Peptide-Functionalized Phase-Transformation Nanoparticles for Low

Nano Lett. , 2018, 18 (3), pp 1831–1841. DOI: 10.1021/acs.nanolett.7b05087. Publication Date (Web): February 8, 2018. Copyright © 2018 American Che...
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Letter Cite This: Nano Lett. 2018, 18, 1831−1841

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Peptide-Functionalized Phase-Transformation Nanoparticles for Low Intensity Focused Ultrasound-Assisted Tumor Imaging and Therapy LeiLei Zhu,†,‡ HongYun Zhao,‡ ZhiYi Zhou,‡ YongHong Xia,‡ ZhiGang Wang,†,‡ HaiTao Ran,†,‡ Pan Li,†,‡ and JianLi Ren*,†,‡ †

Ultrasound Department of the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China Chongqing Key Laboratory of Ultrasound Molecular Imaging, Chongqing 400010, China

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S Supporting Information *

ABSTRACT: In this study, we successfully developed novel tumor homing-penetrating peptide-functionalized drug-loaded phase-transformation nanoparticles (tLyP-1-10-HCPT-PFP NPs) for low intensity focused ultrasound (LIFU)-assisted tumor ultrasound molecular imaging and precise therapy. With the nanoscale particle size, tLyP-1-10-HCPT-PFP NPs could pass through the tumor vascular endothelial cell gap. Induced by tLyP-1 peptide with targeting and penetrating efficiency, tLyP-1-10HCPT-PFP NPs could increase tumor accumulation and penetrate deeply into the extravascular tumor tissue, penetrating through extracellular matrix and the cellular membrane to the cytoplasm. With LIFU assistance, tLyP-1-10-HCPT-PFP NPs could phase-transform into microbubbles and enhance tumor ultrasound molecular imaging for tumor diagnosis. Furthermore, after further irradiation by LIFU, an intracellular “explosion effect” caused by acoustic droplet vaporization, ultrasound targeted microbubble destruction, and release of 10-HCPT could realize physicochemical synergistic antitumor therapy. KEYWORDS: Tumor homing-penetrating peptide, phase-transformation, low intensity focused ultrasound, ultrasound molecular imaging, precise therapy

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membrane through sonoporation, effectively facilitating intracellular transfer of macromolecules.3,17 However, UTMD is passive targeting, and microbubbles in the sound field were all destroyed by UTMD, which did not achieve the precise location in the true sense. Therefore, to enhance ultrasound imaging and achieve precisely targeted microbubble destruction, our lab invented low intensity focused ultrasound (LIFU). Integrating diagnosis, treatment, monitoring, and effect evaluation, LIFU realized imaging and targeted drug release at the same time. LIFU could precisely focus on tumor tissue, which reduced nonspecific phase-transformation and destruction in normal tissues. Compared with high intensity focused ultrasound (HIFU), acoustic intensity in a focal spot of LIFU was only 0.4−3.2 W/cm2, which further prevented damage to the surrounding normal tissues. Additionally, tumor-targeting delivery systems have been developed for precise tumor targeting, increased tumor accumulation, and enhanced cancer therapy.18−20 For the targeting effect to be enhanced further, ligands characterized by specific targeting effects were modified onto the surface of a

ltrasound molecular imaging has made remarkable progress over the past decade, and recent studies have shown that ultrasound contrast agents not only enhance the imaging for diagnosis but also serve as vehicles to carry drugs or genes for therapy.1−5 Although traditional organic microbubbles can significantly enhance the echo signals of ultrasonography due to the fact that they can function as reflectors for the ultrasound by the resonance at the diagnostic frequency,6,7 their extremely large particle sizes (typically several micrometers) restrict them only to image in the blood pool because only particle sizes of less than 700 nm can infiltrate the large pores that are present in the leaky vasculature of tumors.8,9 Comparatively, nanosized bubbles only have very limited contributions for improving contrast-enhanced ultrasound imaging due to the substantial decrease of the nonlinear backscattering with the reduction of their diameters.10−12 Fortunately, nanoparticles (NPs) or microspheres with perfluorocarbon can go through phase-transformation to microbubbles via acoustic droplet vaporization (ADV) technology, which was first proposed by Kripfgans et al.13 Furthermore, the technique of ultrasound targeted microbubble destruction (UTMD) for drug or gene delivery has shown an attractive application in disease treatment, especially in cancer therapy.13−16 UTMD increases the permeability of the cell © 2018 American Chemical Society

