release System. High NO concentration is essential to tumor therapy.1 Herein, we evaluated whether this unique ultrasound-triggered NO release was sufficient for implementing NO-mediated apoptosis of the highly aggressive and lethal Panc-1 pancreatic cancer. Conventional treatment approaches such as chemotherapy, surgery, and radiotherapy are inappropriate for patients, especially for those in advanced stage or with poor tolerance.43,44 This invasive, ionizing radiation-free, and on-demand treatment strategy using such ultrasound-responsive NO release system provides us an avenue to treat those highly lethal tumors with mitigated side-effects. Before evaluation, the cytotoxicity of peptide− HMSN carrier as a function of dose was investigated via CCK8 method, as shown in Figure S11, wherein higher than 95% viability can be observed even though the concentration is up to 2 mg/mL, indicating the low cytotoxicity of peptide−HMSN carrier. The number of apoptotic Panc-1 cells increases as the ultrasound power increases (Figure 4d), which agrees with the variation trend of NO concentrations as a function of ultrasound power, validating that the amount of ultrasoundresponsive NO release is sufficient for inducing Panc-1 apoptosis. Figures S12 and S13 show the variation profiles of Panc-1 viability and corresponding NO release concentration as a function of the mass concentration of peptide−HMSN−LA,

and the results also demonstrate the occurrence of ultrasoundresponsive NO release and NO-mediated Panc-1 apoptosis. Furthermore, the flow cytometry method and laser confocal microscopic imaging were further employed to evaluate the NO-mediated apoptosis of Panc-1 cells. Viable and dead Panc-1 cells could be clearly differentiated after dual-immunofluorescence staining by propidium iodide (PI) and calcein. Since US + peptide−HMSN−LA could generate the largest amount of NO (Figure 4e), this group undoubtedly harvests the largest apoptotic percentage of Panc-1 cells. Moreover, flow cytometry data (Figure 4f) shows that the late apoptotic percentage of Panc-1 cells after treatment with US + peptide−HMSN−LA is 37.51% ± 1.82%, much higher than that treated with peptide− HMSN−LA alone (0.24% ± 0.09%). All above cytotoxicity experiments confirm that ultrasound irradiation can promote the reaction between LA molecules in peptide−HMSN−LA and H2O2 in tumor and generate more NO for killing Panc-1 cells. Mechanism Validation of Ultrasound-Responsive NO Release. The principle of such ultrasound-responsive NO release behavior was investigated in detail. Figure 5c shows the H2O2 concentrations in Panc-1 cells after treatment with different groups, which can be calculated according to the standard curve (Figure 5b). It is found that H 2 O 2 concentrations in Panc-1 cells after US irradiation are much lower than that without US irradiation, for example, control vs US or peptide−HMSN−LA vs US + peptide−HMSN−LA, suggesting H2O2 decomposition in Panc-1 cells once exposed to US irradiation. The ranking of H2O2 concentration is obtained as control > peptide−HMSN−LA > US > US + peptide−HMSN−LA, which indicates that ultrasound can accelerate H2O2 decomposition to generate highly reactive oxygen-containing species that are available and beneficial for oxidizing LA molecules and rapidly producing more NO. Based on these results, the underlying principle of ultrasoundresponsive NO release behavior is obtained that ultrasound irradiation can rapidly generate more ROS via the inertial cavitation-mediated energy transformation from ultrasound mechanical energy to chemical energy,45,18 and these ROS featured high oxidation ability that can oxidize LA molecules to generate NO,46 simultaneously resulting in the decrease of H2O2 concentration, as shown in Figure 5a. To further validate this principle, the variations of ROS and pH in Panc-1 cells after treatments with different groups were explored. In comparison to peptide−HMSN−LA, the considerably decreased fluorescence intensity in Panc-1 cells stained by pH indicator (2′,7′-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, BCECF AM) after treatment with US + peptide−HMSN−LA indicates pH reduction in Panc-1 cells, consequently confirming H2O2 decomposition (Figure 5d). Moreover, the ROS in Panc-1 cells treated with different groups were also monitored. Confocal images of Panc1 cells stained by the ROS indicator (DCFH-DA) definitely reveal that the content of ROS in Panc-1 cells after US irradiation significantly increases via the comparison control vs US alone or peptide−HMSN−LA vs US + peptide−HMSN− LA, and the ranking of ROS content is control < peptide− HMSN−LA < US < US + peptide−HMSN−LA, as demonstrated in Figure 5e. This variation trend is in contrast to that of H2O2 concentration, suggesting the yield of ROS is in positive correlation to H2O2 consumption, which further demonstrates the principle of this ultrasound-responsive NO release. 10822

