Mitochondria-Targeted and Ultrasound-Activated ... - ACS Publications

Subscriber access provided by TULANE UNIVERSITY

Functional Nanostructured Materials (including low-D carbon)

Mitochondria-Targeted and Ultrasound-Activated Nanodroplets for Enhanced Deep-Penetration Sonodynamic Cancer Therapy Liang Zhang, Hengjing Yi, Jiao Song, Ju Huang, Ke Yang, Bin Tan, Dong Wang, Nanlan Yang, Zhi-Gang Wang, and Xingsheng Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21968 • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 8, 2019

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

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

Page 1 of 40 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 Applied Materials & Interfaces

Mitochondria-Targeted and Ultrasound-Activated Nanodroplets for Enhanced Deep-Penetration Sonodynamic Cancer Therapy Liang Zhang,†, ‖Hengjing Yi,†, ‡, ‖ Jiao Song,† Ju Huang,† Ke Yang,§ Bin Tan,§ Dong Wang,# Nanlan Yang,# Zhigang Wang,† Xingsheng Li*, †, ‡ †Institute

of Ultrasound Imaging, Department of Ultrasound, the Second Affiliated

Hospital of Chongqing Medical University, Chongqing 400010, China ‡Department

of Geriatrics, the Second Affiliated Hospital of Chongqing Medical

University, Chongqing 400010, China §Pediatric

Research Institute, Children’s Hospital of Chongqing Medical University,

Chongqing 400010, China #

Department of Ultrasound, the First Affiliated Hospital of Chongqing Medical

University, Chongqing 400010, China

KEYWORDS: ADV, Deep penetration, Nanodroplets, Sonodynamic therapy, Nanomedicine

ABSTRACT Sonodynamic therapy (SDT), a promising alternative for cancer therapy, utilizes a sonosensitizer combined with ultrasound (US) irradiation to damage tumor cells/tissue for therapeutic purposes. The ability for sonosensitizers to specifically accumulate in tumor cells/tissues could greatly influence their therapeutic efficiency. In this work, we report the use of US-activated sonosensitizer (IR780)-based nanodroplets (IR780-NDs) for SDT, which provide numerous benefits for killing cancer cells compared to traditional methods. For instance, IR780-NDs showed effective surface to core diffusion both in vitro and in vivo. In the presence of US, the acoustic droplet vaporization (ADV) effect significantly assisted the conveyance of IR780-NDs from 1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 circulatory system to tumor regions, and the acoustic wave force also increased the penetration depth within tumor tissues. Furthermore, IR780-NDs possesses mitochondrial targeting capabilities, which improves the precision and accuracy of SDT delivery. During in vitro assessment, the overproduction of reactive oxygen species (ROS) was observed following mitochondrial targeting, which rendered cancer cells more susceptible to ROS-induced apoptosis. Additionally, IR780-ND is a suitable candidate for photoacoustic (PA) and fluorescence (FL) imaging, and can also enhance US imaging due to the ADV-generated bubbles, which provides the potential for SDT guidance and monitoring. Therefore, with combined modalities, IR780-NDs can be a promising theranostics nanoplatform for cancer therapy.

INTRODUCTION Sonodynamic therapy (SDT) has emerged as a promising alternative for cancer therapy.1-4 In contrast to light-assisted photodynamic therapy (PDT), SDT is an ultrasound (US)-based approach and possesses many encouraging advantages. For instance, as US is a mechanical wave, it has minimal invasiveness and deep tissuepenetration.1-4 Additionally, US is a cheap and non-radioactive stimulus.3-4 In particular, this therapeutic modality is able to target lesion zones with high precision, hence leaving normal tissue undamaged.1-4 Although the exact mechanism contributing to its extraordinary therapeutic performance has yet to be elucidated, there is a consensus that the response of sonosensitizers to US plays an indispensable role. Upon a high-energy input, sonosensitizers can be activated to transfer energy to nearby oxygen molecules, which subsequently generates reactive oxygen species (ROS) and leads to further cytotoxicity for therapeutic purposes.5 The activation can be accomplished directly from US irradiation, by sonoluminescence, or by a pyrolysis process led by the ultrasonic cavitation effect.6 Moreover, US itself may directly induce cancer cell apoptosis.3 Despite the exceptional potential SDT has presented against cancer cells, the rational incorporation of diagnostic functions deserves attention to optimize for cancer therapy.7 2


Page 2 of 40

Page 3 of 40 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


For nanomedicine in cancer therapy, one of the primary obstacles is the selective localization and accumulation of nanovesicles into tumor regions.8-9 The critical step within this process is the diffusion of nanovesicles from surface tumor regions that could be reached from blood vessels, to poorly perfused inner core regions.8-11 Generally, nanoparticles with the size up to 400 nm can passively accumulate in tumors via the enhanced permeability and retention (EPR) effect resulting from the specific leaky structure of tumor vasculature.12-15 In addition, the extreme heterogeneity of solid tumors circumvents penetration of nanovesicles into the tumor core/parenchyma, leading to a nonuniform intratumoral distribution of the nanovesicles, thus undermining the therapeutic efficacy.16-17 Therefore, there is a pressing need to engineer smart nanovesicles that can bypass this issue.11, 18 Known for its capability to induce vascular disruption, therapeutic US combined with drug-loaded microbubbles (MBs), also known as the ultrasound-targeted microbubble destruction (UTMD) technique, has been extensively studied for improving drug delivery efficiency.19-20 However, the short lifespan of MBs in vivo restricts the duration of therapeutic effects.19 It has recently been reported that acoustic nanodroplets with liquid cores can transform into MBs upon US irradiation. This process is known as acoustic droplet vaporization (ADV), and can create an on-demand production of MBs; vascular disruption and tissue erosion typically follow ADV.9,

19, 21

While ADV

functions on the region of the tumor, vessel-tumor barrier can also become more penetrable, and disturbance within the solid tumor lowers the impedance for drug dissemination.22-24 In the meantime, the acoustic radiation force resulting from an acoustic pressure gradient in tumor tissue media drives the droplets and bubbles away from the wave source and toward the inner tissue.25-26 Other than the advantages resulting from US, some ligands or other compounds for active-targeting can also be integrated into nanovesicles to improve the therapeutic efficacy towards cancer cells.27-28 For instance, mitochondria-targeted drugs can exploit the susceptibility of mitochondria to ROS.29-34 This feature has been exploited via PDT and successfully demonstrated in some preliminary works.35-36 Similarly, while SDT 3



possesses a number of exclusive advantages, it is believed that SDT could also be more effective when incorporating mitochondria-targeted sensitizers.37-38 In addition, imaging-guided therapy has attracted extensive attention for theranostic purposes in combating cancer.39-41 Each imaging modality has its inherent advantages;42-43 for instance, fluorescence (FL) imaging can provide real-time monitoring of the distribution of imaging probes to determine the optimum therapeutic time window.44-45 Comparatively, photoacoustic (PA) imaging is a highly sensitive technique and can provide essential information of tumors.46-48 US imaging is a noninvasive real-time imaging technique that can guide SDT.49-50 Accordingly, a rational combination of various imaging modalities, can improve the precision and accuracy of diagnosis and treatment delivery. Inspired by the cancer nanomedicine research described above, we herein propose an innovative fabrication of a nanoplatform with consideration of incorporating multiple functions for amplified SDT, by concurrently achieving enhanced deep tissue penetration, multimodal imaging (FL/PA/US imaging) and mitochondria-targeting. Specifically, perfluoropentane (PFP) has been employed to construct PFP-based nanodroplets (PFP is encapsulated in liposomes), which can mediate ADV in the presence of US irradiation; the ADV-generated bubbles can further enhance US imaging. With ADV inducing vascular disruption and tissue erosion, the nanodroplets could then readily cross the blood vessel-tumor barrier and deeply penetrate the tumor efficiently. Additionally, IR780, a sonosensitizer with strong absorption in the near infrared region for FL and PA imaging,51-54 was further integrated into the lipid shell of PFP-based nanodroplets (designated as IR780-NDs). Importantly, by virtue of its inherent properties, IR780 itself can penetrate the tumor tissue efficiently and preferentially retained in mitochondria.55-56 Therefore, similar capabilities can also be expected on IR780-NDs. As such, a mitochondria-targeted and multi-modal imagingguided SDT can be achieved, and deep tumor tissue penetration can be accomplished without additional chemical conjugation of target ligands.