Received: December 3, 2017 Revised: January 31, 2018 Published: February 8, 2018 1831

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Figure 1. Schematic diagram of the study. tLyP-1-10-HCPT-PFP NPs consist of four parts: (1) liquid core composed of pefluoropentane (PFP), (2) lipid shell consisted of biocompatible DSPE-PEG3400-tLyP-1, DPPG, DPPC, and cholesterol, (3) 10-HCPT loaded in the phospholipid bilayer of the lipid shell, and (4) tumor homing-penetrating peptide tLyP-1 connected to DSPE-PEG3400-mal present on the outer shell of nanoparticles. With nanoscale particle size, tLyP-1-10-HCPT-PFP NPs can pass through the tumor vascular endothelial cell gap. Induced by tLyP-1 peptide with targeting and penetrating efficiency, tLyP-1-10-HCPT-PFP NPs can increase tumor accumulation and penetrate deeply into the extravascular tumor tissue, penetrating through extracellular matrix and the cellular membrane to the cytoplasm. With LIFU assistance, tLyP-1-10-HCPT-PFP NPs could phase-transform into microbubbles and enhance tumor ultrasound molecular imaging for tumor diagnosis. After further irradiation by LIFU, an intracellular “explosion effect” caused by ADV, UTMD, and release of 10-HCPT could realize physicochemical synergistic antitumor therapy.

camptothecin analogues with powerful antitumor activity against a wide range of tumors in clinical practice. The required dose of 10-HCPT for cancer therapy is 4−6 mg per injection, which is over 20-times lower than that of paclitaxel or doxorubicin.36−38 (4) The tumor homing-penetrating peptide tLyP-1, which connects to DSPE-PEG3400-mal via a maleimide−thiol coupling reaction (Figure S1), is present on the outer shell of nanoparticles. After the reaction, the analysis of HPLC verified that tLyP-1 peptide was successfully connected to DSPE-PEG3400-mal (Figure S2). Our study has shown that FITC-tLyP-1 could target MDA-MB-231 cells and penetrate deep into the MDA-MB-231 spheroids model39,40 (Figures S3 and S4). The nanoparticles mediated by tLyP-1 peptide could penetrate through tumor blood vessels and tumor stroma deep into the tumor tissue,26,41−43 which might realize targeted diagnosis and precise therapy of every tumor cell. This strategy is shown in Figure 1; our novel ultrasound contrast agent with smaller size could arrive in tumor tissues through the tumor vascular endothelial gap (380−780 nm) by the EPR effect. The nanoparticles mediated by tLyP-1 peptide accumulated and penetrated deep into the extravascular tumor tissue. At the target site, the nanoparticles were irradiated by LIFU. Then, nanoparticles were turned into microbubbles by ADV for enhancing tumor ultrasound imaging. Then, 10-HCPT was released accurately from the microbubbles destructed by LIFU for precise targeting antitumor therapy due to the focusing of LIFU, targeting, and penetration of the tLyp-1 peptide. To substantiate our strategy for enhancing ultrasound molecular imaging and precise therapy, we prepared the 10HCPT-loaded phase-transformation nanoparticles mediated by tLyP-1 peptide (tLyP-1-10-HCPT-PFP NPs) using the filmingrehydration method and acoustic vibration method (details see the methods of the Supporting Information). It showed that the size and zeta potential of the tLyP-1-10-HCPT-PFP NPs were (366.30 ± 11.50) nm and (5.70 ± 3.70) mV determined by a Malvern Zeta Sizer (Figure 2a, b), respectively, whereas the size