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Figure 6. In vivo inhibitory outcomes of Panc-1 solid tumor implanted on nude mice using such NO release system. (a) Digital photos of Panc-1 solid tumors implanted on nude mice after different treatments, that is, PBS control, US alone, peptide−HMSN−LA alone, and US + peptide−HMSN−LA. (b−d) Volume variation profiles of Panc-1 tumor as a function of time after above different treatments; the significant differences between the control group and either US + peptide−HMSN−LA group or US + HMSN−LA group were analyzed using the Student’s two-tailed t test (*p < 0.05, **p < 0.01). (c,d) Survival rates (c) and weight variation profiles (d) of nude mice bearing Panc-1 solid tumors as a function of incubation time after above different treatments.

Figure 7. Analysis of pathological mechanism of enhanced inhibitory growth using this ultrasound-responsive NO release system. (a) Optical microscopic images of Panc-1 tumor slices stained by TUNEL and PCNA immunohistochemistry after treatments with different groups, that is, control, US alone, peptide−HMSN−LA alone, and US + peptide−HMSN−LA, and the tumor slices were harvested at the 10th day; scale bar is 100 μm. (b,c) Quantitative statistics of apoptotic Panc-1 cells (b) and proliferating Panc-1 cells (c) obtained from image a; **p < 0.01 and ***p < 0.001. Note, the administering dose of LA is 10 mg/kg; the ultrasound power is 1.0 W−20% duty cycle; the total duration is 20 pulses, and the duration of each pulse is 15 s with a 30 s interval between two adjacent pulses.

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ACS Nano In Vivo Treatment Evaluations Using This UltrasoundResponsive NO Release System. The blood biocompatibility of peptide−HMSN−LA before in vivo treatment experiments was first evaluated. It is found that even though the mass concentration of peptide−HMSN−LA is up to 800 μg/mL, the hemolysis ratio (Hr) remains less than 5%, and the values of activated partial thromboplastin time (APTT), fibrinogen (FIB), and prothrombin time (PT) remain approximately identical to the control group (Figure S14), indicating the excellent blood biocompatibility of peptide− HMSN−LA. Despite the presence of stromal barrier,47 the subcutaneous xenograft studies of Panc-1 solid tumor remained as a general tumor model in independently investigating the treatments of pancreatic cancers using genes or drugs.48−50 Compared to the orthotopic pancreatic tumor model, the subcutaneous xenograft model without the stromal barrier is more favorable for validating the two primary aims, (1) confirming the whole “relay” targeting process in solid tumor from blood vessels to intercellular matrix and to cancer cells and (2) demonstrating the capability of this ultrasound-triggered NO release system to induce apoptosis of tumor cells. Therefore, the subcutaneous xenograft Panc-1 tumor model in nude mice can be regarded as an ideal model to evaluate the in vivo therapy using this ultrasound-responsive NO release system in comparison to the orthotopic pancreatic tumor model in this report. Based on aforementioned in vitro experiments, it is expected that the validated enhanced retention via the aforementioned “relay” targeting strategy and ultrasound-triggered NO release will cooperatively benefit the in vivo treatment of Panc-1 xenografted solid tumor. According to the digital photos and the volume variation profiles of Panc-1 solid tumors subcutaneously implanted on nude mice after treatment with different groups (Figure 6a,b), it is clear that the variation trend of treatment outcomes is control < US + peptide−HMSN < peptide−HMSN−LA < US + HMSN−LA < US + peptide− HMSN−LA, completely corresponding to that of NO concentration. It is noteworthy that the largest retention of peptide−HMSN−LA in tumor via the relay targeting after US irradiation (US + peptide−HMSN−LA) contributed to the most excellent therapeutic effect against Panc-1 tumor in comparison to US + HMSN−LA. From the pathological examination images of hematoxylin and eosin (H&E) staining (Figure S15), the characteristics associated with necrosis, such as collapsed cytoskeleton and nucleus disintegration, can be clearly observed. More significantly, after treatment by US + peptide−HMSN−LA, the survival rate is significantly augmented from 0% to 80% at the 30th day, as confirmed in Figure 6c. Additionally, during the experiments, all nude mice remained healthy (Figure 6d), implying that neither starvation nor diseases occurred. To investigate the molecular mechanism of this in vivo treatment, TUNEL and proliferating cell nuclear antigen (PCNA) immunohistochemistry were systematically investigated. After treatment with US + peptide−HMSN−LA, the least proliferating cells via PCNA assay and the most apoptotic cells via TUNEL assay are observed (Figure 7a). Concurrently, the corresponding integral optical density (IOD) values (Figure 7b,c) extracted from above optical images also quantitatively confirm this point. Western blot was further carried out to analyze the apoptotic signaling pathway of Panc-1 cells treated with aforementioned different groups, as shown in Figure S16, wherein P53 and caspase-3 proteins exhibit an up-regulation