4


Page 4 of 40

Page 5 of 40 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


RESULTS AND DISCUSSION IR780-NDs, unique US-activated nanodroplets with a core/shell structure (PFP as the core and liposome/IR780 as the shell) were rationally constructed with enhanced deep penetration, mitochondrial targeting and for SDT with concurrent FL/US/PA imaging guidance. These nanodroplets can readily accumulate in tumor regions from blood circulation system via the typical EPR effect (Scheme 1). Upon US irradiation, acoustic nanodroplets transferring into microbubbles is known as ADV, it can induce vascular disruption and tissue erosion, allowing more droplets to leave the systemic circulation to enter the tumor stroma and penetrate into inner tissue farther from the blood vessels. The diffusion of nanodroplets to deeper tumor area can also be assisted by the loaded IR780. Importantly, the inherent mitochondria-targeting capability further increases the ROS cytotoxicity during the SDT because susceptibility of mitochondria towards to ROS. Aiming at the complexity and particularity of tumors, the “IR780-NDs” system was rationally designed with enhanced US imaging induced by ADV-generated bubbles upon US irradiation, as well as PA and FL imaging enhancement resulting from IR780, providing the potential for comprehensive SDT guidance and monitoring of tumor tissue.

Design, synthesis and characterization of IR780-NDs. IR780-NDs were synthesized via a single-step emulsion method, in which lipophilic IR780 was integrated into the lipid bilayer and PFP was encapsulated in the core. 1,2-distearoylsn-glycero-3-phosphoethanol-amine-N-[methoxy(polyethyleneglycol)-2000] (DSPEmPEG2000) was simultaneously conjugated onto the NDs for an enhanced colloidal stability.57-58 As shown in Figure 1a, an apparent color change from white to green after the loading of IR780 verified the successful loading of IR780. The as-fabricated IR780-NDs displayed a desirable dispersity and uniform spherical morphology under optical microscopy (Figure 1b). Figure 1c and S1 present a transmission electron microscopy (TEM) images of IR780-NDs with diameters of approximately 300 nm and a spherical structure. Dynamic light scattering (DLS) measurements were performed to 5



probe the hydrodynamic size and surface zeta potential of both NDs and IR780-NDs. The diameters of IR780-NDs and NDs were 354.5 ± 156.3 nm and 346.4 ± 139.0 nm (Figure 1d), respectively. The identical size distribution of ND before and after loading indicated IR780 was homogeneously dispersed among NDs. The resulted size allows the NDs to accumulate into the tumor region via the EPR effect.15 IR780 is a positively charged molecule,59 thus, the zeta potential of IR780-NDs was higher than that of the pristine NDs (Figure S2). The UV–vis spectrum shows that the IR780-NDs exhibited a characteristic band at λ = 780 nm, and the intensity of which shows a concentrationdependent manner (Figure 1e). In contrast, no obvious absorption was detected for pristine NDs (Figure 1f), which further demonstrates the efficient loading of IR780 onto the NDs. Moreover, according to the standard curve and relative absorbance intensity of IR780 in the UV–vis spectrum (Figure S3), the loading efficiency of IR780 in IR780-NDs was calculated to be 82.6%.

In vitro ADV capability, US/PA imaging performance and ROS generation of IR780-NDs. Upon US irradiation, While IR780-NDs gathered in tumor tissue via EPR effect, on-demand US irradiation can mediate a ND to microbubble phase transformation, namely the ADV effect. This “nano-to-micro” protocol can not only eliminate the drawbacks of traditional MBs, and also resolve the critical issue of poor MB stability. These capabilities are essential for biomedical applications. One of the applications it the PFP core-endowed US imaging by virtue of ADV.27 To investigate the ADV capability, the phase-transformation of IR780-NDs was induced at different US intensities and visualized a with optical microscopy. As shown in Figure 2a, no obvious MBs were detected at the intensity of 0.8 W/cm2 due to insufficient US intensity. In contrast, MBs were gradually generated when the intensity reached 1.6 W/cm2, and the number of bubbles increased along with US intensity. At an intensity of 2.4 W/cm2, substantial amount of IR780-NDs were converted into MBs. And with an increasing US intensity, the generated bubbles collapsed before the observation point (3 min). After 3 min of US treatment, the average sizes of resultant 6


Page 6 of 40

Page 7 of 40 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


MBs among were approximately 2 μm (Figure S4). Analysis of the in vitro US imaging performance of IR780-NDs revealed a similar tendency to the phase transformation, as detailed above (Figure 2b). When the US intensity reached 2.4 W/cm2, marked US enhancement was observed with the contrast mode of US imaging. Comparatively, negligible contrast-enhanced signal was observed at 0.8 or 4.0 W/cm2. The corresponding echo intensities were determined by a US analysis software, and the result was consistent with the in vitro performance. quantitative results of echo intensities measured by US analysis software were also in accordance with the in vitro US findings (Figure 2c). These results imply that the PFP encapsulated-IR780-NDs have an efficient ADV response and subsequently enhance US imaging performance. Besides the enhanced US imaging, IR780-NDs also demonstrated excellent PA imaging capability. For light excitation wavelengths ranging from 680 nm to 970 nm, IR780-NDs showed the strongest PA signal at 780 nm (Figure S5). It was also clearly observed that the PA signal was linearly increased as a function of IR780-NDs concentrations upon irradiation by a PA laser at λ = 780 nm (Figure 2d). The multiple functions of IR780-NDs was affiliated to the synergistic effect of the PFP core and loaded IR780. In a theranostics regard, other than the imaging guidance capability provided by the ADV, IR780-NDs can also induce extraordinary therapeutic performance. One of the causes for IR780-NDs-induced cell damage was proposed resulted from the overproduction of ROS that respond to US irradiation. To explore the potential of IR780-NDs as a sonosensitizer, SOSG, a widely used ROS detection probe, was applied to confirm the ROS generation in vitro. The produced ROS reacted with SOSG, inducing an increase in FL intensity.60 The FL intensity of the SOSG solution containing IR780 at a concentration of 5 μg/mL increased drastically with prolonged irradiation (Figure 2e). The test was also performed with other concentrations (2.5 and 3.75 μg/mL) of IR780-NDs, while the overall intensity increases with elevated IR780ND concentration, the US treatment duration-dependent manner was also observed (Figure S6a-f). Furthermore, we have employed electronic spin resonance (ESR) 7



spectroscopy to verify the generated radical species. As shown in Figure S7, characteristic signals of both hydroxyl radicals (•OH) and singlet oxygen (1O2) were observed with IR780-NDs after US treatment, while no obvious signal was found in the absence of IR780-NDs or US.

Deep penetration of NDs at the tissue level assisted by ADV. Nanotherapeutics involving nanoparticles exploit the specific cumulation induced by leaky vascular structures. However, the over-large size of injected nanocarriers, rarely leaky vascular structures in the early stage of tumors, and the finite tumor vascular gap hinder the convey of nanovesicles towards the tumor.8, 18 In addition, the nanoparticles that leak through blood circulation often suffer from endocytosis by cells located on the surface of the tumor, leading to poor penetration into the core cancer sites.16 Thus, it is highly desirable to exploit a strategy that has the ability to accelerate the leakage of nanoparticles, which would ensure the effective accumulation in tumors, and also the ability to promote the movement of nanoparticles into the core of tumors. For this purpose, not only known by its imaging capability, ADV is also recognized as a wellprogrammed process, that can address the intractable problem of effective accumulation and intratumoral diffusion. The loaded PFP can induce effective ADV upon US irradiation, thus disrupt tumor vascular endothelium and cause tissue erosion, which is beneficial to solve above-mentioned issues. To evaluate the penetration depth of nanoparticles assisted by ADV, the tumor tissues were subjected to fluorescence assays after different treatments. Models of intertissueNDs distribution after US stimulation were established (Figure 3a). In contrast to PFP, perfluorooctyl bromide (PFOB) cannot be converted into gas upon US irradiation, namely PFOB-based liposomes cannot transform into bubbles, thus ADV will not occur. As shown in Figure 3b, green fluorescence represents tumor vasculature and red represents nanoparticles. Result in the control group tracks NDs that gathered to tumor area via only EPR effect. In the US (PFOB-based liposomes + US) group, an acoustic radiation force resulting from an acoustic pressure gradient in tumor tissue media upon 8


Page 8 of 40

Page 9 of 40 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


US irradiation pushed the droplets and bubbles away from the wave source, thus led to the NDs reached into a deeper area. In the case of the ADV group (NDs + US), significant red fluorescence was distributed farther away from the blood vessels compared to other groups. The distributions of red fluorescence away from vessels were analyzed, as shown in Figure 3c, and exhibited the same tendency. The results indicated that the ADV effect played an indispensable role for the deep-penetration.