vehicle that could be recognized by specific receptors, carriers, and so forth.21−23 However, nanoparticles mediated by the ligands with poor penetration effect only enter tumor tissue by the enhanced permeability and retention (EPR) effect and remain near blood vessels. Although angiogenesis is one of the main features of tumors, there are regions that lack microvessels in the tumors that restrict the ability of targeting delivery systems. Therefore, the penetration of targeting delivery systems is extremely significant for the effective treatment of solid tumors. For overcoming this problem, phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels and penetrate deep into the extravascular tumor tissue,24,25 which is called tumor homing-penetrating peptide. tLyp-1 (CGNKRTR), a truncated form of LyP-1 (CGNKRTRGC) with 7 amino acids, was reported as a ligand targeted to the NRP receptor with high affinity and specificity.26 Studies showed that the neuropilin-1 (NRP-1) was frequently overexpressed in several human tumor types, including carcinomas (e.g., pancreas, prostate, breast, ovarian, colon, and kidney), melanoma, glioblastoma, leukemias, lymphomas and others.27−34 tLyp-1 peptide was a kind of targeting peptide with the function of penetrating tumor vessels and tumor stroma. Herein, the aim of this study was to develop novel tumor homing-penetrating peptide-functionalized drug-loaded phasetransformation nanoparticles for LIFU-assisted tumor ultrasound molecular imaging and precise therapy. As shown in a schematic diagram (Figure 1), our nanoparticles consist of four parts: (1) The liquid core is composed of pefluoropentane (PFP), a hydrophobic fluorocarbon compound with an adequate boiling point (∼29 °C),35 which has been extensively used clinically. (2) The lipid shell consists of biocompatible DSPE-PEG3400-tLyP-1, DPPG, DPPC, and cholesterol. (3) 10Hydroxycamptothecin (10-HCPT) is loaded in the phospholipid bilayer of the lipid shell, which is one of the natural 1832

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Figure 2. Characterization of tLyP-1-10-HCPT-PFP NPs. (a) Size distribution, (b) zeta distribution, (c) TEM image of tLyP-1-10-HCPT-PFP NPs, and (d) inverted fluorescence microscope image of DiI-labeled tLyP-1-10-HCPT-PFP NPs. The red fluorescence was DiI.

and zeta potential of 10-HCPT-PFP NPs were (247.50 ± 9.80) nm and (−42.70 ± 5.50) mV (Figure S5). Interestingly, compared with 10-HCPT-PFP NPs, the zeta potential of tLyP1-10-HCPT-PFP NPs was raised to positive due to the tLyP-1 peptide with positive charge. The nanoparticles displayed spherical shape with moderate uniform size and particle size measured from TEM images was in good agreement with that measured by inverted fluorescence microscope (Figure 2c, d). With the help of the particle size advantage, tLyP-1-10-HCPTPFP NPs could pass through the tumor vascular endothelial gap (380−780 nm) to the extravascular tumor tissue. They thus overcame the first barrier of drug delivery systems for effective tumor treatment. Furthermore, the envelopment rate and 10HCPT-loading rate of our nanoparticles were (86.04 ± 4.27)% and (7.82 ± 0.38)%, respectively. The targeting and penetrating efficiency of tLyP-1-10HCPT-PFP NPs was evaluated in vitro. After coincubation with monolayer MDA-MB-231 cells at 37 °C for 1 h, confocal laser scanning microscopy (CLSM) images showed that DiIlabeled tLyP-1-10-HCPT-PFP NPs obviously accumulated around the cytomembrane of MDA-MB-231 cells in which NRP-1 was overexpressed. Meanwhile, a part of the tLyP-1-10-

HCPT-PFP NPs penetrated into the cytoplasm. We also found that the amount of tLyP-1-10-HCPT-PFP NPs around the cytomembrane and into the cytoplasm of MDA-MB-231 cells was higher than that of unmodified 10-HCPT-PFP NPs. However, there was no significant accumulation of the two NPs around human umbilical vein endothelial cells (HUVEC) (Figure 3a). Therefore, this showed that the targeting efficiency of tLyP-1-10-HCPT-PFP NPs to MDA-MB-231 cells was high. The results of quantitative analysis by flow cytometry were in accordance with the results presented above (Figure 3b−d). Some studies have found that in vitro three-dimensional tumor spheroids are a sufficient model to mimic the microenvironment of solid tumors because of several similarities, among which are poor drug penetration, drug resistance, altered protein expression, and enzyme activity and a viable rim with gradients of oxygen tension, nutrients, catabolites, and cell proliferation. In this study, we established three-dimensional spheroids of MDA-MB-231 cells successfully, and after coincubation with DiI-labeled tLyP-1-10-HCPT-PFP NPs or 10-HCPT-PFP NPs at 37 °C for 24 h, it was found that the red fluorescence of tLyP-1-10-HCPT-PFP NPs were distributed into tumor spheroids more evidently than that of 10-HCPT1833

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incubated with DiI-labeled 10-HCPT-PFP NPs or tLyP-1-10-HCPTPFP NPs. (d) Quantitative fluorescence intensity of DiI-labeled 10HCPT-PFP NPs and tLyP-1-10-HCPT-PFP NPs in HUVEC and MDA-MB-231 cells; untreated cells served as a control (n = 3). *P < 0.05.

