Efficient siRNA Delivery Using Novel Cell-Penetrating Peptide-siRNA Conjugate-Loaded Nanobubbles and Ultrasound

Ultrasound in Med. & Biol., Vol. 42, No. 6, pp. 1362–1374, 2016 Copyright Ó 2016 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter

http://dx.doi.org/10.1016/j.ultrasmedbio.2016.01.017

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Original Contribution EFFICIENT SIRNA DELIVERY USING NOVEL CELL-PENETRATING PEPTIDE-SIRNA CONJUGATE-LOADED NANOBUBBLES AND ULTRASOUND XIANGYANG XIE,* WEN LIN,y MINGYUAN LI,z YANG YANG,z JIANPING DENG,y HUI LIU,* YING CHEN,* XUDONG FU,* HONG LIU,* and YANFANG YANGx * Department of Pharmacy, Wuhan General Hospital of Guangzhou Military Command, Wuhan, China; y Department of Clinical Laboratory, Huangshi Love & Health Hospital of Hubei Province, Huangshi, China; z Beijing Institute of Pharmacology and Toxicology, Beijing, China; and x State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (Received 31 May 2015; revised 13 January 2016; in final form 27 January 2016)

Abstract—Because of the absence of tolerable and effective carriers for in vivo delivery, the applications of small interfering RNA (siRNA) in the clinic for therapeutic purposes have been limited. In this study, development of a novel siRNA delivery system based on ultrasound-sensitive nanobubbles (NBs, nano-sized echogenic liposomes) and cell-permeable peptides (CPPs) is described. A CPP-siRNA conjugate was entrapped in an NB, (CPPsiRNA)-NB, and the penetration of CPP-siRNA was temporally masked; local ultrasound stimulation triggered the release of CPP-siRNA from the NBs and activated its penetration. Subsequent research revealed that the (CPP-siRNA)-NBs had a mean particle size of 201 ± 2.05 nm and a siRNA entrapment efficiency .85%. In vitro release results indicated that .90% of the encapsulated CPP-siRNA was released from NBs in the presence of ultrasound, whereas ,1.5% (30 min) was released in the absence of ultrasound. Cell experiments indicated higher cellular CPP-siRNA uptake of (CPP-siRNA)-NBs with ultrasound among the various formulations in human breast adenocarcinoma cells (HT-1080). Additionally, after systemic administration in mice, (CPP-siRNA)NBs accumulated in the tumor, augmented c-myc silencing and delayed tumor progression. In conclusion, the application of (CPP-siRNA)-NBs with ultrasound may constitute an approach to selective targeted delivery of siRNA. (E-mail: [email protected]) Ó 2016 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Nanobubbles, Cell-penetrating peptides, siRNA delivery.

INTRODUCTION

short peptide sequences that are rich in lysine or arginine (Derossi 1998). These cationic peptides can facilitate the cellular internalization of therapeutic agents, which is attributed to the interaction between the negatively charged plasma membrane and the positively charged CPP (Lindgren and Langel 2011; Zorko and Langel, 2005). Some studies have reported that siRNA/CPP complexes, formed via electrostatic interactions between the cationic CPP and anionic siRNA, could facilitate cellular import and elicit RNAi, which results in the silencing of endogenous genes (Simeoni et al. 2003). Researchers reported that entrapment of the gene and CPP into microbubbles is an effective strategy for gene transfection (Ren et al. 2009, 2014). However, the depressed cell penetrating ability of CPPs neutralized by anionic siRNA and the discounted gene silencing efficacy resulting from an unpacked carrier (caused by strongly electrostatic interaction) hinder the potential use of such complexes. An alternative strategy is

Small interfering RNA (siRNA), suppressing the expression of oncogenes (e.g., c-myc) closely related to tumor growth, proliferation, invasion and expansion, has been studied as a potential candidate for cancer treatment for decades. However, the application of siRNA in the clinic would encounter a series of hurdles, such as the rapid degradation by nuclease, renal clearance and poor cellular uptake. Thus, development of a siRNA delivery vehicle that can reinforce specificity for the tumor and has efficient cellular uptake is desirable. To overcome these challenges, a new approach employing cell-penetrating peptides (CPPs) for payload delivery seems promising. CPPs are positively charged,

Address correspondence to: Yang Yang, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, China. E-mail: [email protected] 1362

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covalent siRNA-CPP conjugates of CPPs and siRNA linked by reducible disulfide bonds. This conjugate could dissociate naked siRNA in the cytoplasm in response to intra-cellular glutathione. However, because CPP is a non-specific functional molecule that can penetrate any cell on encountering it, this drawback was limited the utilization of siRNA-CPP in drug delivery systems (Vives 2005). With the focus of addressing such a problem, the concept of stimulus-responsive nanocarriers was imported to assemble an ‘‘off–on’’ switch, which is based on sensitivity to endogenous triggers (e.g., pH and enzymatic activity) and external triggers (e.g., light and ultrasound) to control the penetrating activity of CPPs. For example, Xiang et al. (2013) developed an ‘‘activatable cell-penetrating peptide’’ system for the targeted delivery of siRNA to prostate-specific antigen–positive prostate tumors. In this system, the activity of CPP was blocked by masking the arginine positive charges with a polyanionic segment (polyglutamate). Between the two peptide segments, there was a prostate-specific antigen cleavable sequence linking them. Thus, the overexpressed matrix metalloproteinases in the vicinity of the tumor cells could initiate the cell penetration of CPPs. In another example, by using different pH levels between tumor tissue and healthy tissue, He’s group designed long-circulating pH-detachable polyethylene glycol (PEG)-shielded CPP-liposomes to realize site-specific drug delivery (Zhang et al. 2013a). These liposomes were constructed with PEG chains attached to their surface, and the outside segment of the PEG chain linked to a pH-sensitive bond cloaked the CPP on the surface of the liposome. The slightly acidic tumor environment could trigger removal of the PEG chain, thus activating the cell crossing ability of CPP. However, with respect to the reliance on the endogenous stimulus, the aforementioned triggered release mechanisms may suffer some disadvantages, mainly because of the large interindividual variability in the expression levels of enzymes or intra-tumoral pH. In addition, as the CPPs are immobilized on the surface of the nanocarriers in these strategies, they may not be well protected from enzymatic degradation in vivo before approaching the targeting sites. To overcome the drawback of endogenous stimulustriggered CPP, some researchers began to investigate the general triggered release methodology, which was independent of the tumor microenvironment and could realize targeted drug delivery (Yang et al. 2014). In this strategy, the non-specific function of CPPs was sterically shielded during the first phase of drug delivery; after the CPPS reached the target site, their exposure to external triggers would activate their cell penetration and, therefore, enhance the intracellular delivery of their payloads. Among the various external triggers, ultrasound is

