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Low-Intensity Focused Ultrasound Responsive PhaseTransitional Nanoparticles for Thrombolysis without Vascular Damage: A Synergistic Nonpharmaceutical Strategy Yixin Zhong, Yu Zhang, Jie Xu, Jun Zhou, Jia Liu, Man Ye, Liang Zhang, Bin Qiao, Zhi-Gang Wang, Hai-Tao Ran, and Dajing Guo ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09277 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 11, 2019

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Low-Intensity Focused Ultrasound Responsive Phase-Transitional Nanoparticles for Thrombolysis without Vascular Damage: A Synergistic Nonpharmaceutical Strategy Yixin Zhong,† ‡ Yu Zhang,† ‡ Jie Xu,† Jun Zhou,† Jia Liu,† Man Ye,† Liang Zhang,‡ Bin Qiao,‡ Zhi-gang Wang,‡ Hai-tao Ran,*,‡ Dajing Guo*,†

†Department

of Radiology, Second Affiliated Hospital of Chongqing Medical University,

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

‡Chongqing

Key Laboratory of Ultrasound Molecular Imaging & Department of

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

Corresponding authors E-mail: [email protected] (D.G.); [email protected] (H.R.)

ABSTRACT: Multimodal molecular imaging has shown promise as a complementary approach to thrombus detection. However, the simultaneous noninvasive detection and lysis of thrombi for cardiovascular diseases remain challenging. Herein, perfluorohexane

ACS Paragon Plus Environment

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(PFH)-based

biocompatible

nanostructure

was

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fabricated,

namely,

as-prepared

Fe3O4-poly (lactic-co-glycolic acid)-PFH-CREKA nanoparticles (NPs), which combine phase transition (PT) thrombolysis capabilities with properties conducive to multimodal imaging. This well-developed PT agent responded effectively to low-intensity focused ultrasound (LIFU) by triggering the vaporization of liquid PFH to achieve thrombolysis. The presence of the CREKA peptide, which binds to the fibrin of the thrombus, allows targeted imaging and efficacious thrombolysis. Then, we found that, compared with thrombolysis using a non-phase-transition (NPT) agent, PT thrombolysis can produce a robust decrease in the thrombus burden regardless of the acoustic power density of LIFU. In particular, the reduced energy for LIFU-responsive PT during the lysis process guarantees the superior safety of PT thrombolysis. After injecting the NPs intravenously, we demonstrated that this lysis process can be monitored with ultrasound and photoacoustic imaging in vivo to evaluate its efficacy. Therefore, this nonpharmaceutical strategy departs from routine methods and reveals the potential use of PT thrombolysis as an effective and noninvasive alternative to current thrombolytic therapy.

Key words: thrombolysis, CREKA peptide, low-intensity focused ultrasound, phase transition, multimodal imaging Thrombosis is the main pathogenesis of acute ischemic cardiovascular disease and the leading cause of unstable angina, myocardial infarction and stroke.1,2 For decades,


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patients with thrombosis have been treated primarily with recombinant tissue plasminogen activator (rtPA), which is conducive to the dissolution of blood clots when given within 4.5 h of ischemic stroke onset; however, this restrictive time window limits its clinical application, and even intra-arterial administration of rtPA can expand the time window at most to 6 h.3 Meanwhile, when rtPA treatment is used beyond the time window of thrombolysis4,5 or when the balance of the blood-brain barrier is disrupted by rtPA,6 life-threatening symptomatic intracranial hemorrhages and neurotoxicity can occur, increasing the risk of rtPA in clinical therapy. Thus, it is necessary to develop a cost-effective, dose-controlled and targeted-delivery approach to treating thrombotic diseases and explore strategies for amplifying the effect of rtPA to address the concerns described above. Thus far, many experimental studies have attempted to optimize drug delivery systems to ensure rtPA efficacy at the site of the thrombus.7 Typically, drug delivery nanocarriers, such as magnetic nanomotors,8 liposomes,9 and synthetic polymers,10 are used for drug release via different mechanisms in lysis therapy. These methods can hold great promise for delivering the encapsulated drug to the desired site, resulting in an increased therapeutic benefit and the alleviation of side effects. Regardless of the position where rtPA is loaded in the nanocarrier,10,11 the integration of rtPA with a nanocarrier can affect the activation of plasminogen to plasmin due to the limited loading rate and unsatisfactory drug release of nanocarriers.11 Considering the above problems regarding