PFP NPs (Figure 4a). The tLyP-1-10-HCPT-PFP NPs could reach more than 80 μm away from the bottom of the tumor spheroids and were distributed throughout the whole spheroids, whereas 10-HCPT-PFP NPs were distributed only in the surface layer of the tumor spheroids, which indicated that tLyP-1 peptide could facilitate the transport of NPs into the tumor spheroids (Figure 4b, c). For the active targeting efficiency of tLyP-1-10-HCPT-PFP NPs to be further validated in vivo, fluorescence imaging was performed in MDA-MB-231 xenograft-bearing nude mice using DiR (a near-infrared dye) to track the NPs. As shown in Figure 5a, red fluorescence was seen in the tumor area 1∼12 h after the injection of DiR-labeled tLyP-1-10-HCPT-PFP NPs, and the fluorescence intensity and range peaked at 1 h. However, there was no obvious red fluorescence in the tumor area 1∼12 h after the injection of DiR-labeled 10-HCPT-PFP NPs. The fluorescence intensity had no obvious difference in other organs between tLyP-1-10-HCPT-PFP NPs and 10-HCPT-PFP NPs. Correspondingly, the conclusion was further determined by ex vivo imaging of the tumor and other organs (Figure 5b). It suggested that modification with tLyP-1 peptide improved the active tumor-targeting efficiency of NPs in vivo. Meanwhile, 12 h after the injection, tissue frozen sections were prepared, and the images were captured using CLSM. The fluorescent intensity of DiI-labeled tLyP-1-10-HCPT-PFP NPs in the tumor was considerably higher than that of DiI-labeled 10HCPT-PFP NPs. In most of the normal tissues, the fluorescence intensity also had no obvious difference between tLyP-1-10-HCPT-PFP NPs and 10-HCPT-PFP NPs (Figure 5c). It indicated that tLyP-1-10-HCPT-PFP NPs could only increase the targeting effect to tumors rather than normal tissues. These results should be attributed to the tLyP-1 peptide, which could specifically combine with NRP-1 overexpressed on MDA-MB-231 cells and contained a cryptic CendR motif ((R/K)XX(R/K)), which was responsible for cell internalization and tissue penetration.44−46 For the phase-transformation effect of tLyP-1-10-HCPT-PFP NPs by heating to be tested, NPs were treated with different temperatures. From the optical microscopy observations, it was found that tLyP-1-10-HCPT-PFP NPs realized phase-transformation when the temperature reached ∼45 °C (Figure 6a), whereas there were no obvious changes in size or shape when the temperatures were approximately 37 and 40 °C. This proved that PFP was successfully loaded into the core of the NPs. As far as we know, the boiling pint of PFP was only 29 °C. In this study, we found that the phase-transformation temperature of tLyP-1-10-HCPT-PFP NPs was ∼45 °C. It demonstrated that the phase-transformation temperature of tLyP-1-10-HCPT-PFP NPs increased with the cover of the lipid shell. Furthermore, tLyP-1-10-HCPT-PFP NPs could not turn into microbubbles at physiological temperature (37 °C), which avoided embolism. For confirming that tLyP-1-10-HCPT-PFP NPs could enhance ultrasound imaging by ADV in vitro and in vivo, NPs were irradiated by LIFU. As shown in Figure 6b, tLyP-1-

Figure 3. Tumor targeting and penetrating efficiency of DiI-labeled tLyP-1-10-HCPT-PFP NPs in vitro. (a) CLSM image of tumor targeting and penetrating efficiency of DiI-labeled tLyP-1-10-HCPTPFP NPs or 10-HCPT-PFP NPs in vitro after incubation for 1 h at 37 °C in 5% CO2 atmosphere. The blue fluorescence was DAPI, and red fluorescence was DiI. Bar represents 20 μm. (b) Fluorescence intensity of HUVEC incubated with DiI-labeled 10-HCPT-PFP NPs or tLyP-110-HCPT-PFP NPs. (c) Fluorescence intensity of MDA-MB-231 1834