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especially attractive, as it can penetrate deeply into tissues and can be focused on regions of tumor growth to effectively activate sonosensitizers while preserving peripheral healthy tissue. Ultrasound-mediated drug delivery can be amplified by the acoustic disruption of microbubble carriers that undergo cavitation (Escoffre et al. 2013). Recently, microbubbles have been successfully used in preclinical research for drug delivery (de Saint Victor et al. 2014; Tsu-Yin et al. 2013). However, microbubbles (1–10 mm) are incapable of targeting specific tissue in vivo because of large particle sizes that limit them from penetrating the vessel wall and entering target tissues (Son et al. 2014). On the other hand, nanocarriers (10–1000 nm) are more suitable for in vivo drug delivery to target sites, because they can extravasate from the bloodstream and, because of their smaller size, enter the desired tissues (Cavalli et al. 2013). To date, the use of nanobubble (NB) systems combined with ultrasound for drug delivery has been studied extensively, and this strategy has been found to have a variety of merits, including: non-invasiveness, local applicability, targeted release, high transfection efficiency and proven tolerability (Kantarci and Cavalli 2012; Kwan et al. 2015; Suzuki et al. 2011). Among the various types of NBs, liposomes, which are temperature sensitive, have attracted increasing attention in drug delivery research for their sonosensitive features (Evjen et al. 2013; Lin et al. 2014; Rizzitelli et al. 2015); thus, they are also called echogenic or sonosensitive liposomes. Therefore, by using the ascendance of NBs, a new strategy of encapsulating cell-permeable peptides-small interfering RNA (CPP-siRNA) conjugates into NBs to mask the activity of CPPs in circulation was adopted in this work. Here, we planned to verify a new RNA delivery strategy by constructing a nanocarrier, (CPP-siRNA)NBs, that was sensitive to ultrasound. Figure 1 is a schematic of the design. CPP (CKRRMKWKK), derived from Penetratin, which has increased membrane translocation efficiency (Fischer et al. 2000), is first conjugated to siRNA to form CPP-siRNA via a chemical reaction; then, the CPP-siRNA is encapsulated in NBs (also called echogenic liposomes). After systemic administration, (CPP-siRNA)-NBs pass or accumulate in tumor sites through the enhanced permeability and retention (EPR) effect. As the target site is exposed to ultrasound, CPPsiRNA is released from the NBs, which have been burst by ultrasound irradiation. Then, the CPPs deliver the siRNA directly and actively through cellular membranes into the cytoplasm to silence the target gene. In this work, the physicochemical and in vitro biological characters of the NBs loaded with CPP-siRNA were investigated, and the in vivo tumor therapy efficiency of (CPP-siRNA)NB was explored.

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siRNA with CPP via disulfide bonds. Briefly, c-mycsiRNA, CPP and diamide (thiol oxidant) were dissolved in Hepes buffer solution (10 mM Hepes, 1 mM ethylenediaminetetraacetic acid, pH 8.0) at a final equimolar concentration of 6 mM in a reactor with gentle stirring for 1 h at 40 C. The formed conjugates were purified via a minicolumn centrifugation method with a dextran gel (Sephadex G-50) column. The final CPP-siRNA (yield of 81.5%) was confirmed by matrix assisted laser desorption ionization mass spectrometry. Fig. 1. Schematic of our proposed design for nanobubbles containing CPP-siRNA conjugate, (CPP-siRNA)-NBs, for delivering siRNA to cancer cells under ultrasound stimulation. C3F8 5 perfluorobutane; CPP 5 cell-penetrating peptide; siRNA 5 small interfering RNA.

METHODS Materials and cell lines The CPP (CKRRMKWKK) was custom-synthesized by Shanghai GL Biochem (Shanghai, China). 1,2Dipalmitoyl-snglycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) (ammonium salt) (DSPEmPEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoe thanolamine-N-maleimide(polyethylene glycol) (DSPEPEG2000-Mal) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Anti-c-myc siRNA (sense strand: 50 -AAC GUU AGC UUC ACC AAC AUU TT-30 , antisense strand: 50 -AAU GUU GGU GAA GCU AAC GUU TT-30 ), scramble siRNA (sense strand: 50 -UUC UCC GAA CGU GUC ACG UTT-30 , anti-sense strand, 50 ACG UGA CAC GUU CGG AGA ATT-30 ) as the negative control (NC), and thiol group- or FAM-labeled siRNA (50 end of one strand) were synthesized and purified by highpressure liquid chromatography by GenePharma (Shanghai, China); Cy3-siRNA was purchased from RiboBio (Guangzhou, China). All chemicals were of reagent grade and were obtained from Sigma-Aldrich, unless otherwise stated. Human fibrosarcoma cells (HT-1080 cells) purchased from the Institute of Basic Medical Science, Chinese Academy of Medical Sciences (Beijing, China), were maintained in culture medium consisting of modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin and 100 mg/mL streptomycin. The cells were maintained in a 37 C humidified incubator in a 5% CO2 atmosphere. Synthesis of CPP-siRNA The CPP-siRNA conjugate was synthesized as reported by Muratovska and Eccles, (2004) by coupling