trend, indicating a DNA-damage apoptotic pathway.51 More significantly, in this “relay” targeting NO delivery, ultrasound irradiation was confined to the tumor, and normal organs or tissues failed to receive US irradiation. As a result, despite the ubiquitous presence of H2O2 in human body,52 NO generation in normal organs or tissues is trivial because of US-free irradiation and low dose, which can avoid the possible side effects of systemic release of LA (NO donor) upon using US triggering. In vivo pathological examinations (Figure S17) demonstrate this point, wherein no evident differences in pathological slices of normal organs stained by H&E between control group and US + peptide−HMSN−LA is observed.

CONCLUSIONS In summary, a targeting NO release system with the natural amino acid (L-arginine) as the NO donor and exogenously local ultrasound as the trigger was successfully constructed. Such NO release system could respond to ultrasound irradiation and release more NO, realizing an ultrasound-responsive NO release behavior. The principle of ultrasound-responsive NO release behavior is demonstrated in that the local ultrasound activated H2O2 to generate more ROS through the energy transformation for oxidizing the entrapped LA molecules and generating much more NO molecules. Additionally, a “relay” targeting strategy consisting of two successive “relay” deliveries, that is, intravascular matrix to intercellular matrix and intercellular matrix to intracellular matrix, has been established and validated, through which over 7-fold increase of NO release system retention in Panc-1 tumor was realized. This ultrasound-responsive NO release system integrates local ultrasound, targeting delivery, and on-demand NO release into a whole, resulting in an excellent in vivo curative effect in inhibiting the subcutaneously implanted Panc-1 tumor. Nevertheless, to practically apply this NO system in pancreatic cancer, adequate delivery of the nanoparticle-based NO release system to the pancreas as a prerequisite is indispensible. In future work, the orthotopic xenograft model will be employed to investigate how to overcome the stromal barrier using this “relay” targeting ultrasound-responsive NO delivery system. Additionally, this targeted ultrasound-triggered NO release system as a general method holds great potential in other tumors or diseases (e.g., antibacterial/antibiofilms). MATERIALS AND METHODS Synthesis of HMSN-NH2, HMSN-COOH, HMSN-N3, Peptide− HMSN, FITC-Labeled Peptide−HMSN (peptide−HMSN−FITC), and Dil-Labeled Peptide−HMSN (Peptide−Dil@HMSN). Amino group modified HMSNs (namely, HMSN-NH2) were obtained via a simple modification method.36 In brief, 100 mg of HMSNs were dispersed in 45 mL of ethyl alcohol solution via sonication and placed on flask heating mantles at 78 °C. After that, 20 μL of 3aminopropyltriethoxysilane (APTES) was added and reacted for a whole night, and then the particles were collected via centrifugation and washed three times with ethyl alcohol, yielding HMSN-NH2 nanoparticles. After that, the HMSN-NH2 nanoparticles were dried via the lyophilization method and used for measurements in dynamic light scattering (DLS), FTIR, and ζ potential. Above HMSN-NH2 were redispersed in dimethylformamide (DMF) and then added dropwise to a flask containing 50 mL of 0.1 M succide anhydride in DMF. The mixture was stirred for 48 h, and the resulting nanoparticles with carboxylic-function groups on their surface (HMSN-COOH) were cleaned and collected with DMF as the washing medium. The HMSN-COOH nanoparticles were then dried under vacuum and used for measurements in DLS, FTIR, and ζ potential. 10824