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drug delivery systems for thrombolysis, nonpharmaceutical thrombolysis strategies based on ultrasound (US),12 alternating magnetic fields,13 and lasers14 have been proposed in basic research with the hope of optimizing early recanalization rates or increasing the collateral blood supply to improve survival rates. Nevertheless, because of their potential mechanical damage and limited effect, these nonpharmaceutical methods do not fully satisfy the requirements of safety or thrombolytic effect. Therefore, modified approaches to promoting thrombolytic efficacy without causing proximal vascular or tissue damage need to be explored. Recent advances in nanobiotechnology and nanomaterials have demonstrated that a functional agent, perfluorohexane (PFH), holds great potential in imaging-based approaches via a "liquid-to-gas” phase transition (PT) in the tumor region.15-17 In contrast to conventional US microbubbles, PFH droplets, as a liquid phase, are generally enclosed in liposomes, poly (lactic-co-glycolic acid) (PLGA) or albumin shells18,19 to enhance their stability and avoid air embolism at the beginning of the administration. Furthermore, the small dimensions of PFH-containing nanoparticles (NPs) enhance their ability to permeate the internal regions of a tumor and elude capture by the mononuclear phagocyte system (MPS) to further prolong their circulation in the bloodstream.19 In contrast, when PFH-containing NPs are triggered by US to enter the gaseous phase, they possess significantly augmented US contrast due to their volume expansion, and this expansion


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process, along with the subsequent explosion of the NPs, eventually destroys the surrounding tissue. Such findings have inspired considerable reformation in thrombosis treatment, which often utilize urgent and expensive pharmaceuticals or interventional surgery to prevent thrombi from causing infarctions. Hence, we hope that PFH used with a low-intensity focused ultrasound (LIFU) device will serve as an alternative to current thrombolytic therapy for controlled local recanalization with superior convenience and without potential side effects. We preliminarily applied this PT method as a proof of concept to lyse blood clots, which was the first application in thrombolytic treatment. This approach depends largely on NPs that are based on PLGA, iron oxide, PFH and India ink and conjugated with EWVDV peptides; these NPs display a capacity for both multimodal imaging and controlled local mechanical thrombolysis.20 The discovery of cavities around the boundary of a blood clot is encouraging. However, it remains unclear that which mechanism the clots are dissolved by, and additional work is needed to demonstrate applications of this technique in practical scenarios. Notably, CREKA (Cys-Arg-Glu-Lys-Ala) peptide is an emerging pentapeptide that can bind to fibrin specifically.21 A recent study demonstrated the potential of CREKA to facilitate the specific targeting of NPs to fibrin and subsequently promote local accumulation and microthrombus detection.22 During thrombus formation, the structure of the thrombus changes, with cross-linked fibrin gradually trapping platelets or


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erythrocytes. In addition, fibrin not only plays a necessary role by enhancing the activity of rtPA but also acts as a substrate for plasmin. In particular, sonothrombolysis activates fibrinolysis by virtue of its mechanical effect.23,24 For these reasons, we selected CREKA peptide to target fibrin and postulated that this peptide would facilitate the use of these NPs for monitoring or amplifying the efficacy of thrombolysis. In this study, we formulated a versatile type of NP, namely, the as-prepared Fe3O4-PLGA-PFH-CREKA NP. This NP depends on the highly biocompatible compound PLGA as a vehicle. Through the use of ferric oxide, this NP can be applied for magnetic resonance (MR) and photoacoustic (PA) imaging. Additionally, we investigated this nonpharmaceutical treatment strategy of PT thrombolysis. Figure 1 schematically illustrates the process of disrupting a blood clot using these as-prepared targeted thrombolytic NPs. More importantly, building upon previous studies,20 we sought to investigate whether this fibrin-specific PT agent would respond well to LIFU in dissolving arterial thrombi and, subsequently, whether this promising type of NP could enable multimodal imaging for thrombolysis monitoring.