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Figure 4. Tumor targeting and penetrating efficiency of DiI-labeled tLyP-1-10-HCPT-PFP NPs in MDA-MB-231 spheroids model. (a) Multilevel scan started at the bottom of the spheroid in 10 μm intervals for the penetration of DiI-labeled tLyP-1-10-HCPT-PFP NPs or 10-HCPT-PFP NPs after incubation for 24 h at 37 °C in 5% CO2 atmosphere. (b) Three-dimensional reconstruction of the MDA-MB-231 spheroids model incubated with DiI-labeled tLyP-1-10-HCPT-PFP NPs or 10-HCPT-PFP NPs. (c) Quantitative analysis for the penetration depth of DiI-labeled tLyP-1-10HCPT-PFP NPs and 10-HCPT-PFP NPs. The red fluorescence was DiI.

Image Analyzer (DFY-II type, Institute of Ultrasound Imaging, Chongqing Medical University, China), which was an independent research and development product of our lab. The analysis results conformed with ultrasound imaging in the B-mode and CEUS mode (Figure 6c, d). It indicated that the grayscale values of the B-mode and CEUS mode imaging of tLyP-1-10-HCPT-PFP NPs were obviously enhanced after being irradiated by LIFU at 2 W/cm2 for 3 min (P < 0.05). At the same time, PBS and tLyP-1-10-HCPT NPs were irradiated by LIFU under the same conditions. This showed that the ultrasound images of PBS and tLyP-1-10-HCPT NPs had no obvious change, which indirectly suggested that PFP was the critical factor for the phase-transformation and enhancement effect of ultrasound imaging (Figure S7). The enhancement effect of ultrasound imaging in vivo was further evaluated. tLyP1-10-HCPT-PFP NPs or 10-HCPT-PFP NPs were injected into MDA-MB-231 xenograft-bearing nude mice through the tail vein at a dose of 200 μL. Before the injection of tLyP-1-10HCPT-PFP NPs, tumor nodules showed regular hypoecho. After injection for 0 and 1 h, ultrasound imaging of the tumor showed no statistical differences from that before injection.

10-HCPT-PFP NPs also realized phase-transformation by LIFU in vitro. With the increase in acoustic intensity and time, the B-mode and contrast-enhanced ultrasound (CEUS) mode imaging of tLyP-1-10-HCPT-PFP NPs were enhanced after being irradiated by LIFU. Interestingly, this phenomenon described above was only observed within the scope of 0.4−2 W/cm2, whereas the B-mode and CEUS mode imaging of tLyP-1-10-HCPT-PFP NPs receded at 2.8 W/cm2. The light microscopy images of tLyP-1-10-HCPT-PFP NPs after irradiation by LIFU showed that the size of nanoparticles increased when acoustic intensity was within the scope of 0.4− 2 W/cm2, whereas the phase-transformation nanoparticles decreased at 2.8 W/cm2 (Figure S6). This demonstrated that some of the phase-transformation nanoparticles exploded at 2.8 W/cm2, which provides a basis for drug release triggered by LIFU. The drug release experiment in vitro showed that the drug release rate of the LIFU group was ∼4-times that of the control group at 12 h; the drug release rates of the LIFU group were also higher than those of the control group at 24, 48, 60, and 72 h (Figure S8). As for ultrasound imaging, quantitative analysis of grayscale values was calculated using an Ultrasound 1835

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Figure 5. In vivo targeting efficiency and tissues distribution of DiR-loaded tLyP-1-10-HCPT-PFP NPs to MDA-MB-231xenograft-bearing nude mice. (a) In vivo fluorescence images of the nude mice at 1, 2, and 12 h after intravenous administration of DiR-labeled tLyP-1-10-HCPT-PFP NPs or 10-HCPT-PFP NPs. (b) Ex vivo fluorescence images of tumor and main normal tissues at 12 h after the administration of DiR-labeled tLyP-1-10HCPT-PFP NPs or 10-HCPT-PFP NPs. (c) DiI-labeled tLyP-1-10-HCPT-PFP NPs and 10-HCPT-PFP NPs distribution in tumor and main normal tissues. Nuclei were stained by DAPI. Bar represents 200 μm.