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ACS Nano

were maintained in a humidified atmosphere with 5% CO2 at 37 °C. Because of lacking a thymus, nude mice cannot generate mature T lymphocytes and thus are unable to produce adaptive immune responses. Moreover, the absence of functioning T cells allows the nude mice to accept the grafts including allografts and xenografts without any immunological rejection. Therefore, nude mice (Balb/C nu/nu) are the popular one of the few model animals appropriate for xenograft studies such as solid tumors, and numerous reports employed nude mice as the animal model to subcutaneously implant exogenous solid tumors including human pancreatic carcinoma.48−50,53 Nude mice (Balb/C nu/nu) with an average body weight of about 20 g were supplied by Laboratory Animals Center of Tenth Peoples’ Hospital of Tongji University and were kept in sterilized cages with a supply of filtered air and sterile food and water. For developing solid tumors in nude mice, the procedures were carried out as described previously.48,49 In detail, 0.1 mL of cell suspension (1 × 106 cells) in PBS was injected subcutaneously into the flank of nude mice using a 1 mL injector. After 1 week, the solid tumor could be observed, and tumors were allowed to grow to 120 ± 20 mm3 (experimental day 0) prior to any experiment. Tumor burden associated with general wellbeing, weight, tumor volume, survival rate, and tumor metastasis was monitored via direct observation and ultrasound imaging. All in vivo animal experiments were performed according to protocols approved by the Laboratory Animal Center of Shanghai Tenth Peoples’ Hospital and were in accordance with the policies of National Ministry of Health. Observation of in Vivo Enhanced Permeability of Blood Vessels after Ultrasound Irradiation. Panc-1 xenografted tumorbearing nude mice were anaesthetized, and the skin at the site of tumor was cleaned with 75% ethyl alcohol. Subsequently, 0.1 mL of Dillabeled nanoparticles (peptide−DLM/Dil@HMSN, 0.8 mg/mL) was intravenously injected into the nude mice at 25 °C. After 20 s, images of superficial blood vessels that could be clearly observed were captured by optical microscopy. Subsequently, in situ US irradiation at 1.0 W−20% duty cycle for 10 s was carried out, and common optical microscopic images of the corresponding blood vessels were subsequently captured again. In Vivo Retention Evaluations of Relay Targeting. Panc-1 xenografted tumor-bearing nude mice were randomly sorted into four groups. The corresponding four treatments, HMSN−FITC, US + HMSN−FITC, peptide−HMSN−FITC, and US + peptide−HMSN− FITC, were carried out, and the injection dose of LA was 10 mg/kg; the ultrasound parameter wa 1.0 W−20%−15 s per cycle and 20 cycles with a 30 s interval between two cycles in total; the injection method was tail intravenous. Quantitative Distribution of Si Atoms in Tumor and Normal Organs. After treatments with above-mentioned four groups and subsequent 12 h incubation, the treated mice were dissected, and all organs were harvested for ablation by the mixed solution of nitric acid and perchloric acid (volume ratio: 3:1). After that, quantitative analysis for Si atom was carried out via inductively coupled plasma-optical emission spectrometry (ICP-OES) method. Microscopic Observations via Laser Confocal Microscopy. After treatments with above-mentioned four groups and subsequent 12 hincubation, the nude mice were euthanized via injecting excess anesthetics (2.5% pentobarbital). Subsequently, the tumor was isolated and cut into slices via freezing microtome section and then observed on the laser confocal microscopy. Microscopic Observations via Laser Confocal Microscopy. In this experiment, the samples HMSN−FITC and peptide−HMSN−FITC were replaced by Dil@HMSN and peptide−Dil@HMSN, respectively, and other experimental procedures were the same as above. After treatments with above-mentioned four groups (i.e., HMSN−FITC, US + HMSN−FITC, peptide−HMSN−FITC, and US + peptide− HMSN−FITC) and subsequent 12 h incubation, the tumors were isolated, and tumor slices were obtained via freezing microtome section and then stained by DAPI and CD31−FITC immunofluorescence for confocal microscopic observations. In Vitro Tests of NO Release and LA Release. Forty milligrams of the above-mentioned peptide−HMSN−LA was dispersed in 50 mL