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Figure 1. Schematic representation of the PT thrombolysis process, wherein LIFU pulses are applied remotely from outside the blood vessel. RESULTS AND DISCUSSION Characteristics of the NPs. To generate the NPs for the targeting of thrombi, we used CREKA, a linear 5-amino-acid peptide, to bind to fibrin at the thrombus site. After 12 h of incubation, dehydration condensation occurs, and PLGA binds covalently to the CREKA peptide by forming an amide bond (Figure S1). Flow cytometry quantitatively confirmed that the conjugation rate of CREKA was a strikingly high 98.72 % (Figure S2) by demonstrating that 98.72 % of the 10,000 randomly selected NPs were changed in wavelength. Inverted fluorescence microscopy of the Fe3O4-PLGA-PFH-CREKA-FITC


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NPs demonstrated that the CREKA-FITC was successfully conjugated with the NPs (Figure

S3a).

Transmission

electron

microscopy

(TEM)

of

the

Fe3O4-PLGA-PFH-CREKA NPs and PLGA-PFH-CREKA NPs showed their spherical morphology and different structures, where oleic acid-modified iron oxide was distributed in the former NPs (Figure 2a), in contrast to the lack of iron oxide in the structure of the latter NPs (Figure 2b). Elemental line-scan mapping across the NPs revealed that the Fe and fluorine (F) were present in this nanoplatform (Figure 2c). The distribution of Fe3O4 within the NPs is clearly displayed by high-resolution TEM imaging, and the elemental mapping images of Fe and F demonstrated that the Fe3O4 and PFH were loaded in the NPs (Figure 2d). We expected that, for imaging applications, Fe3O4 embedded in the PLGA NPs could endow them with desirable imaging efficacy because of its nontoxicity and ability to modify reactive surfaces,25 thus making them a promising multimodal imaging agent for both PA and MR imaging to improve the early diagnosis of diseases.26 In addition, Fe3O4-PLGA-PFH-CREKA NPs with high water monodispersity and homogeneity (size of 311.3 ± 4.3 nm and polydispersity index [PDI] of 0.094 ± 0.048; Figure S3b) were successfully obtained. Since single-channel fluorescence did not effectively determine whether CREKA peptides were attached to the NPs, we decided to perform an enzymolysis assay to describe

the

CREKA

peptide

conjugation

in

detail.

The

prepared

Fe3O4-PLGA/DiI-PFH-CREKA-FITC NPs are displayed in Figure 2e. Then, we


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employed carboxypeptidase B to achieve the desirable result of hydrolysis in an alkaline environment. As expected, after 3 h of incubation with carboxypeptidase B, CREKA-FITC (green) decreased in signal intensity on fluorescence imaging (Figure 2f), suggesting that the CREKA-FITC peptides had already been hydrolyzed. In contrast, DiI-labeled NPs (red) were structurally similar to what was observed in the fluorescence image before enzymolysis. All these observations indicated that the CREKA peptides were successfully conjugated with Fe3O4-PLGA-PFH NPs. This feature forms the basis of the targeting ability of the NPs in the subsequent section. Compared with that of the non-Fe3O4 NPs, the mean diameter of the NPs with iron oxide was increased slightly (Figure S3c), indicating that iron was successfully embedded in the NPs and that the 10-nm iron oxide components influenced the size of the NPs. We also used Fe3O4-PLGA-CREKA NPs as the control, and the differences in the

above

properties

between

the

Fe3O4-PLGA-CREKA

NPs

and

Fe3O4-PLGA-PFH-CREKA NPs were not statistically significant. As shown in Figure S3d, the quantification of the iron carrier rate revealed no significant differences between the three groups of NPs (F=0.302, P>0.05), indicating that neither material loading nor peptide conjugation influenced the iron carrier rate. Interestingly, the negative charge of the nanocarriers decreased gradually when they were carrying the CREKA peptide (Figure S3e), which was similar to an observation from a previous study,22 further verifying the successful conjugation of peptides from another perspective. Meanwhile, no


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significant size or zeta potential change was observed during the period of 9 days (Figure S3f and S3g), which demonstrated the stability of the NPs.