Preparation of NBs All lipid compositions consisted of PEGylated species, DSPC and DPPC at a molar ratio of 10:10:90. NBs were prepared as previously described with some modifications (Zhou et al. 2013; Zhu et al. 2015). In brief, the lipids (10 mg) were mixed and dissolved in chloroform (1 mL), then dried on a rotary evaporator for 15 min at 40 C. The lipid film obtained was hydrated using 3 mL of Hepes buffer (20 mM, 150 mM NaCl, pH 7.0) containing 10 mM CPP-siRNA (CPP-cmyc-siRNA, CPP-NC-siRNA, CPP-FAM-siRNA or CPP-Cy3-siRNA) at 40 C (in a rotating flask at the speed of 150 rpm) for 30 min to obtain a homogeneous dispersion. The liposome dispersion (4 mg/mL lipid or 4 mmol/mL CPP-siRNA) was homogenized by three circulations under 10,000 psi pressure in a high-pressure homogenizer (EmulsiFlex-C3; Avestin, Ottawa, ON, Canada) and was then extruded through polycarbonate membrane (200 nm, Whatman, Shanghai, China) using an extruder (EmulsiFlex-C3, Avestin). The precursor solution was transferred to 3-mL vials and sealed, and the headspaces were exchanged with perfluorobutane (C3F8, Flura, Newport, TN, USA). Finally, NBs were formed by shaking the sealed vials with a VialMix (ImaRx Therapeutics, Tucson, AZ, USA) for 45 s. NBs containing CPP-siRNA were then diluted to 10 mL Hepes buffer and washed three times by centrifugation flotation in a bucket-rotor centrifuge at 300g for 3 min. The supernatant (1 mg/mL lipid or 1 mmol/mL CPP-siRNA) was collected and stored at 4 C for further use. Photon correlation spectroscopy (Nanophox, Sympatec, Germany) was used to measure the diameter of the NB samples. The NB suspension was diluted with distilled water and then analyzed at a laser intensity of 50%–60% at 25 C. The measuring mode was crosscorrelation. The morphology of the samples was observed by transmission electron microscopy (TEM, Hitachi, H-7650, Japan). CPP-siRNA encapsulation efficiency of each formulation The CPP-FAM-siRNA encapsulation efficiency of NBs was determined by ultrafiltrating the NBs entrapping

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CPP-FAM-siRNA using Amicon Ultra-4 centrifugal filter devices (100,000 NMWL, Millipore, Billerica, MA, USA) (Gao et al. 2012). The fluorescence of CPP-FAMsiRNA was determined with the spectrofluorometer (Synergy 4, Biotek, Winooski, VT, USA) using excitation and emission wavelengths of 495 and 525 nm, respectively. The encapsulation efficiency of CPP-FAM-siRNA was calculated using the formula: (Mi2MU)/Mi 3 100%, where MU and Mi are the masses of unencapsulated CPP-FAM-siRNA and initially added CPP-FAM-siRNA, respectively. Ultrasound-triggered release of payload The CPP-FAM-siRNA was released from NBs by insonation using an ultrasound instrument. Briefly, the samples were diluted 20-fold with phosphate-buffered saline (PBS, pH 7.4) in small glass disks (the outside bottom of the disk is coated with 1.0-cm-thick gel interfaces, [EcoGel 100 Imaging Ultrasound Gel, Eco-Med Pharmaceutical, Mississauga, ON, Canada]), incubated at 37 C for 5 min and immediately treated with an ultrasound probe from the HUT-105 sonication system (Institute of Biomedical Engineering, Huazhong University of Technology, Wuhan, China). The ultrasound treatment conditions were as follows: 1 MHz, spatial-peak temporalaverage intensity (Ispta) of 1 W/cm2, and a 10-s sonication with a 10-s pause for a total of different times. The choice of acoustic parameters was based mainly on the reference description with some modifications (Du et al. 2011). After insonation exposure, the released CPP-FAM-siRNA was measured. In this study, the samples treated with ultrasound were labeled (CPP-siRNA)-NBs (1US). Control groups were not sonicated. After ultrasonic processing for the pre-determined time intervals, the sample was thoroughly ultrafiltered, and the released CPPFAM-siRNA was collected and determined using the spectrofluorometer, as previously described. Analysis of cellular uptake Flow cytometry. HT-1080 cells were seeded in sixwell plates at a density of 2 3 105 cells per well overnight. After the attachment period (24 h), the cells were rinsed with PBS and incubated with pre-treated fresh cell culture medium containing free CPP-FAM-siRNA, free FAMsiRNA or an equivalent concentration of NBs containing CPP-FAM-siRNA for 4 h. The autofluorescence of the cells was used as the control. In the NB group, the corresponding culture media were pre-treated with or without ultrasound (1 MHz, Ispta 5 1 W/cm2, 10-s sonication with 10-s pause for a total of 60 s). After incubation with these formulations for 4 h at 37 C, the cells were trypsinized, washed twice with cold PBS and then immediately analyzed using a flow cytometer (BD FACSCalibur, USA).