As-prepared HMSN-COOH particles were dispersed in deionized water, and then 20 mg of N-hydroxysuccinimide (NHS) and 50 mg of excess N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide (EDC) was added into the dispersion and stirred for 1 h so as to activate -COOH, and NH2-PEG-N3 molecules were added to react for 12 h to generate HMSN-N3 via the amidation reaction. Afterward, the samples were washed with deionized water to remove the excessive EDC and collected via centrifugation. Subsequently, the HMSN-N3 nanoparticles were dried under vacuum and used for measurements in DLS, FTIR, and ζ potential. As for peptide−HMSN, the procedures were carried out as described previously.34 Typically, CuSO4·5H2O (5 mg) and ascorbic acid as catalyst were first added into tert-butyl alcohol/H2O mixed solution (95/5 v/v, 50 mL), followed by adding CLT1-G-propargyl ligands (20 mg), and then above as-prepared HMSN-N3 nanoparticles were added and reacted with for 3 days, yielding peptide−HMSN. The as-prepared peptide−HMSN nanoparticles were washed with tert-butyl alcohol and collected via centrifugation. Then, the HMSN-NH2 nanoparticles were dried under vacuum and used for measurements in DLS, FTIR, and ζ potential. Peptide−HMSN−FITC. Fluorescein isothiocyanate (FITC) isomer (20 mg) first reacted with excess APTES (200 μL) for 12 h at room temperature, yielding FITC−APTES. Then, the preparation procedures of peptide−HMSN−FITC were the same as that for preparing peptide−HMSN, the sole difference lying in that APTES was replaced by the mixture of APTES and APTES−FITC. Peptide−Dil@HMSN. Dil and LM were first loaded in HMSNs via a well-documented method, generating Dil@HMSN. The preparation procedures for peptide−HMSN−FITC were the same as that for preparing peptide−HMSN, the sole difference lying in that Dil@ HMSN replaced HMSNs. Peptide−HMSN−LA. Two grams of LA was dissolved into 40 mL of deionized water via stirring, and then the as-prepared peptide−HMSN was added and stirred for 24 h so as to sufficiently adsorb L-arginine molecules, obtaining peptide−HMSN−LA. Ultimately, the samples were collected via centrifugation and dried under vacuum. The loading amount of LA molecules and peptide can be quantified via the TGA and ICP-AES methods, respectively. Release Measurement of LA Molecules from Peptide− HMSN−LA. Ten milligrams of dried peptide−HMSN−LA was placed in the dialysis bag (cutoff, 3000) and sealed with a plastic clip, and the whole dialysis bag was placed in an opaque centrifugal tube containing 20 mL of PBS. Finally, the centrifugal tube was fixed in an electronic shaker at an oscillation rate of 100 rpm/min at 37 °C. At certain intervals, 3 mL of PBS solution was taken out for UV−vis measurement, and the intensity of the LA characteristic peak ranging from 205 to 210 nm was recorded. The release amount of LA can be calculated via introducing the obtained peak intensity into the standard curve as a function of mass concentration of LA molecules. As for ultrasound-triggered LA release, at each recording time point, ultrasound irradiation was carried out. The absorbance intensities before and after ultrasound irradiation were recorded. In Vitro Retention Evaluations of Relay Targeting. Two groups of treatments, peptide−HMSN−FITC and US + peptide− HMSN−FITC, were carried out, wherein the mass concentration of peptide−HMSN−FITC was 50 μg/mL and the ultrasound parameter is 1.0 W−20%−15 s per cycle and 6 cycles with a 30 s interval between two cycles in total. Panc-1 cells seeded in special culture dishes with a 0.5 mm-thick rounded groove of 1 cm in diameter were cultured at 37 °C under 5% CO2 in Roswell Park Memorial Institute medium (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. After aforementioned treatments and subsequent 2 h incubation, cells were stained with 4′,6-diamidino-2phenylindole (DAPI) solution in PBS and then were observed via laser confocal microscope. Model Establishment of Nude Mice Bearing Melanoma Tumor. Human pancreatic carcinoma cells, Panc-1 cells, were supplied by China Infrastructure of Cell Line Resources and cultured in DMEM containing 10% heat-inactivated fetal bovine serum and 1% antibiotics (streptomycin and penicillin) (Invitrogen). The cultures 10825