Intracellular uptake, penetration depth detection, mitochondria-targeting ability and ROS production of IR780-NDs in cancer cells. The efficient penetration and accumulation of the sonosensitizer IR780-NDs in tumor tissue/cells is a prerequisite for exerting a therapeutic effect, which is essential for the in vivo SDT performance. Hence, the intracellular uptake of IR780-NDs with prolonged incubation time (1, 2, 3, and 4 h) was visualized by confocal laser scanning microscopic (CLSM) images and quantified by flow cytometry. As expected, the CLSM image shows that IR780-NDs could be efficiently internalized into 4T1 cancer cells, as evidenced by the presence of obvious red fluorescence originating from DiI-labeled IR780-NDs after 1 h of coincubation (Figure 4a). Comparatively, while without IR780, minimal NDs accumulated onto the 4T1 cells. The fluorescence intensity measured by flow cytometry also significantly enhanced as the coincubation duration increased to 4 h, which is similar to the CLSM results (Figure 4b, S8, S9). To simulate the penetration of IR780NDs within tumor tissue, a 3D culture model with heterogeneous tumor perfusion, high cell density, and increased interstitial pressure was constructed to mimic the tumors.61 After 12 h of incubation, numerous DiI-labeled IR780-NDs (red ﬂuorescence) penetrated into tumor core spheroids and distributed throughout the whole spheroid. However, pristine NDs adhered only to the surface of the tumor spheroids (Figure 4c, d, S10-11, Movie S1-4). In addition to selective accumulation in tumor cells, IR780-NDs also demonstrated the ability to target mitochondria, which can further enhance the SDT efficiency. To illustrate the mitochondria targeting capability of IR780-NDs, the internalization and 9



distribution of IR780-NDs in mitochondria and lysosomes were monitored and compared. As depicted in Figure 5a, S12, S13, the mitochondria and lysosome were stained with Mito-Tracker and Lyso-tracker, respectively. Significant red fluorescence (represents DiI-IR780-NDs) was detected in regions of mitochondria. When the images were overlapped, the red fluorescence merged well with the green fluorescence (represents mitochondria), whereas a relatively poorer overlap was detected in lysosomes regions. The Pearson Correlation (PC) coefficients of Mito-Tracker was 0.87, which was higher than that of Lyso-tracker 0.41, indicated the mitochondria-targeting behavior of IR780-NDs. To illustrate the active role of IR780 contributing to the mitochondria-targeting, pristine NDs were used to compare with IR780-NDs. As a result, no obvious red fluorescence occurred in mitochondrial regions in the presence of NDs without IR780 loading, which verified the IR780 capability of mitochondria targeting. This behavior is favorable for therapeutic purposes as mitochondria play fundamental roles in cell apoptosis, programmed cell death, and signaling through mitochondrial ROS.29-30 It has been reported that mitochondria are susceptible to ROS,62-63 the overexpression of which may induce mitochondrial permeability transition pore (mPTP).64 Besides, another possible mechanism involved in STDinduced macrophage apoptosis and necrosis is the loss of mitochondrial membrane potential (MMP).65 Hyo Sung Jung et al. reported a mitochondria-targeted sensitizer rendered more efficacy in photodynamic therapy, which is based in ROS generation.35 Similarly, while SDT possesses a number of exclusive advantages, it is believed that SDT could also be more effective when incorporating mitochondria-targeted sensitizers. Meng Jia Chen et al. grafted mitochondria-targeted ligand, triphenylphosphonium bromide (TPP), to sonosensitizer-based liposomes, achieving greater cancer cell inhibition efficacy in SDT.37 Based on our result and the reported, we consider the mitochondrial targeting behavior can be a strong cause for cell damage. Given the satisfactory ROS generation of IR780-NDs in aqueous solution, the intracellular ROS production of IR780-NDs was further evaluated with a reactive oxygen species assay kit DCFH-DA (20,70-dichlorofluorescin diacetate). As shown in 10


Page 10 of 40

Page 11 of 40 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 5b, positive control group demonstrated the strongest fluorescence intensity. Other than that, strongest green fluorescence appeared only in the IR780-NDs + US group revealed the consequential production of ROS in the simultaneous presence ofIR780-NDs and US irradiation. Contrarily, no obvious fluorescence can be detected in all rest groups.

In vitro sonotoxicity of IR780-NDs against 4T1 cells. Following the investigation of the cellular uptake, in vitro sonotoxicity of IR780-NDs against 4T1 cells was assessed using a standard Cell counting kit-8 (CCK-8) assay. Cells were co-incubated with IR780-NDs at various IR780 concentrations (0, 1, 2, 3, 4, and 5 μg/mL) and then treated with US irradiation for different durations (0, 30, 60, and 90 s). In the presence of US irradiation, IR780 displayed a concentration-dependent cytotoxicity towards 4T1 cells (Figure 6a). The cell viability also dropped more significantly with a longer US duration. Other than the US irradiation, both ND-mediated ADV and the IR780induced STD can contribute to the cell damage. When NDs were uptaken by cancer cells, ADV can be induced by US irradiation, namely the bubble conversion and expansion inside cells with a high wall velocity will subsequently cause mechanical bio-effects including cellular damage.66 To clarify the mechanism of the cell death, we have investigated the influence of pristine NDs with US treatment. As shown in Figure S14, in the absence of IR780, statistically significant cell viability decrease was only observed while with NDs concentration of 96.8 μg/mL or higher. For instance, in the presence of a US irradiation, with equivalent NDs concentration at 121 μg/mL, NDs induced less than 10% cell viability decrease, while with IR780-NDs, the cell viability drastically dropped more than 60%. As a result, the IR780-induced SDT was the predominant factor inducing the cell damage. The cell-damaging of IR780-NDs was also assessed by a flow cytometry (Figure 6b, S15). Significant cell damage was observed after US irradiation for 90 s with various IR780 concentrations (0, 1, 2, 3, 4 and 5 μg/mL). Additionally, after various treatments, the cytotoxicity of IR780-NDs to cancer cells was directly observed by CLSM. Calcein11



AM (CAM)-PI costaining was used to differentiate live and dead cells, respectively. As shown in Figure 6c, massive cell deaths were found in the IR780-NDs + US group. The results indicated that extensive apoptosis and necrosis occurred under the assistance of SDT.

In vivo FL imaging and biodistribution of IR780-NDs. Due to the strong fluorescence signal and selective accumulation of IR780-NDs in tumor cells, FL imaging was further carried out using IR780-NDs as a contrast agent to investigate its biodistribution and aggregation in vivo. FL imaging was performed in 4T1 tumorbearing mice at various time points after intravenous injection of IR780-NDs. As shown in Figure 7a, the tumor area at 0.5–24 h post injection were highlighted with FL signal, and the signal strengthened over time (Figure 7b). Twenty-four hours after the administration of IR780-NDs, the tumor nodes and major organs and tumors were collected for ex vivo FL imaging (Figure 7c). The result revealed a notable accumulation of IR780-NDs in tumor tissue, where the fluorescence signal was stronger than that of other major organs (heart, liver, spleen, lung, and kidney, Figure 7d). Additionally, the major organs and tumors were also harvested for pathology examination (Figure 7e). In the IR780-NDs group, a large amount of these nanodroplets accumulated in the tumor tissue, while in the pristine ND group, the NDs mainly accumulated in the liver and spleen (Figure 7f). These results demonstrate the efficient tumor accumulation of IR780-NDs and the desirable features as a contrast agent for FL imaging.

In vivo US/PA imaging performance of IR780-NDs. The functionality of IR780NDs as a multimodal imaging agent was tested on breast cancer 4T1 nude mice xenografts after intravenous injection of IR780-NDs aqueous solution. First, the capability of enhanced US imaging of IR780-NDs was evaluated. Twenty-four hours after injection of IR780-NDs, US irradiation was applied, and a bright US signal occurred at the tumor site in CEUS mode (Figure 8a). The corresponding echo intensity 12


Page 12 of 40

Page 13 of 40 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


in the tumor area after injection was substantially higher than that before injection (Figure 8b). Meanwhile, the intratumoral PA signal increased over time and reached a maximum value approximately 24 h post injection (Figure 8c). In addition, the PA signal intensity was also consistent with the PA images (Figure 8d, Figure S16).