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For each analysis, more than 105 cells were used, and 104 cells were collected. Confocal laser scanning microscopy. After culture of HT-1080 cells (2 3 105 cells per well) for 24 h on a Petri dish, free FAM-siRNA, free CPP-FAM-siRNA and NBs containing CPP-FAM-siRNA were added to each dish and incubated at 37 C. Control cells were incubated in medium under the same conditions. In the NB group, the corresponding culture media were pre-treated with or without ultrasound, as mentioned above. After incubation, the medium was removed, and the cells were washed with cold PBS followed by fixation with 4% paraformaldehyde in PBS. After fixation, the cells were treated with Hoechst 33258 for 10 min. Fluorescence images of the cells were analyzed using confocal laser scanning microscopy (CLSM, UltraVIEW Vox, Perkin Elmer, USA). In vitro siRNA transfection and analysis of gene expression. HT-1080 cells were seeded at a density of 2.0 3 106 cells/well in a 35-mm dish. After 24 h of culture in a humidified atmosphere of 5% CO2 at 37 C, the medium was exchanged with fresh serum-free medium containing various samples. Among these samples, the (CPPsiRNA)-NB group was pre-treated with or without ultrasound, as mentioned above. The final concentration of siRNA (c-myc-siRNA or NC-siRNA) used in the experiment was 100 nM. The cells were incubated for 48 h (for mRNA assays) at 37 C. Subsequently, c-myc mRNA was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). These methods have been described in our previous study (Yang et al. 2015). Cell apoptosis assay. HT-1080 cells were placed on 25-cm2 tissue culture flasks at 2.0 3 106 cells per flask with 6 mL of complete modified Eagle’s medium. After 24 h culture, the cells were washed with PBS (0.1 M, pH 7.4) and exposed to fresh serum-free medium containing free c-myc-siRNA, free CPP-c-myc-siRNA or c-myc-siRNA-loaded NBs. Among these samples, the (CPP-siRNA)-NB group was pre-treated with or without ultrasound, as mentioned previously. After incubation for 6 h, the cells were collected and stained with the Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer’s instructions and were immediately analyzed using the FACScan flow cytometer with 10,000 events collected (Xiang et al. 2013). Animal model. Female BALB/c nude mice (weighing 18–22 g, 5 wk old) were purchased from Vital River Laboratories (Beijing, China). All procedures involving animal housing and treatment were approved by the Animal Care and Use Ethics Committee of the Academy of Military Medical Sciences. The xenograft tumor models

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were produced via subcutaneous injection of 100 mL HT1080 cells as described previously (Gao et al. 2012; Zhao et al. 2012). Each of the HT-1080 cells (5 3 106) was inoculated into the hypoderm of the mouse armpit. Tumor sizes were measured with calipers across two perpendicular diameters. The estimated tumor volume was calculated using the formula volume (mm3) 5 (length 3 width2)/2. In vivo imaging. When the tumor volume reached approximately 200 mm3, the HT-1080 xenografted mice were injected in the tail vein with 200 mL of 5% glucose aqueous injections (control), free Cy3-siRNA, free CPPCy3-siRNA, or NBs containing CPP-Cy3-siRNA at 1.2 mg/kg. Thirty minutes after administration, the tumor-xenografted mice treated with (CPP-Cy3siRNA)-NBs were fixed, and the surface of the tumor sites was covered with 1.0-cm-thick gel interfaces (EcoGel 100 Imaging Ultrasound Gel, Eco-Med Pharmaceutical); each tumor site was then treated with an ultrasound probe from the HUT-105 sonication system (Institute of Biomedical Engineering, Huazhong University of Technology) with a sonication area of 0.8 cm2 (Zhao et al. 2012), given ultrasound (1 MHz, Ispta 5 1 W/cm2, 10-s sonication with 50-s pause for a total of 30 min). Under such sonication conditions, the temperature of exposed tumor tissue was below 39 C, near the normal body temperature of mice (37 C–39 C). Another group of mice injected with (CPP-Cy3siRNA)-NBs not exposed to ultrasound served as the control. Subsequently, in vivo fluorescence imaging was performed with an IVIS Lumina II in vivo imaging system (IVIS Lumina II In Vivo Imaging System, Caliper Life Sciences, USA) at the indicated times (1, 6 and 12 h). After in vivo imaging, the mice were sacrificed by cervical dislocation, and the tumors were excised and imaged. In vivo anti-tumor efficacy. When tumor volume reached approximately 200 mm3, the HT-1080 tumorxenografted mice were intravenously injected via the tail vein with 5% glucose (control), 10 mg/kg of c-mycsiRNA, CPP-c-myc-siRNA and (CPP-c-myc-siRNA)NBs on the 6th, 9th, 12th and 15th days. Thirty minutes after administration, the tumor-xenografted mice treated with (CPP-c-myc-siRNA)-NBs were exposed to ultrasound, as mentioned previously. Meanwhile, another group of mice injected with (CPP-c-myc-siRNA)-NBs not exposed to ultrasound served as a control. Tumor volumes were determined, and mice were weighed. The treatment-to-control ratio (T/C) was calculated as T/C 5 (mean RTV of treated group)/(mean RTV of control group) 3 100%, where RTV 5 relative tumor volume, calculated as RTV 5 (tumor volume at day n)/ (tumor volume at day 0).