However, after the tumor was irradiated by LIFU, the B-mode and CEUS mode imaging of the tumor were enhanced, whereas there was no obvious change in echo after injection of 10HCPT-PFP NPs and irradiation by LIFU (Figure 7a). This suggested that tLyP-1-10-HCPT-PFP NPs accumulated to the tumor effectively and turned into microbubbles by ADV. Quantitative analysis results of the Ultrasound Image Analyzer were in accordance with the results of ultrasound imaging (Figure 7b, c). tLyP-1-10-HCPT-PFP NPs could realize ultrasound molecular imaging of tumor cells, which were better than the general ultrasound contrast agent. Different from ordinary ultrasound, LIFU was focused, and the focusing area was 0.4 cm2. Therefore, LIFU could be used for early diagnosis of tumors but not suitable for advanced tumors with large volume. This peculiarity of LIFU increased the accuracy and security of phase-transformation. The range of enhancement in the tumor was in agreement with the focus area of LIFU. That was also the reason why ultrasound imaging in just a part of the tumor tissue was enhanced. For the antitumor efficacy to be assessed in vitro and in vivo, the MDA-MB-231 cells and tumor-bearing mice were treated by the following eight groups: control, 10-HCPT, 10-HCPTPFP NPs, tLyP-1-10-HCPT-PFP NPs, control+LIFU, 10HCPT+LIFU, 10-HCPT-PFP NPs+LIFU, and tLyP-1-10HCPT-PFP NPs+LIFU. Cellular apoptosis in vitro was detected by flow cytometry. It was found that the tumor cell apoptosis rate in the tLyP-1-10-HCPT-PFP+LIFU group was the highest (Figure 8a−h). Quantitative flow cytometry data showed that the early and late apoptosis rate of tLyP-1-10HCPT-PFP NPs+LIFU was (58.55 ± 2.06)% (Figure 8i). In addition, the cell viabilities in vitro were evaluated by CCK-8 assay. As shown in Figure 8j, With LIFU assistance, the cell viabilities of 10-HCPT-PFP NPs, tLyP-1-10-HCPT-PFP NPs, and 10-HCPT were declined, especially the tLyP-1-10-HCPTPFP NPs. This showed that the 10-HCPT in 10-HCPT-PFP NPs or tLyP-1-10-HCPT-PFP NPs was released by LIFU.

Furthermore, LIFU irradiation increased the cell membrane permeability. Most importantly was that the tLyP-1 peptide could deliver 10-HCPT directly and actively through cellular membranes into cells. This distinctly increased the antitumor efficacy of tLyP-1-10-HCPT-PFP NPs. Targeted antitumor efficacy in vivo was evaluated in MDAMB-231 xenograft-bearing nude mice. During the treatment, we found that the tumor volume of the saline group increased rapidly, and treatment with 10-HCPT, 10-HCPT-PFP NPs, and tLyP-1-10-HCPT-PFP NPs could suppress the growth of xenografts. After treatment with LIFU, they could significantly suppress the growth of xenografts, especially in the tLyP-1-10HCPT-PFP NPs group (Figure 9a−i). On day 14, the tumor volumes of nude mice treated with 10-HCPT-PFP NPs, 10HCPT, and tLyP-1-10-HCPT-PFP NPs were approximately 86.28, 67.47, and 60.12% of that of the saline group, respectively. The treatment with LIFU further improved the antitumor effect. The tumor volume of the nude mice treated with tLyP-1-10-HCPT-PFP NPs+LIFU was only 34.95% that of the saline group, which was significantly lower than those of the 10-HCPT-PFP NPs+LIFU (76.89%) and 10-HCPT+LIFU (62.79%) groups (Figure 9i). At the same time, the antitumor effect of all groups was assessed by immunohistochemical methods. HE examinations of tumor tissue revealed that large amounts of cells were destroyed due to the absence of cell nuclei, demonstrating the remarkable therapeutic efficacy of tLyP-1-10-HCPT-PFP NPs with LIFU assistance (Figure 10a− h). Cell apoptosis in tumor tissue sections was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end-labeling assay (TUNEL) in which apoptotic cells showed brown nuclear staining (Figure 10a−h). In contrast to the proliferation assay, the index of TUNEL in tLyP-1-10-HCPT-PFP NPs+LIFU group was higher than in other groups (P < 0.05) (Figure 10j). In the tLyP-1-10-HCPTPFP NPs+LIFU group, the expression of proliferating cell nuclear antigen (PCNA) was absent or reduced, whereas other 1836