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Figure

2.

Characteristics

of

the

NPs.

Representative

TEM

images

of

Fe3O4-PLGA-PFH-CREKA (a) and PLGA-PFH-CREKA NPs (b). (Scale bar: 1 μm for large view; 200 nm for small view.) (c) Elemental line-scan mapping of Fe3O4-PLGA-PFH-CREKA NPs. (d) High-resolution TEM image and the corresponding elemental mapping of Fe, F, and Fe+F combined. (Scale bar: 200 nm.) Fluorescence images of Fe3O4-PLGA/DiI-PFH-CREKA-FITC NPs before (e) and after (f) an enzymolysis assay. (Scale bar: 5 μm.) Multimodal imaging of the NPs in vitro. MRI was applied in the acute thrombus detection due to its outstanding spatial resolution. In addition, the use of molecular imaging in MRI allows for the direct detection of thrombus, and further improves the image quality.27 Therefore, to confirm the MRI properties of Fe3O4-PLGA-PFH-CREKA NPs, T2* images of various concentrations of NPs in agarose gel were recorded. As shown in Figure S4a, as the NP concentration increased, the MR signal steadily declined, suggesting that these Fe3O4-PLGA-PFH-CREKA NPs generated a high magnetic field gradient. The relaxation rate (R2*) of the Fe3O4-PLGA-PFH-CREKA NPs, which corresponds to the slope of the fitted line in Figure S4b, was calculated to be 50.98 mM−1 s−1 at increasing iron concentrations from 0 to 2.6286 mM; this rate was slightly higher than that of free iron oxide NPs28 and confirmed the outstanding MR properties of the Fe3O4-PLGA-PFH-CREKA NPs. The relaxation rate of free iron oxide is reported to


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be between 20 and 40 mM−1 s−1, which enables an MR signal decrease.29 Additionally, with the Fe3O4-PLGA-PFH NPs as the comparison group, similar phenomena were discovered, demonstrating that Fe3+ retains an MR contrast-enhancing property after conjugation with the CREKA peptide. As a result, we can extrapolate that conjugation with CREKA peptide would not influence the MR properties. The pseudocoloring of the MR images (Figure S4c), a mirror of the grayscale map (Figure S4d), also reflects the homogeneity of the relevant MR signal intensity. These results demonstrate that the Fe3O4-based NPs are a suitable MR contrast agent and have further potential for diagnosing thrombosis. In view of the potential use of Fe3O4-PLGA-PFH-CREKA NPs as a PA agent, the PA signal of the NPs in vitro was tested in aqueous solution at increasing iron concentrations from 0.1643 to 2.6286 mM (Figure S4e). As presented in Figure S4f, 680 nm is the optimal excitation wavelength, as it is the wavelength at which maximum PA absorbance occurs. Both iron oxide and India ink were selected as imaging agents for PA imaging in previous experiments because of their synergistic effect.20 This time, in order to acquire similar imaging results upon increasing the loading dose of PFH to further fortify the thrombolytic effect, we chose to use iron oxide alone as the PA imaging agent. By adding a quantity of iron oxide, we found that our NP formulation, which provided an improved linear correlation of PA signals with the increase in the NP concentrations (R2=0.9958) (Figure S4g), displayed behavior similar to that of mixed materials. We speculated that


