Phase Transition Nanoparticles as Multimodality Contrast Agents for

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Phase Transition Nanoparticles as Multimodality Contrast Agents for the Detection of Thrombi and Targeting Thrombolysis: in vitro and in vivo experiments Jie Xu, Jun Zhou, Yixin Zhong, Yu Zhang, Jia Liu, Yuli Chen, Liming Deng, Danli Sheng, Zhigang Wang, Haitao Ran, and Dajing Guo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12689 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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ACS Applied Materials & Interfaces

Phase Transition Nanoparticles as Multimodality Contrast Agents for the Detection of Thrombi and Targeting Thrombolysis: In Vitro and In Vivo Experiments Jie Xu,† Jun Zhou,†Yixin Zhong,† Yu Zhang,† Jia Liu,† Yuli Chen,‡ Liming Deng, ‡ Danli Sheng, ‡ Zhigang Wang, ‡ Haitao Ran,‡ and Dajing Guo*,†

†

Department of Radiology, the Second Affiliated Hospital of Chongqing Medical

University, No. 74 Linjiang Rd, Yuzhong District, Chongqing, 400010, P. R. China

‡

Institute of Ultrasound Imaging, Department of Ultrasound, the Second Affiliated

Hospital of Chongqing Medical University, No. 74 Linjiang Rd, Yuzhong District, Chongqing, 400010, P. R. China

Abstract Thrombotic disease is extremely harmful to human health, and early detection and treatment can improve the prognosis and reduce mortality. Multimodal molecular imaging can provide abundant information about thrombi, but to date, few studies have used multimodal and multifunctional nanoparticles (NPs) for thrombus detection and targeting thrombolysis. In this study, phase transition multimodal and multifunctional NPs (EWVDV-Fe-Ink-PFH NPs) were constructed for the first time using a three-step emulsification and carbodiimide method, and the physical and chemical properties of the NPs were investigated. The targeting abilities of the NPs and multimodal imaging, i.e., photoacoustic, magnetic resonance and ultrasound imaging, were successfully achieved in vitro and in vivo. The ability of the EWVDV

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on the NPs to effectively target the P-selectin of thrombi was confirmed by multimodal imaging and pathology, and the penetration depths of the NPs into the thrombi were far deeper than the previously reported depths. Moreover, a perfluorohexane (PFH) phase transition induced by low-intensity focused ultrasound (LIFU) irradiation enabled the EWVDV-Fe-Ink-PFH NPs to cause thrombolysis in vitro. In summary, EWVDV-Fe-Ink-PFH NPs are a theranostic contrast agent that will provide a simple, effective, and noninvasive approach for the diagnosis and treatment of thrombosis.

Keywords: perfluorohexane, EWVDV peptide, thrombosis, multimodal imaging, phase transition

1. Introduction Thrombosis, which is a leading cause of myocardial infarction, ischemic stroke, pulmonary embolism, and other various disorders, is a major cause of morbidity and mortality. The early detection and effective evaluation of thrombus formation is clinically urgent for both diagnosis and therapy, but it remains practically challenging. Various imaging modalities, such as ultrasound (US), nuclear medicine, magnetic resonance imaging (MRI), computed tomography angiography (CTA) and digital subtraction angiography (DSA), cannot readily detect early-stage thrombi or provide molecular and/or cellular information about thrombus composition, which is conventionally visualized only by pathological examination upon autopsy or biopsy.


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Molecular imaging is an attractive technology that greatly enhances our understanding of the pathophysiology and treatment of various types of thrombi, especially early-stage thrombi. Molecular imaging is a novel, multidisciplinary technique that can be defined as the real-time visualization and in vivo characterization and qualification of biological processes at the molecular and cellular levels.1,