In vivo SDT assisted by IR780-NDs. After confirmation of the deep penetration into tumor tissue and mitochondria-targeting capability of IR780-NDs, the in vivo therapeutic efficacy was further evaluated to address its clinical translation potential. Twenty 4T1 tumor-bearing mice were randomly divided into four groups: the (i) control group, (ii) US only group (only exposed to US), (iii) IR780-NDs only group (only intravenous injection of IR780-NDs), and (iv) US + IR780-NDs group (US irradiated after intravenously administrated with IR780-NDs). To reduce thermal effects on the tumor sites, US was applied in 4 cycles of 3 min on and 3 min off. The US irradiation was repeated during the therapeutic process. The digital photos of mice during the therapeutic period were taken every other day during the therapeutic period (16 d) (Figure 9a), and the corresponding tumor volumes (Figure 9b) were recorded. As a result, the tumors in the control, US only, and IR780-NDs only groups grew rapidly as almost none therapeutic effect was exerted. In comparison, the tumor growths in the treatment group (US + IR780-NDs) were greatly inhibited. After 16 d of treatment, the tumors were excised for weight measuring. The result also followed similar comparisons, as also shown by the tumor volume and in digital photos (Figures 9c, S17). The hematoxylin and eosin staining (H&E), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and proliferating cell nuclear antigen (PCNA) staining results further confirmed the therapeutic effect of US-activated IR780-NDs (Figures 10a, S18-20). As shown the H&E- and TUNEL-stained tumor sections, there was negligible change in cell morphology or status of the control group, US only group, and IR780-NDs only group, while the US + IR780-NDs group exhibited cell morphology changes, including karyopyknosis, karyorrhexis, and karyolysis, 13



indicating the substantial necrosis of cancer cells. The PCNA staining results were in consistent with the H&E and TUNEL findings and indicated a significantly lower proliferation index in the treated groups (US + IR780-NDs). The development of innovative therapeutic nanoplatforms was established upon not only the theranostics capabilities but also the potential for clinical translation. In this regard, a satisfactory biosafety is a prerequisite. After the administration of IR780-NDs, all mice in every group showed neglectable weight fluctuations (Figure S21), demonstrating the negligible adverse effect of this nanoagent. Besides, H&E staining of the main organs of mice, including the heart, liver, spleen, lung and kidney, was conducted at the end of different treatments. As shown in Figure 10b, no significant adverse effects on major organs were found during the treatment period, suggesting the desirable biosafety of our nanosystem. In addition, the potential toxicity of IR780-NDs in vivo was further evaluated in mice to ensure its high biocompatibility. No detectable changes in blood biochemical indexes (Figures S22-23) or histopathological results from H&E staining of the main organs (Figure S24) were observed for the tested dose, demonstrating the high safety in vivo.

CONCLUSION In summary, we have successfully constructed, for the first time, a mitochondriatargeted and US-activated multifunctional IR780-NDs nanosystem for enhancing deeppenetration SDT, guided and monitored by multimodal imaging (US/PA/FL imaging). Notably, this nanosystem entered tumor regions with deep penetration as shown by enhanced arrival from systemic circulation, tissue penetration induced by ADV, and surface-to-core diffusion of IR780-NDs. Importantly, IR780-NDs can selectively accumulate in mitochondria, which are susceptible to ROS, further enhancing the SDT performance. In addition, based on the unique ADV effect of PFP and the physicochemical properties of the sonosensitizer, these nanodroplets could act as a concurrent multimodal imaging agent for enhanced FL, US, and PA imaging, improving the precision and accuracy of SDT delivery. This work paves the way for 14


Page 14 of 40

Page 15 of 40 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


improving the therapeutic efficacy of SDT by a rational combination of multiply enhanced deep penetration of the sonosensitizer, organelle-specific ROS production, and multimodal imaging guidance/monitoring.

Experimental Section Materials

and

reagents.

1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine

(DPPC), DSPE-mPEG2000 were purchased from Xi’an Ruixi Biological Technology Co., Ltd (China). Cholesterol, PFP, PFOB, sepharose gel and IR780 were obtained from Sigma Aldrich (St. Louis, MO, USA). 4’,6-Diamidino-2-phenylindole (DAPI), DiI, Hoechst 33342 and DCFH-DA were purchased from Beyotime Biotechnology (China). Singlet oxygen sensor green (SOSG) reagent, MitoTracker™ Deep Red FM and LysoTracker™ Deep Red were obtained from Thermo Fisher (USA). CCK-8, CAM, PI, TEMPO and DMPO were obtained from Dojindo (Japan). Chloroform (CHCl3) was purchased from Chongqing Chuan Dong Chemical Co., Ltd. (China). Design, synthesis and characterization of IR780-NDs. The liposomes loaded with IR780 and PFP were synthesized by an emulsion method. First, the lipid compounds, DPPC, DSPE-mPEG2000, cholesterol and IR780, were mixed together (at a weight ratio of 12:4:4:1) and then dissolved in 5 mL of CHCl3. The above solution was transferred to a round flask for rotary evaporation to form a lipid film. After the complete evaporation of CHCl3 (~ 2h), the resulted film was hydrated by PBS. Next, 400 μL of PFP was added to the above suspension for emulsion by a sonicator (Sonics & Materials Inc., Newtown, CT, USA) at an intensity of 125 W for 5 min (pulse duration, 5 s; pulse interval, 5 s). Finally, IR780-NDs was obtained after centrifugation (4629 g, 5 min) (5804R; Eppendorf, Germany). The fabrication of NDs was similar to the above process except without the addition of IR780. The properties of the IR780-NDs aqueous solution were evaluated. The morphology and structure of IR780-NDs were characterized with an optical microscopy. For TEM imaging, approximately 10 μL of sample at concentration of 0.2 mg/mL was drop onto an 500-mesh carbon-filmed copper grid. Then the specimen was subjected to TEM test 15



after the complete evaporation of the solvent. The images were taken with an Hitachi 7500 electronic microscope (Tokyo, Japan) at an acceleration voltage of 200 kV. The size distribution, zeta potential and particle size of IR780-NDs and NDs were analyzed using a Zetasizer Nano ZS instrument (Malvern Instruments, Malvern, UK). A UV–vis spectrophotometer (UV-3600, Shimadzu, Japan) was used to determine the UV–vis absorption spectrum of IR780, NDs and IR780-NDs. The concentration of IR780 (c1) was measured by a standard curve method using a UV-vis spectrometer. The encapsulated IR780 (m1) and encapsulation efficiency was calculated as follows. m1 = minitial - c1•vsupernatant Loading efficiency of IR780 = m1 / minitial Where supernatant represents the volume of the supernantant and minitial for the initial weight of dosed IR780.

In vitro ADV capability, US/PA imaging performance and ROS generation of IR780-NDs. The IR780-NDs emulsion (2 mg/mL) was irradiated by an ultrasound transducer instrument (LM.SC051 ACA; Institute of Ultrasound Imaging of Chongqing Medical Sciences, Chongqing, China) at different intensities (0.8, 1.6, 2.4, 3.2 and 4.0 W/cm2) for 3 min. The ultrasound parameters employed for treatment purposes were set as follows: Frequency: 650 KHz, focal length, 1.5 cm; pulse wave mode, 50% duty cycle, pulse duration: 1s. After US irradiation, optical microscopy images of IR780NDs were collected, and US imaging of IR780-NDs was performed on a US system (MyLab 90, Esaote, Italy) equipped with high frequency linear array probe (LA523, Frequency: 12 MHz, Mechanical index (MI): 0.06). Finally, a US imaging software was used to analysis the echo intensities of an regions of interest (ROI). Also, the sizes of generated bubbles were measured by the optical microscopic software. For PA imaging, IR780-NDs suspension was scanned within the excitation wavelength range of 680 to 970 nm to detect the Amax (maximum absorbance) using a Vevo LAZR Photoacoustic Imaging System (Visual Sonics Inc., Toronto, Canada). Then, PA images of the IR780-NDs suspension at different concentrations 16


Page 16 of 40

Page 17 of 40 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


(IR780 concentrations: 20, 40, 60, 80, and 100 μg/mL) were obtained at an excitation wavelength of 780 nm, and the corresponding PA signal intensities were analyzed using Vevo LAZR software. The ROS generation after US activation was determined by SOSG. A certain amount of SOSG dissolved in methanol was added to IR780-NDs suspensions (2.5, 3.75, and 5.0 μg/mL). The mixture was sonicated by US (2.4 W/cm2) for various durations (0, 30, 60, 90 and 120 s). Finally, the FL intensities of SOSG were recorded from 500 nm to 700 nm by a fluorescence spectrophotometer (Agilent Technologies). Electron spin resonance (ESR) spectroscopy was applied to further verify the radical species. Prior to the test, sample was thoroughly mixed with spin trap and extracted into a quartz capillary. After a certain time (0, 2, and 5 min) of US (2.4 W/cm2) treatment, spectra were obtained in perpendicular mode on a Bruker EMX-8/2.7 spectrometer with the following settings: microwave frequency = 9.784 GHz and microwave power = 6.375 mW. The modulation frequency was 100.00 kHz and the modulation amplitude was set as 2.00 G. TEMPO and DMPO were used as spin trap to capture singlet oxygen (1O2) and hydroxyl radicals (•OH) respectively. PBS mixed with spin traps were used as negative controls. All tests were conducted at room temperature.