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ACS Nano of H2O2 solution with a molar concentration of 5 μM, and then ultrasound irradiation was carried out. After 30 min, a milliliter of dispersion was taken out for assay via the Griess assay kit. The ultrasound power is 1.0 W, and duty cycle is 20%; the total duration was 6 pulses, and the duration time of each pulse was 15 s with a 30 s interval between two adjacent pulses. As for the release measurement of LA from peptide−HMSN−LA, above-mentioned harvested samples peptide−HMSN−LA were loaded in a dialysis bag (cutoff MW = 3500) and then placed in 20 mL of simulated body fluid (SBF) with different pH values, and at different time points, 3 mL of SBF solution was sent for UV−vis measurement; the peak intensity at 205 nm was recorded. Ultimately, according to the concentration-dependent UV−vis standard intensity curve, the release percentage at different time points can be calculated, and release profile of LA molecules at different pH values could be obtained. Intercellular NO Release and NO-Induced Panc-1 Apoptosis via CCK8 Method, Flow Cytometry, Confocal Observation after PI and Calcein Double-Staining. The irradiation method is pulsed, and in detail, the duration time of each pulse was 15 s, and the interval was 30 s, the number of cycles was 6. Panc-1 cells were cultured at 37 °C under 5% CO2 in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin. NO Release. Panc-1 cells seeded in a 6-well cell-culture plate at a density of 1 × 106 cells per well were cultured in 5% CO2 at 37 °C for 24 h. After letting cells settle for 24 h, cells were incubated with peptide−HMSN−LA, and then ultrasound irradiation was carried out. The ultrasound power was tunable within a range of 0−1.0 W; two duty cycles, 20% and 50%, were employed, and the mass concentration of peptide−HMSN−LA was within 0−1.5 mg/mL. After above treatments and another 30 min-incubation, a milliliter of dispersion was taken out for assay via the Griess assay kit. Detection of H2O2, pH, and ROS. Panc-1 cells seeded in a 6-well cell-culture plate at a density of 1 × 106 cells per well were cultured in 5% CO2 at 37 °C for 24 h. After letting cells settle for 24 h, cells were incubated with peptide−HMSN−LA, and then ultrasound irradiation was carried out. The mass concentration of peptide−HMSN−LA was 0.8 mg/mL, and the ultrasound power was 1.0 W−20% duty cycle. Before and after ultrasound irradiation, H2O2 concentrations were quantitatively determined via the Griess assay kit. ROS in Panc-1 cells treated with different objective groups could be qualitatively observed via confocal microscopic observation after staining by DCFH-DA assay kit, and the variation of pH in Panc-1 cells was qualitatively observed via confocal microscopic observation after staining by BCECF AM assay kit. CCK8 Method. Panc-1 cells seeded in a 6-well cell-culture plate at a density of 1 × 106 cells per well were cultured in 5% CO2 at 37 °C for 24 h. After letting cells settle for 24 h, cells were incubated with peptide−HMSN−LA, and then ultrasound irradiation was carried out. The ultrasound power was tunable within a range of 0−1.0 W; two duty cycles, 20% and 50%, were employed, and the mass concentration of peptide−HMSN−LA was within 0−1.5 mg/mL. After the treatments with different groups, another 24 h-incubation was carried out, and then the upper floating dead cells were discarded. The viable cells were detected via CCK8 assay kit. For cytotoxicity evaluation of peptide−HMSN carrier, the procedures were the same as above, the differences lying in that no US irradiation was employed, the mass concentration range of peptide−HMSN carrier was 0−2 mg/mL, and besides 24 hincubation, another 48 h-incubation was also included. Flow Cytometry. Panc-1 cells seeded in a 6-well cell-culture plate at a density of 1 × 106 cells per well were cultured in 5% CO2 at 37 °C for 24 h. After letting cells settle for 24 h, Panc-1 cells were incubated with peptide−HMSN−LA, and then ultrasound irradiation was carried out. The mass concentration of peptide−HMSN−LA was 0.8 mg/mL, and the ultrasound power was 1.0 W−20% duty cycle. After the treatments with above-mentioned groups, the floating dead cells were instantly collected, and the adherent cells were also harvested, and then all the cells were integrated together for staining. Next, the collected cells were stained with propidium iodide (PI) and annexin