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Detection of c-myc expression in tumor tissues. For analysis of c-myc expression in vivo, tumor tissues were excised 24 h after the last administration. Tumor fragments were processed for total mRNA extraction followed by qRT-PCR. To analyze c-myc mRNA, the extracted mRNA samples were individually normalized to the same 260-nm absorbance value and detected by qRT-PCR as described in a previous report (Zhang et al. 2013b). Briefly, the selected tumor tissues (100 mg) were excised and homogenized in 1 mL of TRNzol A1 reagent (Tiangen, China) in an ice bath. Then the homogenized samples were centrifuged at 11,000g for 10 min, and the supernatant was handled according to the protocol of the manufacturer. The extracted mRNA samples were treated with SuperReal Premix SYBR Green kit (Tiangen, China) and analyzed on the IQ5 real-time PCR detection system (Bio-Rad). Relative gene expression was quantified with IQ5 Optical System Software, Version 2.0 (Bio-Rad). Statistical analysis The data are presented as means 6 standard deviations. The difference between any two groups was determined via analysis of variance. Survival cures were compared with the log-rank (Mantel–Cox) test. p-Values , 0.05 were considered to indicate statistical significance. The statistical software SPSS 10.0 (IBM, Armonk, NY, USA) was employed to process the experiment data. RESULTS AND DISCUSSION Synthesis of functional conjugates The procedure for synthesis of CPP-siRNA is illustrated in Figure 2a. The thiol group at the 50 end of the chemically modified siRNA strand was able to react with the free thiol group in the cysteine amino acid on the CPP. As illustrated in Figure 2b, the observed molecular weight of siRNA was 8197.86 Da, which was equal to the theoretical mass-charge ratio of 8194.5 Da. Thus, it was suggested that the siRNA-CPP conjugate was successfully synthesized. The disulfide bond of siRNACPP has the merit of releasing naked siRNA into the cytosol of cancer cells and, therefore, can minimize the interference of CPP with the anti-sense process of siRNA (Lundberg et al. 2007). However, three challenges involving siRNA-CPP had to be addressed before its successful employment: (i) the non-specific penetration of siRNA-CPP; (ii) the instability of the CPP in the blood circulation caused by extracellular proteases; (iii) unwanted reduction of the disulfide bond before the conjugate reached the target cell. To solve these problems, encapsulation of siRNA-CPP in targeted NBs seemed to be a good option. It should be noted that it was difficult

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Fig. 2. (a) Procedure for synthesis of CPP-siRNA conjugate. (b) and MALDI-TOF mass spectrum of CPP-siRNA conjugate. CPP 5 cell-penetrating peptide; siRNA 5 small interfering RNA.

to discriminate the chemical environment of the hydrogen nucleus in polymers and peptides, so only the mass spectrum was used as an alternative to identified CPP-siRNA. Nanobubble characteristics The CPP-siRNA encapsulation efficiency of NBs was 87.23 6 1.12%. (CPP-siRNA)-NBs measured 201 6 2.05 nm, as assessed with photon correlation spectroscopy. As no bubble-like structure was found in the visual field of TEM, it was assumed that the bubbles were destroyed in the sample preparation and observation process. According to a reference, the NBs could not be observed by TEM because perfluorobutane escaped from the vesicles and destroyed their structures under high vacuum (Du et al. 2011). As an alternative, to investigate the possible surface morphology of NBs, liposomes, (CPP-siRNA)-LPs, without perfluorobutane were used to represent the NBs and observed under a TEM. As illustrated in Figure 3a, TEM of (CPPsiRNA)-LPs revealed that the prepared blank NBs were round particles and their sizes were close to the values measured using the laser particle analyzer (Fig. 3b). Figure 3c illustrates that no microbubbles formed in the prepared system. In addition, the prepared NBs could be stable at room temperature for 6 d (approximately 75% of the entrapped payload could be released from NBs after insonation); afterward, ultrasound could not efficiently trigger the drug release (,50% release after insonation). After 6 d, the NBs increased in size by about 5 nm. The size of the prepared NBs would increase around 4–6 nm after incubation at 37 C for 10 h.

Microbubbles have been used clinically as ultrasound contrast agents for decades (Stride, 2015), and researchers have developed many new methods for preparing microbubbles during this time (Mahalingam et al. 2015; Parhizkar et al. 2014). It is commonly thought that nanoparticles of around 200 nm can take advantage of the EPR effect, and thus, we prepared NBs in this work. The NBs described here were prepared using methods reported in previous studies with some modifications (Lundberg et al. 2007; Stride 2015), and their structure was composed of liposome with perfluorobutane molecules in the bilayer membrane (please see the schematic in Fig. 1). In fact, the prepared NBs could be considered thermosensitive liposomes (because their formulation was similar to that of thermosensitive liposome, but their release mechanism was independent of heat here for the low temperature used during ultrasound treatment [please see next paragraph]) containing the ultrasound-sensitive agent perfluorobutane. However, the structure of the prepared NBs requires further study, which we will report in the future. Dependence of release of CPP-siRNA from NBs on ultrasound As the frequency of ultrasound applied in delivery of genes was in the range 1–3 MHz (Cavalli et al. 2013), here 1 MHz was selected to stimulate the payload release. The Ispta (1 W/cm2) and duty cycle (50%) were based on the Du et al. (2011) report. Percentage release was determined as a function of sonication time. It was found that the cumulative payload release increased as the

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Fig. 3. (a) Morphologic appearance of samples based on transmission electron microscopy (without perfluorobutane). (b) Particle size distribution of (CPP-siRNA)-NBs. (c) Microscopic image of the prepared samples. CPP 5 cell-penetrating peptide; siRNA 5 small interfering RNA; NB 5 nanobubbles.