Deep penetration of IR780-NDs at the tissue level assisted by ADV. All animal experiments were approved by the Animal Ethics Committee of Chongqing Medical University. Female immunodeficient BALB/c nude mice (4–5 weeks, 18–24 g) were bred and housed at 20 oC with certain humidity. For the establishment of 4T1 tumorbearing mouse model, 4T1 cells (4 ×106 cells per mL) suspended in PBS (150 μL) were injected subcutaneously into the left back flank. PFOB-based liposomes were synthesized in a manner similar to the process of NDs fabrication except that PFP was replaced with PFOB. The mice were randomly separated into three groups, including a control group (DiI-labeled NDs injection without US irradiation), a US group (DiI-labeled PFOB-based liposome with US irradiation), and an ADV group (DiI-labeled NDs injection with US irradiation). After 17



Page 18 of 40

the above treatments, tumor nodes were collected for fluorescence evaluation. The blood vessels were stained with CD31. Then, the slices were observed by fluorescence microscopy and the corresponding relative FL signal of nanoparticles from the vasculature towards the tumor center was measured using Image J software.

Intracellular uptake, surface-to-core penetration, and in vitro ROS production of IR780-NDs. The 4T1 cells were seeded at a density of 1 × 105 cells per CLSM flask and incubate overnight, Cells were then divided into two groups (NDs group and IR780-NDs group). DiI-labeled NDs and DiI-labeled IR780-NDs (1 mL, 50 μg/mL) were added to the two different groups. After 4 h of coincubation, the cells were rinsed with PBS for 3 times fixed with 4% paraformaldehyde (PFA, 1 mL) for 15 min, and stained by 200 μL of DAPI (1 μg/mL) for 15 min. Finally, the uptake of NDs and IR780NDs by 4T1 cells was observed by CLSM. To assess surface-to-core penetration, 3D spheroid models of 4T1 tumor cells was established. 4T1 cells were seed at a density of 5 × 104 cells per well and cultured for 6 d in spheroid microplates (Corning). The 4T1 cells were assigned into

two groups

(NDs group and IR780-NDs group). After coincubation with DiI-labeled NDs or DiIlabeled IR780-NDs, the cells were stained by Hoechst 33342 for 12 h. After washing with PBS, the multicellular spheroids were observed by CLSM. To evaluate the mitochondria-targeting ability of IR780-NDs, 4T1 cells (1 ×104 cells per dish) were cultured in CLSM specified dishes for 24 h.And a certain amount of DiIlabeled NDs or the the DiI-labeled IR780-NDs nanoemulsion were added into the above dishes.

After 4h incubation, cells were washed with PBS and stained with

MitoTracker or Lyso Tracker were applied for another half an hour to label mitochondria or lysosomes. The mitochondrial lysosomes localization of IR780-NDs or NDs was confirmed using CLSM. The resulting PC coefficients were measured. In addition to the SOSG assay, a DCFH-DA-based ROS assay kit was also adopted to detect intracellular ROS production. CLSM dishes seeded with 4T1 cells (1 × 105 cells in each dish) were randomly distributed into five groups: the blank or control 18


Page 19 of 40 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


group (without treatment), US Only group, IR780-NDs Only group, IR780-NDs + US group and positive control group (treated with Rosup). After 24 h incubation, corresponding treatments were conducted on each group. Then, DCFH-DA (30 μM) was added to each dish. Finally, the ROS production was determined by CLSM after 3 times PBS washing.

In vitro sonotoxicity of IR780-NDs against 4T1 cells. To evaluate cytotoxicity in vitro, 4T1 cells were seeded in a 96-well plate at a density of 1 × 105 cells per well and cultured for 24 h. Then, the culture medium above was replaced with fresh medium containing IR780-NDs at different concentrations (IR780 concentration: 0, 1, 2, 3, 4 and 5 μg/mL). After 4 h incubation, the standard CCK-8 assay was performed to determine cell viability, wells were seeded in quintuplicate. To demonstrate the effect of ADV alone on cell viability, the 4T1 cells cultured in a 96-well plate were coincubated with different concentrations of pristine NDs (0, 24.2, 48.4, 72.6, 96.8 and 121 μg/mL, which were based on the total amount of lipid). After that, US was applied to irradiate the NDs, then the cell viabilities were evaluated by CCK-8 assay. To further evaluate the SDT efficiency of IR780-NDs, CLSM seeded with 4T1 cells were designated as control group, US only group, IR780-NDs only group and IR780-NDs + US group respectively. To assess the degree of apoptosis, cells were seeded in 6-well plates and cultured for overnight, and then different concentrations of IR780-NDs (0, 1, 2, 3, 4 and 5 μg/mL) were added into corresponding wells and coincubated for 4 h. After US irradiation for 90 s, cells in each well were stained with 5 μL of Annexin V and 5 μL of fluorescein isothiocyanate (FITC) and then harvested for flow cytometry. Next, different treatments were applied to each group. Lastly, the cells were stained with CAM /PI dye to differentiate dead (red) and live (green) cells by CLSM.

In vivo biodistribution of IR780-NDs. In vivo FL imaging was performed on a Xenogeny IVIS Spectrum imaging system (Perkin Elmer, USA). Before the injection of IR780-NDs, FL images of each mouse were collected as a control. Then, the tumor19



bearing mice were intravenously injected with the IR780-NDs nanoemulsion (200 μL, 3.0 mg/mL) via the tail vein. Images were acquired at 0.5, 1, 2, 4, 6, and 24 h post injection, respectively. The FL intensities at the tumor sites were calculated by the IVIS Spectrum imaging system. Finally, the tumor nodes and major organs (heart, liver, spleen, lung, kidney) were collected for ex vivo FL imaging. The average FL intensities were also recorded. For in vivo biodistribution detection by fluorescence microscopy, mice were treated similarly as the abovementioned with the exception that the IR780-NDs were labeled with DiI. After 24 h of administration, the tumor nodes and the major organs, sent for ultrathin sectioning immediately after extraction from mice. The cell nucleuses were stained with DAPI. Fluorescence images were collected by a fluorescence microscopy and the relative fluorescence signal of nanoparticles using Image J software.

In vivo US/PA imaging performance of IR780-NDs. Mice were intravenously injected with IR780-NDs emulsion (200 μL) at a dose of 3.0 mg/mL. Twenty-four hours later, US was used to stimulate the tumor tissue at an intensity of 2.4 W/cm2. Then US imaging of tumor regions was performed with a US system mentioned above. To evaluate the PA imaging performance of IR780-NDs in vivo, mice were intravenously injected with the IR780-NDs nanoemulsion (200 μL) at a dose of 3.0 mg/mL. Then, PA images were acquired at 0.5, 6, and 24 h post injection. Furthermore, the corresponding PA signal values were measured.

In vivo SDT assisted by IR780-NDs.