2

A variety of single-mode molecular probes have been reported in the

literature.3-9 In our previous studies, an MRI molecular probe loaded with cRGD, Fe3O4 and a thrombolytic drug (rtPA) was synthesized to both target and treat thrombi.8 Further assessments enabled improvements. Because each single imaging modality exhibits unique advantages along with intrinsic limitations, such as insufficient sensitivity or spatial resolution, acquiring accurate and reliable information about early thrombosis is difficult. To overcome many of these limitations, multimodal imaging can be employed. The combination of multiple imaging modalities can yield complementary diagnostic information and offer synergistic advantages over single modalities. Therefore, multimodal imaging is theoretically an ideal candidate for the detection and localization of thrombi. A large number of tumor-associated multimodal molecular probes has been reported, 10, 11 but research about the multimodal imaging of thrombi is limited, and these multimodal molecular probes lack intrinsic therapeutic properties and only function as diagnostic carriers.12-14 Recently, a bimodal molecular probe

was reported for the

fluorescence/photoacoustic imaging of thrombi and the suppression of thrombus formation.15 However, to the best of our knowledge, three-mode molecular probes



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with thrombolytic functionality for the detection of thrombosis and thrombolysis have not yet been reported in the literature. Therefore, in this study, we envisaged the integration of diagnostic and therapeutic multifunctional NPs to detect and destroy thrombi. Safe and effective thrombolysis methods have been pursued by both doctors and researchers. Pharmacological thrombolysis, such as that mediated by the use of anti-platelet or fibrinolytic drugs, is associated with several side effects, such as potentially fatal hemorrhagic complications and neurotoxicity, among which increased bleeding is the main cause of mortality.16 Recently, phase-transitional agents were successfully introduced to US molecular imaging to provide a platform with desirable US contrast properties together with a potential therapeutic functionality in cancer therapy due to the rapid and large increase in volume as the droplets transform into gas bubbles.17-20 Liquid fluorocarbon was selected as the ultrasonic material to obtain nanomolecular probes.18 However, to the best of our knowledge, PFH droplets have not yet been used as an US imaging agent for thrombi in thrombotic diseases. In our study, the NPs were expected to penetrate the thrombi to generate an US image due to the “liquid-to-gas” phase transition of PFH. Further, the phase transition produces a series of biological effects (such as sonoporation and cavitation effects)

21, 22

to achieve both the destruction of the thrombus and the

integration of diagnosis and treatment. Once coupled to an effective phase transition agent, such NPs could supply an improved clinical diagnoses and therapeutic effects.


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Targeted delivery can increase the bioavailability of NPs to thrombi while reducing the drug dosage and the risk of bleeding. Platelet membrane glycoprotein GP IIb/IIIa4, 7-8, 14, 23 and P-selectin24-25 have attracted widespread attention as targets for activated platelets. In our previous studies, we reported that NPs coupled to RGDS and cRGD target glycoprotein GP IIb/IIIa on activated platelets in vitro and in vivo. 8-9, 26

The short expression time of GPIIb/IIIa27 restricted its application for targeting

various types of thrombi, and the ability of these NPs to target thrombi in vivo requires improvement, especially under the high shear stress of arterial blood flow. For P-selection, researchers have identified high expression levels of P-selectin on activated platelets28 with an expression time that is longer than that of GPIIb/IIIa.27 P-selectin exhibits a persistently high expression on each platelet membrane surface of 10,000 P-selectin molecules. P-selectin mediates leukocyte rolling on vascular surfaces through the targeting effect of P-selectin and its ligand.27 Therefore, we assume that P-selectin can mediate molecular probe rolling and adhesion on vascular surfaces via the targeting of its ligand and thus provide an ideal target for multifunctional probes. The short peptide EWVDV has a high affinity and specificity for P-selectin. Gallic-acid-modified EWVDV (GA-EWVDV) exhibits an 800-fold higher affinity than EWVDV without gallic acid.29 Therefore, GA-EWVDV was selected as the targeting component of the probe because it can resist the high shear force due to blood flow and maintain the ability to effectively target thrombi in vivo. Additionally, P-selectin plays an important role in thrombosis, and P-selectin inhibitors29-33 are being extensively studied as antithrombotic drugs. In theory, the