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the simplified NP fabrication procedures could improve material utilization and thereby enhance the ability to conduct imaging in real time using the NPs. The PT of the PFH-loaded NPs might have contributed to the observed contrast-enhanced ultrasound (CEUS) signal or thrombolysis efficacy. Thus, we explored whether the Fe3O4-PLGA-PFH-CREKA or Fe3O4-PLGA-CREKA NPs intensified the contrast signal in vitro. As expected, the NPs loaded with PFH, which is known to be readily triggered by irradiation, gave rise to a strong signal during the course of vaporization. In contrast, the Fe3O4-PLGA-CREKA NPs did not generate CEUS signal in the irradiated areas. Crucially, no difference was observed in B-mode or CEUS imaging of the non-PFH-loaded group before or after irradiation, whereas the B-mode and CEUS image contrast intensities of the PFH-loaded group were increased 12 times and 6.6 times respectively, after 30 min of threshold irradiation; these increases are attributable to microbubble formation by LIFU irradiation (Figure 3a and 3b). Sensitivity to US was confirmed when vaporization occurred (Figure 3c). These phenomena support the feasibility of applying these NPs in multimodal imaging. Similarly, during the process of LIFU-responsive PT, we observed the variations in PFH-loaded NPs every 10 min via light microscopy. Unlike laser-responsive PT, which can induce thermal damage to adjacent tissue, the mildness of LIFU-responsive PT guarantees the safety of this process.16 In addition, LIFU was manifested as the most appropriate technique to transform droplets into bubbles, and importantly, this


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threshold-based process would provide a spatial and temporal trigger point for improved control over the transmission of thermal and mechanical energy remotely, contributing to the convenient and noninvasive nature of the entire therapeutic process.30,31 The volume of the NPs increased nearly 300-fold (from the nanometer scale to the micron scale) from their original size, which conferred on them the capacity for nonpharmaceutical thrombolysis (Figure 3d). As time elapsed, an increasing number of NPs vaporized; nonetheless, we noticed that the PT NPs would not explode immediately after inflating to a certain size. This relatively slow process may enhance microstreaming around the thrombus, possibly loosening the structure. At this point, additional PT NPs would permeate into the thrombus and continue disrupting the surrounding composition because of their targeting ability, improving the effect.32 Interestingly, although PFH-loaded NPs have always been regarded as a temperature-responsive nanoplatform, in our experiment, it was the acoustic droplet vaporization that rose temperature approximately 8°C instead of the thermal impact of US (Figure 3e), might resulting in the enhancement of plasminogen activity.23 However, there would not be so much local temperature increase in vivo because of the heat dispersion by the bloodstream. On the other hand, LIFU creates a lower thermal impact due to the low intensity, which is save enough for the treatment. Therefore, this local temperature increase would not be expected to cause thermal damage.33


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Figure 3. US imaging of the NPs in vitro. Quantitative echo intensity of Fe3O4-PLGA-PFH-CREKA and Fe3O4-PLGA-CREKA NPs on B-mode (a) and CEUS imaging (b) at different time points under LIFU irradiation. (c) Corresponding CEUS and B-mode images of the PT process of the Fe3O4-PLGA-PFH-CREKA NPs, as observed in


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vitro. (d) Light-microscopy images of the LIFU-responsive PT process at different time points. (Scale bar: 50 μm.) (e) The time course of the temperature change. Targeting ability in vitro and in vivo. Binding performance in vitro was investigated by inverted fluorescence microscopy of thrombus sections. After 2 h of incubation with the frozen thrombus sections, the attachment of Fe3O4-PLGA/DiI-PFH-CREKA NPs was significantly greater than that of Fe3O4-PLGA/DiI-PFH NPs, with the fluorescence signal appearing strong in the targeted group but dark in the nontargeted group (Figure S5). The specificity of the targeted NPs was also validated by an in vivo experiment in which an artery thrombus model was established and injected with Fe3O4-PLGA/DiI-PFH-CREKA or Fe3O4-PLGA/DiI-PFH NPs. In the targeted group, visualization by fluorescence imaging revealed the presence of the targeted NPs (red) and their distribution with most of the fibrin (green), which was derived from Alexa 488-conjugated human fibrinogen after thrombus formation.34 In the nontargeted group, however, the red fluorescence was observed not to distribute exactly with the fibrin (green) (Figure S6). These results demonstrate the potential targeting ability of our NPs. LIFU-responsive PT lysis in vitro. The thrombolytic effect was successfully verified by examination of hematoxylin and eosin (H&E)-stained slices or digital images. In the NPT group, there were no obvious changes in the thrombus edges after 2 h of sonothrombolysis (Figure 4a). Meanwhile, in the PT group, small holes were found on