Twenty tumor-bearing mice were separated

into four groups (control, US Only, IR780-NDs Only, IR780-NDs + US). Twenty-four hours after the IR780-NDs administration, US (on 3 min, off 3 min, 4 cycles) at 2.4 W/cm2 was applied to irradiate the tumor regions. Same treatment was repeated after 24 h. The mice body weights and the tumor volume were recorded during the treatment periods. Major organs and tumor tissues were dissected from the mice at the end of treatment and then sent for H&E, PCNA and TUNEL staining. 20


Page 20 of 40

Page 21 of 40 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


In vivo toxicity. Twenty healthy BALB/c mice were intravenously administrated with IR780-NDs (200 μL, 3.0 mg/mL). The control group (n=5) was injected with saline. The mice were euthanized

after certain time (3, 7, 14 and 30 d post-injection),

and blood of mice was collected for biomedical indexes and blood cells measurement. The major organs of mice were excised and then fixed with PFA (4%) for H&E staining. Statistical analysis. Statistical analysis was performed with Origin software. All the data were presented as the mean ± standard deviation. The significance of the data is analyzed according to a Student’s t test: *P < 0.05.

ASSOCIATED CONTENT Supporting Information TEM image of IR780-NDs; Zeta potential of NDs and IR780-NDs; Sizes of generated bubbles induced by ADV; Tomography, movies of the 3D spheroid models incubated with IR780-NDs or NDs; Mitochondrial location of IR780-NDs; ESR spectra; Photographs of tumors dissected from mice in four groups after various treatments; Quantitative analysis of TUNEL and PCNA staining of tumor sections; In vivo toxicity.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] Author Contributions ‖

Liang Zhang and Hengjing Yi contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We greatly acknowledge the financial support from the National Natural Science Foundation of China (Grant No. 81771845, 31630026, 81630047, 81771847, 81601513). 21



Page 22 of 40

REFERENCES (1) Pan, X.; Bai, L.; Wang, H.; Wu, Q.; Wang, H.; Liu, S.; Xu, B.; Shi, X.; Liu, H. Metal–Organic

Framework

Derived

Carbon

Nanostructure

Augmented

Sonodynamic Cancer Therapy. Adv. Mater. 2018, 1800180. (2) Zhu, P.; Chen, Y.; Shi, J. Nanoenzyme-Augmented Cancer Sonodynamic Therapy by Catalytic Tumor Oxygenation. Acs Nano 2018, 12, 3780-3795. (3) Qian, X.; Zheng, Y.; Chen, Y. Micro/Nanoparticle-Augmented Sonodynamic Therapy (SDT): Breaking the Depth Shallow of Photoactivation. Adv. Mater. 2016, 28, 8097-8129. (4) Ping, H.; Qian, X.; Yu, C.; Yu, L.; Han, L.; Wang, L.; Zhu, Y.; Shi, J. Metalloporphyrin-encapsulated Biodegradable Nanosystems for Highly Efficient MRI-Guided Sonodynamic Cancer Therapy. J. Am. Chem. Soc. 2016, 139, 12751284. (5) Chen, H.; Zhou, X.; Gao, Y.; Zheng, B.; Tang, F.; Huang, J. Recent Progress in Development of New Sonosensitizers for Sonodynamic Cancer Therapy. Drug Discovery Today 2014, 19, 502-509. (6) Costley, D.; Ewan, C. M.; Fowley, C.; Mchale, A. P.; Atchison, J.; Nomikou, N.; Callan, J. F. Treating cancer with Sonodynamic Therapy: A review. Int. J. Hyperthermia 2015, 31, 107-117. (7) Chen, H.; Zhang, W.; Zhu, G.; Xie, J.; Chen, X. Rethinking cancer nanotheranostics. Nat. Rev. Mater. 2017, 2, 17024. (8) Kim, J.; Jo, C.; Lim, W.; Jung, S.; Lee, Y. M.; Lim, J.; Lee, H.; Lee, J.; Kim, W. J. Programmed Nanoparticle-Loaded Nanoparticles for Deep Penetrating 3D Cancer Therapy. Adv. Mater. 2018, 1707557. (9) Simona, M.; Julien, N.; Patrick, C. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12, 991-1003. (10)Ya, L.; Di, Z.; Zeng-Ying, Q.; Guo-Bin, Q.; Xing-Jie, L.; Xi-Guang, C.; Hao, W. A Peptide-Network Weaved Nanoplatform with Tumor Microenvironment 22


Page 23 of 40 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


Responsiveness and Deep Tissue Penetration Capability for Cancer Therapy. Adv. Mater. 2015, 27, 5034-5042. (11)Cliff, W.; Triantafyllos, S.; Jian, C.; John, M.; Chauhan, V. P.; Wen, J.; Zoran, P.; Jain, R. K.; Bawendi, M. G.; Dai, F. Multistage Nanoparticle Delivery System for Deep Penetration into Tumor Tissue. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 2426-2431. (12)Torchilin, V. P. Recent Advances with Liposomes as Pharmaceutical Carriers. Nat. Rev. Drug Discovery 2005, 4, 145-60. (13)Chauhan, V. P.; Triantafyllos, S.; Martin, J. D.; Zoran, P.; Ou, C.; Kamoun, W. S.; Bawendi, M. G.; Dai, F.; Jain, R. K. Normalization of Tumour Blood Vessels Improves the Delivery of Nanomedicines in a Size-Dependent Manner. Nat. Nanotechnol. 2012, 7, 383. (14)Matsumura, Y.; Maeda, H. A New Concept for Macromolecular Therapeutics in Cancer Chemotherapy: Mechanism of Tumoritropic Accumulation of Proteins and the Antitumor Agent Smancs. Cancer Res. 1986, 46, 6387-6392. (15)Bae, Y. H.; Park, K. Targeted Drug Delivery to Tumors: Myths, Reality and Possibility. J. Controlled Release 2011, 153, 198-205. (16)Nakamura, Y.; Mochida, A.; Choyke, P. L.; Kobayashi, H. Nano-drug Delivery: Is the Enhanced Permeability and Retention (EPR) Effect Sufficient for Curing Cancer? Bioconjug Chem. 2016, 27, 2225-2238. (17)Jun, F.; Hideaki, N.; Hiroshi, M. The EPR effect: Unique Features of Tumor Blood Vessels for Drug Delivery, Factors Involved, and Limitations and Augmentation of the Effect. Adv. Drug Delivery Rev. 2011, 63, 136-151. (18)Chen, J.; Ding, J.; Wang, Y.; Cheng, J.; Ji, S.; Zhuang, X.; Chen, X. Sequentially Responsive Shell-Stacked Nanoparticles for Deep Penetration into Solid Tumors. Adv. Mater. 2017, 29, 1701170. (19)Ho, Y. J.; Yeh, C. K. Theranostic Performance of Acoustic Nanodroplet Vaporization-Generated Bubbles in Tumor Intertissue. Theranostics 2017, 7, 14771488. 23



(20)Chertok, B.; Langer, R.; Anderson, D. G. Spatial Control of Gene Expression by Nanocarriers Using Heparin Masking and Ultrasound-Targeted Microbubble Destruction. ACS Nano 2016, 10, 7267-7278. (21)Liandong, H.; Dong, K.; Tan, H.; Zhuang, Z.; Xue, Y., Isolation and Characterization of Plasmid Psr336 from Glucose Isomerase Producing Strain Streptomyces Roseoruber 336. Angew. Chem. 2012, 124, 7637-7640. (22)Skyba, D. M.; Price, R. J.; Linka, A. Z.; Skalak, T. C.; Kaul, S., . Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue. Circulation 1998, 98, 290-293. (23)Joo Ha, H.; Juan, T.; Brayman, A. A.; Matula, T. J.; Crum, L. A. Correlation between inertial cavitation dose and endothelial cell damage in vivo. Ultrasound Med. Biol. 2006, 32, 1611-1619. (24)Ho, Y. J.; Chang, Y. C.; Yeh, C. K. Improving Nanoparticle Penetration in Tumors by Vascular Disruption with Acoustic Droplet Vaporization. Theranostics 2015, 6, 392-403. (25)Dayton, P. A.; Allen, J. S.; Ferrara, K. W. The magnitude of radiation force on ultrasound contrast agents. J. Acoust. Soc. Am. 2002, 112, 2183-2192. (26)Dayton, P.; Klibanov, A.; Brandenburger, G.; Ferrara, K. Acoustic Radiation Force in vivo : a Mechanism to Assist Targeting of Microbubbles. Ultrasound Med. Biol. 1999, 25, 1195-1201. (27)Zhu, L. L.; Zhao, H. Y.; Zhou, Z. Y.; Xia, Y. H.; Wang, Z.; Ran, H.; Li, P.; Ren, J. L. Peptide-Functionalized Phase-transformation Nanoparticles for LIFU-Assisted Tumor Imaging and Therapy. Nano Letters 2018, 18, 1833-1841. (28)Zhao, H.; Wu, M.; Zhu, L. Cell-penetrating Peptide-modified Targeted Drugloaded Phase-transformation Lipid Nanoparticles Combined with Low-intensity Focused Ultrasound for Precision Theranostics against Hepatocellular Carcinoma. Theranostics 2018, 8, 1892-1910. (29)Li, X.; Fang, P.; Mai, J.; Choi, E. T.; Wang, H.; Yang, X. F. Targeting mitochondrial reactive oxygen species as novel therapy for inflammatory diseases 24