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Figure 6. Phase-transformation imaging and enhanced ultrasound imaging of tLyP-1-10-HCPT-PFP NPs in vitro. (a) Light microscope images of phase-transformation by heating. tLyP-1-10-HCPT-PFP NPs were turned into microbubbles at 45 °C (100×). (b) Ultrasound images (B-mode and CEUS mode) of tLyP-1-10-HCPT-PFP NPs after being irradiated by LIFU under different conditions. (c) Grayscale values of B-mode imaging of tLyP-1-10-HCPT-PFP NPs after being irradiated by LIFU. (d) Grayscale values of CEUS mode imaging of tLyP-1-10-HCPT-PFP NPs after being irradiated by LIFU.

groups were positive for PCNA expression (Figure 10a−h). Moreover, as shown in Figure 10, the index of PCNA in the tLyP-1-10-HCPT-PFP NPs+LIFU group was lower than in other groups (P < 0.05) (Figure 10i). The antitumor efficacy of tLyP-1-10-HCPT-PFP NPs+LIFU was the best compared with other groups in vivo mentioned above, which might be due to the fact that tLyP-1-10-HCPT-PFP NPs with active targeting and penetrating property could pass through the tumor vascular endothelial cell gap and tumor stroma and then reach the deep tumor tissue where there was a lack of vessels. Furthermore, tLyP-1-10-HCPT-PFP NPs penetrated the cell membrane into the cytoplasm. With LIFU assistance, the tumor cells were killed by a synergistic “explosion effect” caused by ADV and UTMD and release of 10-HCPT. This intracellular killing effect was more efficient. For the detection of systemic toxicity, the body weights were recorded, and serum markers were detected. As shown in

Figure 9j, there were no significant differences in the mouse weights over the treatment course in all groups. This revealed that the NPs and LIFU did not exhibit obvious toxicity. Furthermore, the result of systemic toxicity indicated that there were no significant differences in ALB, ALT, ALP, CRE, and BUN between the tLyP-1-10-HCPT-PFP NPs+LIFU and control groups, whereas ALT and BUN were improved in 10-HCPT and 10-HCPT+LIFU groups compared with those of the control group. This indicated that 10-HCPT and 10-HCPT +LIFU groups had side effects in liver and kidney. Simultaneously, tLyP-1-10-HCPT-PFP NPs were irradiated by LIFU and that 10-HCPT was released at the tumor site; thus, it might reduce the systemic toxicity compared to solo 10HCPT therapy (Table S1). In conclusion, we successfully developed smart 10-HCPTloaded phase-transformation nanoparticles modified by tLyP-1 peptide (tLyP-1-10-HCPT-PFP NPs). As a novel targeted 1837

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Figure 7. Enhanced ultrasound imaging of tLyP-1-10-HCPT-PFP NPs in vivo. (a) Ultrasound images of tumor. tLyP-1-10-HCPT-PFP NPs or 10HCPT-PFP NPs were injected into MDA-MB-231 xenograft-bearing nude mice through the tail vein. One hour later, the tumor was irradiated by LIFU (1 s with a 1 s pause for a total of 3 min; 1 MHz; 2.8 W/cm2). (b) Grayscale values of B-mode imaging. (c) Grayscale values of CEUS mode imaging. *P < 0.05.

Figure 8. Antitumor efficacy in vitro. (a−i) Flow analysis of apoptosis: (a) control, (b) 10-HCPT-PFP NPs, (c) 10-HCPT, (d) tLyP-1-10-HCPTPFP NPs, (e) control+LIFU, (f) 10-HCPT-PFP NPs+LIFU, (g) 10-HCPT+LIFU, and (h) tLyP-1-10-HCPT-PFP NPs+LIFU. For common groups (a−d), MDA-MB-231 cells were incubated for 24 h. For LIFU groups (e−h), MDA-MB-231 cells were irradiated by LIFU (1 s with a 1 s pause for a total of 3 min; 1 MHz; 2.8 W/cm2) after being incubated for 1 h. Then, the cells in LIFU groups were incubated for another 23 h. (i) Quantitative analysis of apoptosis. (j) Cell viabilities. *P < 0.05.