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the boundaries of the thrombi (Figure 4b, yellow arrows). The diameters of these small holes were measured to be several hundred microns upon vaporization. The largest hole reached 300 microns and was significantly larger than those observed in vitro (Figure 3d). This result corroborated the hundredfold change in the diameter of our NPs during the liquid-to-gas transition. We presumed that when the PT NPs reached the expansion limit, the strong shear stress and microstreaming induced by the debris and liquid would combine to destroy the thrombus and expose its internal structure as the PT NPs burst. Furthermore, together with the sonoporation and cavitation effects of sonothrombolysis from LIFU, both can subsequently facilitate the penetration of more PT NPs into the thrombus and enhance the effect of thrombolysis. In the nontargeted group, although a small number of holes were observed in the H&E image (Figure 4c, yellow arrowhead), the amount was significantly smaller than that of the PT group. Next, we used the pharmaceutical thrombolytic agent rtPA as a standard of comparison for PT thrombolysis. Interestingly, no holes were found in the boundary of the thrombus after rtPA thrombolysis (Figure S7), demonstrating that its thrombolysis mechanism is different from that of our PT NPs. In gross photographs, the thrombi of the PT groups appeared to be smaller than those of the NPT, nontargeted group, and rtPA group, especially in the 4 time points within the first hour (Figure 4d). Moreover, geographic white plaque was observed on the surface of the thrombus (Figure 4d, yellow arrowhead), and the distribution of the plaque gradually increased over time.


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Additionally, in the 30× magnified images of PT group (Figure 4e), the geographic white plaque was demonstrated to be the expanded PT NPs, implying that the PT NPs improved the effect of thrombolysis with LIFU. The expanded NPs were also found in the nontargeted group (Figure 4e, yellow arrowhead), which was uncommon compared to PT group.

Figure 4. Representative H&E staining of blood clots after 2 h period of 1 W/cm2 LIFU irradiation with Fe3O4-PLGA-CREKA NPs (NPT group) (a), Fe3O4-PLGA-PFH-CREKA


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NPs (PT group) (b) and Fe3O4-PLGA-PFH NPs (nontargeted group) (c). The blood clots were obtained from the marginal ear arteries of New Zealand White rabbits. (Scale bar: 200 μm.) (d) The appearance of blood clots varies at different time points over a 2-h period of 1 W/cm2 LIFU exposure. Thrombolytic efficacy of PT group, NPT group, rtPA or nontargeted group. (Scale bar: 5 mm.) (e) Amplified digital pictures of thrombi of the PT, NPT, rtPA and nontargeted groups. (The magnification is 30×.) To further investigate the therapeutic efficacy of PT NPs, we compared the thrombus weights and thrombolysis rates of the four groups. All data followed the Gaussian distribution. In the PT and NPT groups, the total thrombus burden prior to therapy was not significantly different among the 6 subgroups (F=1.092, P=0.385), but after treatment, the total thrombus burden in the PT groups was lower than that in the NPT groups (Figure 5a). The rates of thrombolysis in the PT groups were higher than those in the NPT groups (Figure 5b). The application of one-way ANOVA indicated a statistically significant difference between the treatment groups in terms of the total thrombus burden and thrombolysis rate (df=5, P=0.0001), and subsequent Bonferroni post hoc statistical tests indicated statistically significant differences between three subgroups in both thrombus weight and thrombolytic rate (P

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