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GA-EWVDV on our NPs can also occupy the binding sites of platelets and fibrin to play an anti-thrombosis role. By hindering thrombus re-expansion to some extent, GA-EWVDV also has a therapeutic effect. In the present study, to solve the problem of poor targeting and limited information about thrombi in single-mode molecular imaging, we selected poly (lactic-co-glycolic acid) (PLGA) as the carrier and Fe3O4, PFH, and India ink as the imaging agents. A three-step process of emulsification and conjugation to the GA-EWVDV peptide yielded thrombus-targeting NPs for multimodal imaging and thrombolytic therapy (Figure 1). The physical and chemical properties of the NPs were characterized, and we confirmed the targeting abilities and effects of photoacoustic (PA), MR and US imaging in vitro and in vivo. Based on the phase transition performance of PFH, the thrombus tissue structure was simultaneously destroyed. To the best of our knowledge, this is the first time that phase transition technology has been used for thrombolytic therapy while being monitored under multimodal imaging. Thus, these NPs could be applied for both these purposes and thereby provide a simple, effective, and noninvasive approach to diagnosing and treating thrombosis.

2. Results and Discussion 2.1. Characteristics of the NPs The EWVDV-Fe-Ink-PFH NPs produced a light gray emulsion when dissolved in double-distilled water, and they had relatively homogeneous sizes, smooth surfaces


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and good dispersion as observed by optical microscopy (Figure 2a). Scanning electron microscopy (SEM) at different magnifications revealed that the NPs were regular and spherical with smooth surfaces and uniform sizes (Figure 2b). Transmission electron microscopy (TEM) revealed that the lipophilic iron oxide particles modified with oleic acid were relatively uniformly distributed in the NP shell, which better prevented burst release than did the hydrophilic substances encapsulated inside the NPs (Figure 2c). Fluorescence microscopy revealed red fluorescent materials around the NPs, indicating that the short peptide EWVDV with rhodamine was successfully linked to the surfaces of the Fe-Ink-PFH NPs (Figure 2d), and flow cytometry revealed that the carrier rate of EWVDV labeled with rhodamine was 63.48 %. Currently, commercially available US contrast agent particle size is on the micron level.34-35 Large particles are easily captured by the reticuloendothelial systems (RES), which shortens the blood circulating duration and reduces their accumulation within lesions.18 In contrast, small particles can easily penetrate the internal region of a thrombus, but difficulties associated with phase transitions increase if the particles become too small. Taking these factors into account, we designed the size of EWVDV-Fe-Ink-PFH NPs to be 387.1±129.1 nm, which was suitable for thrombus imaging in the blood pool. The polydispersity index (PDI) was 0.09 ± 0.032, which indicated that the NPs were well dispersed (Figure 2e), and the zeta potential was -23.2 ± 3.4 mV (Figure 2f) as detected using a laser particle size analyzer. The Fe content was found to be 4.29±0.27 µg Fe/mg PLGA using an atomic absorption spectrometer.

The

above

characteristics

of

the

NPs


illustrated

that

the


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EWVDV-Fe-Ink-PFH NPs were successfully constructed using the three-step emulsification process and carbodiimide method.

2.2. Multimodal Imaging Properties of the NPs This is the first study to demonstrate novel triple-modality imaging platelet-targeted NPs that allow for the combination of PA, MR, and US imaging modalities to obtain complementary information for the direct imaging of thrombi and targeting of thrombolysis. Loaded with different imaging materials, the EWVDV-Fe-Ink-PFH NPs display excellent properties for PA, MR and US imaging. As an optical absorber of the droplets, India ink has been extensively used in the clinic owing to its intrinsic properties, which include outstanding stability, easy accessibility, low cost and nontoxicity.36-37 India ink exhibits strong absorption in the visible and near-infrared regions. Owing to its large absorption coefficient, India ink can offer excellent PA contrast.38 Therefore, to exploit its broad optical absorption range, we selected India ink as the PA imaging material in this study. Additionally, as an MRI contrast agent, Fe3O4 can simultaneously produce PA signals.39-40 Therefore, we studied the difference between the PA intensities of Fe3O4 and India ink. As illustrated in Figure 3a, the absorption range of 50 % of the maximum PA intensities of the ink NPs was 680-880 nm, which was significantly wider than that of NPs with only Fe (680-785 nm). The PA intensities were detected in three groups of NPs after irradiation, and the results are presented in Figure 3b. The ink NP groups (EWVDV-Ink-PFH NPs and EWVDV-Fe-Ink-PFH NPs) exhibited significantly higher PA intensities than the non-ink NPs (EWVDV-Fe-PFH NPs) (both P< 0.001),