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Figure 9. Antitumor efficacy in vivo. (a−h) MDA-MB-231 xenograft-bearing nude mice and ex vivo tumor: (a) saline, (b) 10-HCPT-PFP NPs, (c) 10-HCPT, (d) tLyP-1-10-HCPT-PFP NPs, (e) control+LIFU, (f) 10-HCPT-PFP NPs+LIFU, (g) 10-HCPT+LIFU, and (h) tLyP-1-10-HCPT-PFP NPs+LIFU. For common groups (a−d), tLyP-1-10-HCPT-PFP NPs were injected into MDA-MB-231 xenograft-bearing nude mice through the tail vein. For LIFU groups (e−h), the tumor was irradiated by LIFU (1 s with a 1 s pause for a total of 3 min; 1 MHz; 3.2 W/cm2) at 1 h after injection. The nude mice were treated on days 1, 4, 7, 10, and 13. On day 14, nude mice were sacrificed (n = 6). (i) Tumor volumes and (j) body weights over the treatment course in all groups. *P < 0.05.

tLyP-1 peptide targeting and penetrating, which could realize “real” ultrasound molecular imaging and targeted therapy inside tumor cells. Additionally, the tumor targeting property of tLyP1-10-HCPT-PFP NPs and focusing property of LIFU improved the accuracy of treatment and minimized systematic adverse side effects. Thus, the results obtained in the present study indicate that tLyP-1-10-HCPT-PFP NPs with LIFU assistance might provide a novel strategy for ultrasound molecular imaging and precise therapy of the tumor.

drug-loaded ultrasound contrast agent, tLyP-1-10-HCPT-PFP NPs have multiple advantages. First, as a new developed drug carrier with nanoscale particle size, tLyP-1-10-HCPT-PFP NPs could pass through the tumor vascular endothelial cell gap. Second, induced by tLyP-1 peptide with targeting and penetrating efficiency, tLyP-1-10-HCPT-PFP NPs could increase tumor accumulation and penetrate deeply into the extravascular tumor tissue. Third, with LIFU assistance, tLyP-110-HCPT-PFP NPs underwent phase-transformation by ADV, which enhanced tumor ultrasound molecular imaging. Fourth, after irradiation by LIFU, an intracellular “explosion effect” caused by ADV, UTMD, and release of 10-HCPT resulted in physicochemical synergistic antitumor therapy. More importantly, tLyP-1-10-HCPT-PFP NPs could enter into the target cell directly, actively and efficiently and penetrate through the extracellular matrix and cellular membrane to the cytoplasm via

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ASSOCIATED CONTENT

S Supporting Information *

Detailed materials, methods and additional figures and tables were in Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.7b05087. 1839

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Nano Letters

Figure 10. Antitumor efficacy in vivo by pathological examination. On day 14, nude mice were sacrificed. (a−h) HE (100×), PCNA (400×), and TUNEL (400×) of the tumor: (a) control, (b) 10-HCPT-PFP NPs, (c) 10-HCPT, (d) tLyP-1-10-HCPT-PFP NPs, (e) control+LIFU, (f) 10HCPT-PFP NPs+LIFU, (g) 10-HCPT+LIFU, and (h) tLyP-1-10-HCPT-PFP NPs+LIFU. (i) Quantitative analysis of PCNA. (j) Quantitative analysis of TUNEL. *P < 0.05.

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Detailed materials and methods, chemical constitution and HPLC analyses of DSPE-PEG3400-tLyP-1, tumor targeting effect of FITC-tLyP-1 in vitro, penetrating effect of FITC-tLyP-1 in MDA-MB-231 spheroids model, size and zeta potential distributions of 10HCPT-PFP NPs, light microscopy images of tLyP-110-HCPT-PFP NPs after irradiation by LIFU, ultrasound images of PBS and tLyP-1-10-HCPT NPs, drug release in vitro of tLyP-1-10-HCPT-PFP NPs, and blood indices indicating the function of liver and kidney after therapy (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86-23-63693172. ORCID

ZhiGang Wang: 0000-0002-7281-5845 JianLi Ren: 0000-0002-2293-4600 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (Nos. 81471675, 61471074, 81227801, 81371578, 81630047, 31630026), Fundamental and Frontier Research Program of Chongqing (Nos. cstc2014jcyjA10031, cstc2014jcyjA10096), and College Program of Excellent Young Talents (2016, No. 20).

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