Page 24 of 40

Page 25 of 40 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


and cancers. J. Hematol. Oncol. 2013, 6, 19-19. (30)Green, D. R. Apoptotic pathways: the roads to ruin. Cell 1998, 94, 695-8. (31)Wang, H.; Gao, Z.; Liu, X.; Agarwal, P.; Zhao, S.; Conroy, D. W.; Ji, G.; Yu, J.; Jaroniec, C. P.; Liu, Z. Targeted production of reactive oxygen species in mitochondria to overcome cancer drug resistance. Nat. Commun. 2018, 9, 562. (32)Yang, Y.; Karakhanova, S.; Hartwig, W.; D'Haese, J. G.; Philippov, P. P.; Werner, J.; Bazhin, A. V. Mitochondria and Mitochondrial ROS in Cancer: Novel Targets for Anticancer Therapy. J. Cell. Physiol. 2016, 231, 2570-2581. (33)Zielonka, J.; Joseph, J.; Sikora, A.; Hardy, M.; Ouari, O.; Vasquez-Vivar, J.; Cheng, G.; Lopez, M.; Kalyanaraman, B. Mitochondria-Targeted TriphenylphosphoniumBased Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications. Chem. Rev. 2017, 117, 10043-10120. (34)Armstrong, J. S. Mitochondria: a target for cancer therapy. Br. J. Pharmacol. 2010, 147, 239-248. (35)Jung, H. S.; Lee, J. H.; Kim, K.; Koo, S.; Verwilst, P.; Sessler, J. L.; Kang, C.; Kim, J. S. A Mitochondria-targeted Cryptocyanine-Based Photothermogenic Photosensitizer. J. Am. Chem. Soc. 2017, 139, 9972-9978. (36)Noh, I.; Lee, D. Y.; Kim, H.; Jeong, C. K.; Lee, Y.; Ahn, J. H.; Hyun, H.; Park, J.; Kim, Y. Cancer Treatment: Enhanced Photodynamic Cancer Treatment by Mitochondria-Targeting and Brominated Near-Infrared Fluorophores. Adv. Sci. 2018, 5, 233-237. (37)Chen, M. J.; Xu, A.; He, W.; Ma, W.; Shen, S., Ultrasound Triggered Drug Delivery for Mitochondria Targeted Sonodynamic Therapy. J. Drug Delivery Sci. Technol. 2017, 39, 501-507. (38)Shimamura, Y.; Tamatani, D.; Kuniyasu, S.; Mizuki, Y.; Suzuki, T.; Katsura, H.; Yamada, H.; Endo, Y.; Osaki, T.; Ishizuka, M., 5-Aminolevulinic Acid Enhances Ultrasound-Mediated Antitumor Activity Via Mitochondrial Oxidative Damage in Breast Cancer. Anticancer Res. 2016, 36, 3607-3612. (39)Eun-Kyung, L.; Taekhoon, K.; Soonmyung, P.; Seungjoo, H.; Yong-Min, H.; 25



Page 26 of 40

Kwangyeol, L., Nanomaterials for Theranostics: Recent Advances and Future Challenges. Chem. Rev. 2015, 115, 327-394. (40)Teo, R. D.; Hwang, J. Y.; Termini, J.; Gross, Z.; Gray, H. B., Fighting Cancer with Corroles. Chem. Rev. 2016, 117, 2711-2729. (41)Zhang, L.; Sheng, D.; Wang, D.; Yao, Y.; Yang, K.; Wang, Z.; Deng, L.; Chen, Y., Bioinspired

Multifunctional

Melanin-Based

Nanoliposome

for

Photoacoustic/Magnetic Resonance Imaging-Guided Efficient Photothermal Ablation of Cancer. Theranostics 2018, 8, 1591-1606. (42)Smith, B. R.; Gambhir, S. S., Nanomaterials for in Vivo Imaging. Chem. Rev. 2017, 117, 901-986. (43)Angelique, L., Multimodality Imaging Probes: Design and Challenges. Chem. Rev. 2010, 110, 3146-3195. (44)Gao, S.; Wang, G.; Qin, Z.; Wang, X.; Zhao, G.; Ma, Q.; Zhu, L., OxygenGenerating

Hybrid

Nanoparticles

to

Enhance

Fluorescent/Photoacoustic/Ultrasound Imaging Guided Tumor Photodynamic Therapy. Biomaterials 2017, 112, 324-335. (45)Sheng, D.; Liu, T.; Deng, L.; Zhang, L.; Li, X.; Xu, J.; Hao, L.; Li, P.; Ran, H.; Chen, H.; Wang, Z., Perfluorooctyl Bromide & Indocyanine Green Co-Loaded Nanoliposomes for Enhanced Multimodal Imaging-Guided Phototherapy. Biomaterials 2018, 165, 1-13. (46)Weber, J.; Beard, P. C.; Bohndiek, S. E., Contrast Agents for Molecular Photoacoustic Imaging. Nat. Methods 2016, 13, 639-650. (47)Miao, Q.; Lyu, Y.; Ding, D.; Pu, K., Semiconducting Oligomer Nanoparticles as an Activatable Photoacoustic Probe with Amplified Brightness for in Vivo Imaging of Ph. Adv. Mater. 2016, 28, 3606-3606. (48)Wang, L. V.; Hu, S., Photoacoustic Tomography: In Vivo Imaging from Organelles to Organs. Science 2012, 335, 1458-62. (49)Min, H. S.; Son, S.; Dong, G. Y.; Lee, T. W.; Lee, J.; Lee, S.; Ji, Y. Y.; Lee, J.; Han, M. H.; Park, J. H., Chemical Gas-Generating Nanoparticles for Tumor26


Page 27 of 40 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


Targeted Ultrasound Imaging and Ultrasound-Triggered Drug Delivery. Biomaterials 2016, 108, 57-70. (50)Luke, G. P.; Hannah, A. S.; Emelianov, S. Y., Super-Resolution Ultrasound Imaging in Vivo with Transient Laser-Activated Nanodroplets. Nano Lett. 2016, 16, 2556. (51)Chen, J.; Luo, H.; Liu, Y.; Zhang, W.; Li, H.; Luo, T.; Zhang, K.; Zhao, Y.; Liu, J. Oxygen-Self-Produced Nanoplatform for Relieving Hypoxia and Breaking Resistance to Sonodynamic Treatment of Pancreatic Cancer. Acs Nano 2017, 11, 12849-12862. (52)Li, Y.; Zhou, Q.; Deng, Z.; Pan, M.; Liu, X.; Wu, J.; Yan, F.; Zheng, H. IR-780 Dye as a Sonosensitizer for Sonodynamic Therapy of Breast Tumor. Sci. Rep. 2016, 6, 25968. (53)Shi, S.; Liu, Y.; Chen, Y.; Zhang, Z.; Ding, Y.; Wu, Z.; Yin, J.; Nie, L. Versatile pH-response Micelles with High Cell-Penetrating Helical Diblock Copolymers for Photoacoustic Imaging Guided Synergistic Chemo-Photothermal Therapy. Theranostics 2016, 6, 2170-2182. (54)Cheng, Y.; Cheng, H.; Jiang, C.; Qiu, X.; Wang, K.; Wei, H.; Yuan, A.; Wu, J.; Hu, Y. Perfluorocarbon Nanoparticles Enhance Reactive Oxygen Levels and Tumour Growth Inhibition in Photodynamic Therapy. Nat. Commun. 2015, 6, 8785. (55)Tan, X.; Luo, S.; Long, L.; Wang, Y.; Wang, D.; Fang, S.; Ouyang, Q.; Su, Y.; Cheng, T.; Shi, C. Structure-Guided Design and Synthesis of a MitochondriaTargeting Near-Infrared Fluorophore with Multimodal Therapeutic Activities. Adv. Mater. 2017, 29, 1704196. (56)Zhang, L.; Wang, D.; Yang, K.; Sheng, D.; Chen, Y., Mitochondria-Targeted Artificial “Nano ‐ Rbcs” for Amplified Synergistic Cancer Phototherapy by a Single Nir Irradiation. Adv. Sci. 2018, 5, 1800049. (57)Khan, M. S.; Hwang, J.; Lee, K.; Choi, Y.; Jang, J.; Kwon, Y.; Hong, J. W.; Choi, J. Surface Composition and Preparation Method for Oxygen Nanobubbles for Drug Delivery and Ultrasound Imaging Applications. Nanomater. 2019, 9, 48. 27