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sonication time was extended (20, 40, 60 and 80 s for 44, 71, 90 and 93%, respectively). Therefore, a sonication time of 60 s was chosen in the in vitro experiment to maintain a relatively complete RNA release. The temperature of the sample was consecutively recorded and never exceeded 38 C, much lower than the gel-to-liquid phase transition temperature of the liposome bilayer (41 C), indicating that RNA release was not triggered by heat (Rizzitelli et al. 2015). As a proof of concept study, the selection of in vivo parameters was based mainly on the in vitro results and our experience (low local temperature and high drug release); however, a detailed study is required. Release of CPP-siRNA from the NBs by insonation is depicted in Figure 4. The data revealed that the release of CPP-siRNA from NBs was dependent on ultrasound. About 90% of the entrapped CPP-siRNA was released from NBs after insonation (1 MHz, Ispta 5 1 W/cm2, 10-s sonication with 10-s pause for a total of 60 s). In contrast, without the ultrasound stimulus, the CPPsiRNA-NBs exhibited minimal CPP-siRNA leakage into the serum medium with a cumulative release of less than 1% after incubation at 37 C for 30 min. The resonance frequency of a NB is in the range of 100 MHz to 1 GHz, so it was not possible for the NBs to disrupt their bilayer membranes through a cavitation effect induced by the resonance. On the other hand, as the temperature of the tissue exposed to ultrasound did not rise significantly (,41 C), it is unlikely the drug release was triggered by heat. Similar to pulsed low-intensity non-focused ultrasound, the low energy associated with the ultrasound used in this work did not deliver enough energy to raise the local temperature, and the potential drug release may occur primarily through mechanical in-

Fig. 4. Release behavior of CPP-siRNA from nanobubbles with or without ultrasound. The data are means 6 standard deviations (n 5 3). CPP 5 cell-penetrating peptide; siRNA 5 small interfering RNA; NB 5 nanobubbles.

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teractions between the acoustic waves and the nanocarrier (Afadzi et al. 2013; Rizzitelli et al. 2015; Schroeder et al. 2007). However, the actual release mechanism remains unclear and requires further study. Analysis of cellular uptake using flow cytometry and confocal laser scanning microscopy After confirming that NBs could work properly via an ultrasound stimulus, their ability to deliver siRNA into tumor cells was further assessed. Whether the ‘‘functional molecule’’ CPP could work after ultrasound stimulation was crucial to the success of the NBs. According to the designed strategy, when the NBs are triggered by ultrasound, cellular uptake of the siRNA is expected to be enhanced because of the CPP penetration effect. To verify this hypothesis, we used HT-1080 cells to evaluate the in vitro uptake of CPP-siRNA by tumor cells. As illustrated in Figure 5a, compared with siRNA, CPP-siRNA exhibited significantly enhanced cellular uptake into the test cell lines (p , 0.05). The results indicate that CPPs and their attached molecules could penetrate cell membranes efficiently and thus induce an increase in fluorescence intensity. As illustrated in Figure 5b, the fluorescence intensity was in the order (CPP-siRNA)NBs (1US) . (CPP-siRNA)-NBs. These results indicate that when the NBs were triggered by ultrasound, the cellular uptake of CPP-siRNA was enhanced because of the CPP-mediated cell penetration. Consistent with these findings, CLSM analysis also confirmed the significant synergetic effect of (CPPsiRNA)-NBs (1US) on uptake by HT-1080 cells. As illustrated in Figure 5c, a cellular uptake significantly increased in cells that were treated with (CPP-siRNA)NBs (1US) compared with cells treated with (CPPsiRNA)-NBs, suggesting that ultrasound did work. Furthermore, among all of the samples, CPP-siRNA had the greatest capacity for improving uptake of siRNA, confirming the strength of CPP. In vitro gene silencing and cell apoptosis assay The in vitro gene silencing activity of siRNA against the human c-myc gene delivered by the nanocarriers was further determined by qRT-PCR. As illustrated in Figure 6a, c-myc mRNA expression of HT-1080 cells treated with (CPP-siRNA)-NBs (1US) or CPP-siRNA was significantly inhibited. Compared with CPPsiRNA, free siRNA did not exhibit greater silencing activity. Results also indicated that (CPP-NC-siRNA)NBs (1US) did not exert any gene silencing effect on c-myc. To clarify the gene silencing activity of CPPsiRNA conjugation assisted by glutathione in the cytosol, gene silencing activity was compared between the CPPsiRNA conjugate and CPP/siRNA complex (zeta potential 5 28.02 6 3.11 mV). From the results

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Fig. 5. (a) Uptake of siRNA or CPP-siRNA into HT-1080 cells. (b) Uptake of CPP-siRNA into HT-1080 cells with or without ultrasound stimulation. (c) Confocal laser scanning microscopy analysis of the uptake of various formulations by HT-1080 cells. The data are means 6 standard deviations (n 5 3). *p , 0.05. CPP 5 cell-penetrating peptide; siRNA 5 small interfering RNA; NB 5 nanobubbles; US 5 ultrasound.