V−FITC, and then the stained cells were instantly tested via flow cytometry. Confocal Observation. Panc-1 cells were seeded in special 1.5 mm thickness dishes at a density of 1 × 106 cells per well and were cultured in 5% CO2 at 37 °C for 24 h. After letting cells settle for 24 h, Panc-1 cells were incubated with peptide−HMSN−LA, and then ultrasound irradiation was carried out. The mass concentration of peptide− HMSN−LA was 0.8 mg/mL and the ultrasound power was 1.0 W-20% duty cycle. After another 24 h incubation, the floating cells were instantly collected, and the adherent cells were also harvested, and then all the cells were regarded as a whole for staining. Next, the collected cells were stained by PI and calcein, and then the stained cells were observed via laser confocal microscopy. Inhibitory Capability of Peptide−HMSN−LA in Combination with US (US + Peptide−HMSN−LA) for the Growth of Panc-1 Xenograft Solid Tumor in Nude Mice Model. Panc-1 pancreatic solid tumor-bearing nude mice (36 in sum) were randomly assigned into 6 groups, control, US alone, HMSN−LA, peptide−HMSN−LA, US + HMSN−LA, and US + peptide−HMSN−LA. The experiment was approved ethically and scientifically by Tongji University and complied with Practice for Laboratory Animals in China. The injection volume was 0.1 mL, and the standard was 10 mg LA/kg that was consistent with aforementioned in vitro dose. All the samples were dispersed in PBS with the same LA mass concentration, and thus the mass concentrations of HMSN−LA and peptide−HMSN−LA are ca. 10 mg/mL and ca. 11.76 mg/mL, respectively, since the loading amounts of LA molecules in HMSN−LA and peptide−HMSN−LA were around 20% and 17%, respectively. The administering method was tail vein injection. After injections (day 0), the tumor tissues were radiated for 12 cycles of irradiations per day on portable focused ultrasound therapeutic apparatus (Chattanooga, USA), and the parameter of each cycle was 1.0 MHz−1.0 W−20% for 15 s with a 30 s interval between two cycles, and 20 cycles of ultrasound irradiation were carried out. Between tumors and the therapeutic US transducer, a sound-absorbing panel with a hole whose diameter was consistent with tumor size was added. The experimental procedures were performed as reported previously.16 In detail, the irradiation was carried out every day. The tumor volume, survival rate, and body weight were measured everyday for 10 days from day 0, and the diameter of the tumor was measured in two dimensions at each time point by using an electronic caliper and calculated using the following formula: Tumor volume = length × width2/2. At the 10th day, the nude mice were euthanized via injection of excess anesthetics (2.5% pentobarbital), and tumor and other organs (heart, liver, spleen, lung, and kidney) of each nude mouse were isolated for staining with hematoxylin and eosin (H&E), TUNEL, and PCNA immumohistochemistry for histopathological analysis by optical microscope, and additionally, the tumor slices were stained by TUNEL immunofluorescence staining for detecting the apoptotic cells, and Western blot analysis of related protein was simultaneously carried out for investigating the apoptotic pathway. Additionally, normal organs were also stained by hematoxylin and eosin (H&E) and CD34 immumohistochemical staining for histopathological analysis by optical microscope. Statistical Analysis. All the experiments were performed in triplicate. The obtained data were expressed as the mean value ± standard deviation (SD), and the statistical significance between two groups was analyzed by the Student’s two-tailed t test through SPSS 15.0. Single, double, and triple asterisks represent p ⩽ 0.05, 0.01, and 0.001, respectively, and *p < 0.05 was considered statistically significant, and **p < 0.01 and ***p < 0.001 were extremely significant.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b04921. Materials and characterization, additional experimental details on HMSN synthesis, hemolysis analysis and 10826