(58)Khan, M. S.; Hwang, J.; Seo, Y.; Shin, K.; Lee, K.; Park, C.; Choi, Y.; Hong, J. W.; Choi, J. Engineering Oxygen Nanobubbles for the Effective Reversal of Hypoxia. Artif. Cells Nanomed. Biotechnol. 2018, 23, 1-10. (59)Li, S.; Johnson, J.; Peck, A.; Qian, X. Near infrared Fluorescent Imaging of Brain Tumor with IR780 Dye Incorporated Phospholipid Nanoparticles. J. Transl. Med. 2017, 15, 18. (60)Flors, C.; Fryer, M. J.; Waring, J.; Reeder, B.; Bechtold, U.; Mullineaux, P. M.; Nonell, S.; Wilson, M. T.; Baker, N. R. Imaging the Production of Singlet Oxygen in Vivo Using a New Fluorescent Sensor, Singlet Oxygen Sensor Green®. J. Exp. Bot. 2006, 57, 1725-1734. (61)Markovitz-Bishitz, Y.; Tauber, Y.; Afrimzon, E.; Zurgil, N.; Sobolev, M.; Shafran, Y.; Deutsch, A.; Howitz, S.; Deutsch, M. A Polymer Microstructure Array for the Formation, Culturing, and High Throughput Drug Screening of Breast Cancer Spheroids. Biomaterials 2010, 31, 8436-8444. (62)Murphy, M. P. Understanding and Preventing Mitochondrial Oxidative Damage. Biochem. Soc. Trans. 2016, 44, 1219-1226. (63)Smith, R. A.; Murphy, M. P. Animal and Human Studies with the MitochondriaTargeted Antioxidant MitoQ. Ann. N. Y. Acad. Sci. 2010, 1201, 96-103. (64)Zorov, D. B.; Juhaszova, M.; Sollott, S. J. Mitochondrial Reactive Oxygen Species (ROS) and ROS-Induced ROS Release. Physiol. Rev. 2014, 94, 909-950. (65)Xin, S.; Xu, H.; Jing, S.; Guo, S.; Shi, S.; Dan, J.; Fang, T.; Tian, Y.; Ye, T. RealTime Detection of Intracellular Reactive Oxygen Species and Mitochondrial Membrane Potential in THP-1 Macrophages During Ultrasonic Irradiation for Optimal Sonodynamic Therapy. Ultrason. Sonochem. 2015, 22, 7-14. (66)Chung-Hsin, W.; Shih-Tsung, K.; Ya-Hsuan, L.; Yun-Ling, L.; Yu-Fen, H.; ChihKuang, Y. Aptamer-Conjugated and Drug-Loaded Acoustic Droplets for Ultrasound Theranosis. Biomaterials 2012, 33, 1939-1947.

28


Page 28 of 40

Page 29 of 40 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


Scheme 1. Schematic illustration of the theranostic functions of as-synthesized USresponsive nanodroplets for efficient SDT, including tumor cell/mitochondria-targeting ability, deep penetration, ADV and guidance/monitoring by multimodal (US, PA and FL) imaging.

29



Figure 1. (a) Photographs of NDs and IR780-NDs dispersed in phosphate buffered saline (PBS). (b) Light microscope image of IR780-NDs. (c) TEM image of IR780NDs (scale bar: 0.2 μm). (d) Size distribution of NDs and IR780-NDs as measured by DLS. (e) UV–vis absorbance spectra of IR780 at elevated concentrations. (f) Absorbance spectra of IR780, NDs, and IR780-NDs, as recorded by a UV–vis spectrophotometer.

30


Page 30 of 40

Page 31 of 40 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) Light microscope images of phase transformation by ADV (scale bar = 50 μm). (b) The US images (B-mode and contrast-enhanced ultrasound (CEUS) mode) (b) and corresponding quantitative analysis (c) of IR780-NDs after US irradiation at different intensities. (d) In vitro PA images and PA values of IR780-NDs at different IR780 concentrations. (e) Time-dependent 1O2 generation of IR780-NDs as irradiated by US (2.4 W/cm2). The concentration of IR780 was 5 μg/mL.

31



Figure 3. (a) The model of intertissue NDs movement after US stimulation. (b) Representative FL microscopy images of the nanoparticles’ distribution in histological sections of the ADV (NDs + US), US (PFOB-based liposomes + US) and control (NDs Only) groups (green fluorescence: tumor vasculature, red fluorescence: nanoparticles; scale bar = 200 µm). (c) The corresponding relative FL signal of nanoparticles from the vasculature towards the tumor center.

32


Page 32 of 40

Page 33 of 40 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 4. Intracellular uptake and penetration. (a) Intracellular uptake of NDs and IR780-NDs observed by CLSM after various intervals of coincubation. The scale bars are 25 μm. (b) FL intensities of 4T1 cells incubated with NDs or IR780-NDs for different durations. (c) 3D reconstruction of the 4T1 spheroid models incubated with IR780-NDs or NDs. (d) Quantitative analysis of the penetration of NDs and IR780NDs.

33



Figure 5. (a) Mitochondrial location of NDs and IR780-NDs as monitored by MitoTracker. The scale bars are 25 μm. (b) Confocal images of DCFH-DA-stained 4T1 cells subjected to various treatments and positive control. The scale bars are 50 μm.

34


Page 34 of 40

Page 35 of 40 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. In vitro sonotoxicity of IR780-NDs to 4T1 cells. (a) Relative cell viability of 4T1 cells after SDT with different IR780 concentrations and different US irradiation times. (Values are the mean ± s.d., n = 5). (b) Flow cytometry analysis of tumor cell apoptosis and necrosis after SDT for 90 s at different IR780 concentrations (0, 1, 2, 3, 4 and 5 μg/mL). (c) Confocal images of CAM and PI costained 4T1 cells after various treatments. The scale bars are 100 μm.

35



Figure 7. Biodistribution of IR780-NDs. (a) In vivo FL images of tumors in 4T1 tumorbearing mice after intravenous injection of IR780-NDs at different time points. (b) Changes of FL signal intensities at tumor regions at the corresponding time points. (c) Ex vivo FL images of major organs and tumors dissected from mice 24 h post injection of IR780-NDs. (d) Quantitative biodistribution analysis of IR780-NDs in mice. (e) Distribution of IR780-NDs and NDs in tumor tissue and the major organs. (f) Quantitative biodistribution of IR780-NDs and NDs in mice. (Values are the mean ± s.d., n = 3).

36


Page 36 of 40

Page 37 of 40 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 8. Enhanced in vivo US/PA imaging. (a) B-Mode and CEUS mode imaging before and after US irradiation. (b) Corresponding echo intensities of tumors. (c) In vivo PA images of tumors after intravenous injection of IR780-NDs at different time points. (d) Changes of PA signal intensities at the tumor regions at the corresponding time points. (Values are the mean ± s.d., n = 3).

37



Figure 9. (a) Photographs of 4T1 tumor-bearing mice over a 16-d period after various treatments. (b) Tumor growth curves after various treatments (Values are the mean ± s.d., n = 5, *P < 0.05). (c) Weight of tumors 16 d post various treatments (Values are the mean ± s.d., n = 5, *P < 0.05).

38


Page 38 of 40

Page 39 of 40 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 10. (a) H&E, TUNEL and PCNA staining of tumor sections after various treatments. (b) H&E staining of the major organs (heart, liver, spleen, lung and kidney) of 4T1 tumor-bearing mice after different treatments. The scale bars are 50 μm.

39



TOC graphic

40


Page 40 of 40

Mitochondria-Targeted and Ultrasound-Activated ... - ACS Publications

Recommend Documents