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illustrated in Figure 6a, we can conclude that the CPPsiRNA conjugate enables an advantageous improvement in endogenous gene silencing efficiency compared with its simple electrostatic counterpart CPP/siRNA. These results agreed with the hypothesis that CPPs binding too strongly to siRNA prevent the successful RNAi (Geoghegan et al. 2012). In the treated cells, apoptosis induced by various formulations carrying siRNA and NC-siRNA were also evaluated by flow cytometric analysis. As illustrated in Figure 6b, cells exposed to siRNA-loaded formulations exhibited significant apoptosis, whereas only a slight effect was observed on the control and free siRNA groups, indicating that the apoptosis originated mainly from the downregulated expression of c-myc in HT-1080 cells. Cell apoptosis was in the order CPPsiRNA (51.31%) . (CPP-siRNA)-NBs (1US) (42.64%) . siRNA/CPP (41.8%) . (CPP-siRNA)NB (19.23%) . free siRNA (4.41%) . (CPP-NCsiRNA)-NB (1US) (1.14%), which was consistent with the c-myc mRNA expression results described previously (Fig. 6a). Overall, these results indicate that combination of CPP-siRNA with ultrasound stimulation could markedly facilitate RNAi-mediated gene silencing and growth inhibition. By now, results from cell studies have roughly validated our design. To verify the actual target effect of the drug delivery strategy, in vivo studies are warranted. In vivo distribution of siRNA Whole-animal imaging (Fig. 7a) indicated that for the mice treated with 5% glucose, no fluorescence signals were detected during the experimental period. The fluorescence intensity of free siRNA rapidly decreased after administration, and there was no accumulation in the tumor. The reason for this phenomenon is related to the problems inherent to siRNA, such as its instant degradation by RNase (a type of nuclease) and rapid renal excretion after intravenous injection. CPP-siRNA-NB (1US)treated mice exhibited the most intense tumor distribution during the whole study period. This phenomenon suggested that introduction of ultrasound as well as activated CPPs could enhance the accumulation of RNA in tumors. As the ultrasound treatment lasted 30 min, much of the NBs in the blood passed through the sonication site and released their payload in the tumor site; therefore, the concentration of siRNA-loaded NBs in the blood would have decreased and resulted in low non-specific Fig. 6. (a) Level of c-myc mRNA determined by quantitative real-time polymerase chain reaction. (b) Cell apoptosis after exposure to different formulations. The data are

means 6 standard deviations (n 5 3). *p , 0.05. CPP 5 cellpenetrating peptide; siRNA 5 small interfering RNA; NB 5 nanobubbles; NC 5 negative control; US 5 ultrasound.

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fluorescence (1 h after administration) in the ultrasoundtreated group as assessed by in vivo imaging. In contrast, in mice treated with free CPP-siRNA, fluorescence was discretely distributed in many organs including tumors, which was attributed to the non-specific cell penetration of CPPs. The results also indicated that mice injected with (CPP-siRNA)-NBs but not exposed to ultrasound exhibited less tumor fluorescence intensity because the cell penetrating ability of CPPs was not activated. In addition, the (CPP-siRNA)-NB group displayed significant amount of non-specific fluorescence, suggesting that the specificity of NBs without ultrasound was not marked and these NBs were circulated in the blood and easily accumulated in organs linked to the blood vessel injected. Consequently, isolated organs and tumor tissues were further observed by sacrificing the mice 12 h after administration. As illustrated in Figure 7b, tumors from (CPP-siRNA)-NB (1US)-treated groups exhibited the strongest fluorescence signals, whereas less or no fluorescence was observed in other isolated organs of this group. The results imply that (CPP-siRNA)-NBs (1US) could efficiently target solid tumors and greatly decrease nonspecific accumulation in normal organs. Mice treated with free CPP-siRNA exhibited fluorescence in all isolated organs, more strongly in the liver and kidney, because of the non-specificity of CPPs and reticuloendothelial system uptake, as well as renal excretion in vivo. For the (CPP-siRNA)-NB group not exposed to ultrasound, the fluorescence was distributed mainly in liver, tumor, and kidney, indicating the vesicles could also be subject to reticuloendothelial system uptake and renal excretion. These in vivo imaging data strongly indicate that (CPP-siRNA)-NBs (1US) could potentially transport siRNA into HT-1080 cells with specificity. In vivo anti-tumor efficiency of the targeted NBs As illustrated in Figure 8a, the T/C values of siRNA, CPP-siRNA, (CPP-siRNA)-NBs, (CPP-siRNA)-NBs (1US) and (CPP-NC-siRNA)-NBs (1US) were 96.8%, 92.4%, 87.0%, 39.6% and 102.7%, respectively. The strongest inhibitory effect was observed on day 24 in (CPP-siRNA)-NBs (1US) (T/C 5 39.6%, p , 0.05). In contrast, the tumor-inhibitory effect of (CPP-siRNA)NBs (T/C 5 92.4%) was not ideal. Therefore, ultrasound is the key to achieving a highly enhanced anti-cancer effect. The NBs used combined both passive and physical targeting. After injection into the circulation, the NBs passed or accumulated in tumor sites because the capillary permeability of the endothelial barrier in newly vascularized tumors (EPR effect) is significantly greater than that of normal tissues (passive targeting); then the tumor sites were treated with ultrasound, which triggered release of the loaded RNA into the tumor cells (physical

Fig. 7. Biodistribution of Cy3-siRNA contained in various formulations in mice bearing HT-1080 tumor xenografts. (a) Whole-body imaging at different time points after systemic administration. (b) Fluorescence detection of isolated main tissues and organs from mice at the endpoint of observation. CPP 5 cell-penetrating peptide; siRNA 5 small interfering RNA; NB 5 nanobubbles; US 5 ultrasound.

targeting) to achieve targeted siRNA delivery. From the results illustrated in Figure 7, we could conclude that the EPR effect in our research was not very strong; therefore, the specificity of CPP-siRNA-NBs basically came

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Fig. 8. Anti-tumor activity (a) and weight changes (b) in HT-1080 tumor-bearing mice after treatment with 5% glucose and various formulations carrying c-myc siRNA or NC-siRNA. The data are means 6 standard deviations (n 5 6). *p , 0.05 (n 5 3). CPP 5 cell-penetrating peptide; siRNA 5 small interfering RNA; NB 5 nanobubbles; NC 5 negative control; US 5 ultrasound.

from the in vitro ultrasound stimulus (physical targeting). Furthermore, free CPP-siRNA (T/C 5 92.4%)-treated mice also exhibited less tumor growth inhibition; this might be attributed to the degradation of CPP-siRNA and the reduction of disulfide bonds in the circulation in vivo. Because of the inherent shortfalls of naked c-myc-siRNA mentioned previously, mice in the naked siRNA group (T/C 5 96.8%) and (CPP-NC-siRNA)NB (1US) (T/C 5 102.7%) group did not exhibit significant tumor growth inhibition compared with the control groups, which exhibited almost no growth inhibition. These results indicate the combined effects of ultrasound stimulus and CPPs.