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ACS Nano plasma coagulation study, and supplementary figures (PDF)

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AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Yu Chen: 0000-0002-8206-3325 Hangrong Chen: 0000-0003-0827-1270 Author Contributions

K. Zhang, H. Xu, and H. Chen supervised the project. K. Zhang conceived and designed the experiments. K. Zhang and X. Jia performed the synthesis of different samples and cell experiments. K. Zhang, Y. Chen, M. Ma, and L. Sun performed the animal experiments and related analysis. K. Zhang and H. Chen wrote the manuscript. All authors discussed the results and commented on the manuscript. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We greatly acknowledge financial support from China National Funds for Distinguished Young Scientists (51225202), National Natural Science Foundation of China (Grant Nos. 81501473, 81371570), Fostering and Action Planning of Tongji University for Young Excellences (Grant No. 2015KJ061), and Program of Shanghai Subject Chief Scientist (Grant No. 14XD1403800). We also thank Prof. Mingqian Tan from The Dalian Institute of Chemical Physics, Chinese Academy of Sciences, for providing the CLT1-G-propargyl peptides. REFERENCES (1) Carpenter, A. W.; Schoenfisch, M. H. Nitric Oxide Release: Part II. Therapeutic Applications. Chem. Soc. Rev. 2012, 41, 3742−3752. (2) Zhou, H.-f.; Yan, H.; Hu, Y.; Springer, L. E.; Yang, X.; Wickline, S. A.; Pan, D.; Lanza, G. M.; Pham, C. T. N. Fumagillin Prodrug Nanotherapy Suppresses Macrophage Inflammatory Response via Endothelial Nitric Oxide. ACS Nano 2014, 8, 7305−7317. (3) Hirst, D.; Robson, T. Nitric Oxide in Cancer Therapeutics: Interaction with Cytotoxic Chemotherapy. Curr. Pharm. Des. 2010, 16, 411−420. (4) Wink, D. A.; Vodovotz, Y.; Laval, J.; Laval, F.; Dewhirst, M. W.; Mitchell, J. B. The Multifaceted Roles of Nitric Oxide in Cancer. Carcinogenesis 1998, 19, 711−721. (5) Riccio, D. A.; Schoenfisch, M. H. Nitric Oxide Release: Part I. Macromolecular Scaffolds. Chem. Soc. Rev. 2012, 41, 3731−3741. (6) Riccio, D. A.; Dobmeier, K. P.; Hetrick, E. M.; Privett, B. J.; Paul, H. S.; Schoenfisch, M. H. Nitric Oxide-Releasing S-nitrosothiolModified Xerogels. Biomaterials 2009, 30, 4494−4502. (7) Walford, G.; Loscalzo, J. Nitric Oxide in Vascular Biology. J. Thromb. Haemostasis 2003, 1, 2112−2118. (8) Williams, D. L. H. The Chemistry of S-nitrosothiols. Acc. Chem. Res. 1999, 32, 869−876. (9) Stasko, N. A.; Fischer, T. H.; Schoenfisch, M. H. S-nitrosothiolModified Dendrimers as Nitric Oxide Delivery Vehicles. Biomacromolecules 2008, 9, 834−841. (10) Diring, S.; Wang, D. O.; Kim, C.; Kondo, M.; Chen, Y.; Kitagawa, S.; Kamei, K.-i.; Furukawa, S. Localized Cell Stimulation by Nitric Oxide Using a Photoactive Porous Coordination Polymer Platform. Nat. Commun. 2013, 4, 2684. (11) Wang, P. G.; Xian, M.; Tang, X. P.; Wu, X. J.; Wen, Z.; Cai, T. W.; Janczuk, A. J. Nitric Oxide Donors: Chemical Activities and Biological Applications. Chem. Rev. 2002, 102, 1091−1134. 10827

DOI: 10.1021/acsnano.6b04921 ACS Nano 2016, 10, 10816−10828

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DOI: 10.1021/acsnano.6b04921 ACS Nano 2016, 10, 10816−10828