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which indicated that the PA effect of the India ink in this experiment was superior to that of Fe3O4 at this dose. Moreover, the PA intensities of the Fe and ink NPs (EWVDV-Fe-Ink-PFH NPs) were slightly better than those of the NPs with only ink (EWVDV-Ink-PFH NPs; P< 0.05; Figure 3c). This difference may have been caused by the synergistic effect of the India ink and Fe3O4 with a 706-nm laser. With a wide light absorption range and good PA imaging capability, the Fe-Ink NPs were excellent candidates for PA imaging that might be able to expand the clinical prospects of EWVDV-Fe-Ink-PFH NPs. In addition to being used as PA materials, the Fe NPs were able to reduce the T2*-weighted signal intensity. MR scans revealed that the T2*-weighted signal intensity of the EWVDV-Fe-Ink-PFH NPs was reduced to varying degrees relative to those of the double-distilled water and EWVDV-Ink-PFH NPs (Figure 3d). The signal intensities of the images

were dependent on the concentration

of the

EWVDV-Fe-Ink-PFH NPs and decreased as the NP concentrations increased. There was no significant difference between the signal intensities of the EWVDV-Ink-PFH NPs and double-distilled water (P>0.05), which demonstrated that the non-Fe NPs did not affect the MR signal intensity. Based on the validation curves (Figure 3e), the R2* values increased with increasing Fe concentration, which is consistent with the results of our previous study.8 These findings indicated that the EWVDV-Fe-Ink-PFH NPs could serve as MR contrast agents to effectively reduce the T2*-weighted signal intensity.



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After irradiation with low-intensity focused ultrasound (LIFU), phase transition microbubbles and US signals were not detected in the non-PFH NPs group (EWVDV-Fe-Ink NPs). White phase transition microbubbles with a uniform size were found in the PFH NPs (EWVDV-Fe-PFH NPs and EWVDV-Fe-Ink-PFH NPs) groups at the top of a gel model, and US signals were detected in the B and contrast modes. US images of each group are presented in Figure 3f. The acoustic intensities of the PFH NPs were significantly higher than those of the non-PFH NPs (B mode and contrast mode: all P0.05), which indicated that India ink did not affect the PFH phase transition (Figure 3g). These results demonstrated that LIFU was an effective method for inducing the PFH NP phase transition, which is the basis of the US imaging of thrombolysis.

2.3. Targeted Thrombolysis in vitro Effectiveness and safety are two main considerations for thrombolysis. Regarding effectiveness, abundant holes formed inside the section of the thrombus with PFH NPs, and the sizes of the holes ranged from 15 µm to 130 µm, while no specific holes were found in the non-PFH NP group (Figure 4). As expected, our design of smaller nanoscale NPs resulted in the delivery of more NPs into the thrombus and greater accumulation per unit volume to achieve the "internal bombing" the thrombus. The


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weight of the thrombi in the non-PFH NPs group (EWVDV-Fe-Ink NPs) was reduced by 11 %, and the edges of the thrombi were loose, which indicated that US combined with non-PFH NPs had a slight thrombolytic effect, which is consistent with the results of previous studies.41-42 In the PFH NP group (EWVDV-Fe-Ink-PFH NPs), massive microbubbles adhered to the surfaces of the thrombi. Their weight was reduced by 27 %, and the difference was significant compared with those treated with the non-PFH and PFH NPs (P

Phase Transition Nanoparticles as Multimodality Contrast Agents for

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