The changes in the weights of the animals were recorded as an indication of drug safety. As shown in Figure 8b, there was no significant change in the weight of the various samples of mice during the experimental period (p . 0.05). These results suggest that there was negligible acute or severe toxicity related to the indicated treatment at the test dose. To confirm the gene silencing activity of (CPPsiRNA)-NB (1US) more clearly in vivo, c-myc expression at the mRNA level in tumors was assayed by qRTPCR, after sacrificing mice in each group at the end of the experiment. Consistent with the aforementioned results, (CPP-siRNA)-NBs (1US) induced the best gene silencing effect compared with the other formulations (Fig. 9). As for free siRNA and (CPP-NC-siRNA)-NBs, the expression of c-myc mRNA exhibited no obvious alterations compared with the 5% glucose-treated group. Compared with (CPP-siRNA)-NBs (1US), (CPPsiRNA)-NBs exhibited a smaller gene silencing effect but had a higher downregulating effect than other groups. Taken together, these results are consistent with the antitumor growth data mentioned previously, supporting the direct causality between delayed tumor progression and silencing of the c-myc gene. CONCLUSIONS

Fig. 9. Expression of c-myc mRNA in tumors was detected 24 h after the last administration. The data are means 6 standard deviations (n 5 6). *p , 0.05 (n 5 3). CPP 5 cell-penetrating peptide; siRNA 5 small interfering RNA; NB 5 nanobubbles; US 5 ultrasound.

Cell-penetrating peptide has a potent ability to aid the penetration of drug carriers into cells, but its nonspecific affinity limits its application in drug delivery systems. In this study, (CPP-siRNA)-NBs combining the specific targeting effect of the efficient cell penetrating ability of CPPs and the ultrasound-triggered siRNA release characteristics of NBs were successfully developed. The constructed vesicle had a suitable particle size and drug entrapment efficiency. The entrapped CPP-siRNA was released under ultrasound stimulation.

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Furthermore, the prepared carriers exhibited strong tumor-inhibitory activity both in vitro and in vivo. Compared with the group not exposed to ultrasound treatment, (CPP-siRNA)-NBs (1US) had increased tumor inhibitory efficacy. The salient advantage of this delivery system is that it overcomes the drawback of the CPPs by ‘‘cloaking’’ them in the carriers and triggers their penetrating function with external ultrasound. Although preliminary, the results of this study indicate the tremendous potential of the NB system for efficient delivery of siRNA for oncotherapy. Acknowledgments—We are grateful for the financial support from the National Natural Science Foundation of China (Grants 81202466 and 81402874).

REFERENCES Afadzi M, Strand SP, Nilssen EA, M asøy SE, Johansen TF, Hansen R, Angelsen BA, de L Davies C. Mechanisms of the ultrasoundmediated intracellular delivery of liposomes and dextrans. IEEE Trans Ultrason Ferroelectr Freq Control 2013;60:21–33. Cavalli R, Bisazza A, Lembo D. Micro- and nanobubbles: A versatile non-viral platform for gene delivery. Int J Pharm 2013;456:437–445. Derossi D. Trojan peptides: The penetratin system for intracellular delivery. Trends Cell Biol 1998;8:84–87. De Saint Victor M, Crake C, Coussios CC, Stride E. Properties, characteristics and applications of microbubbles for sonothrombolysis. Expert Opin Drug Deliv 2014;11:187–209. Du L, Jin Y, Zhou W, Zhao J. Ultrasound triggered drug release and enhanced anticancer effect of doxorubicin loaded poly(D,Llactide-co-glycolide) methoxy poly(ethylene glycol) nanodroplets. Ultrasound Med Biol 2011;37:1252–1258. Escoffre JM, Mannaris C, Geers B, Novell A, Lentacker I, Averkiou M, Bouakaz A. Doxorubicin liposome-loaded microbubbles for contrast imaging and ultrasound-triggered drug delivery. IEEE Trans Ultrason Ferroelectr Freq Control 2013;60:78–87. Evjen TJ, Hagtvet E, Moussatov A, Røgnvaldsson S, Mestas JL, Fowler RA, Lafon C, Nilssen EA. In vivo monitoring of liposomal release in tumours following ultrasound stimulation. Eur J Pharm Biopharm 2013;84:526–531. Fischer PM, Zhelev NZ, Wang S, Melville JE, F ahraeus R, Lane DP. Structure–activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin. J Pept Res 2000;55:163–172. Gao J, Yu Y, Zhang Y, Song J, Chen H, Li W, Qian W, Deng L, Kou G, Chen J, Guo Y. EGFR-specific PEGylated immunoliposomes for active siRNA delivery in hepatocellular carcinoma. Biomaterials 2012;33:270–282. Geoghegan JC, Gilmore BL, Davidson BL. Gene silencing mediated by siRNA binding fusion proteins is attenuated by double-stranded RNA-binding domain structure. Mol Ther Nucleic Acids 2012;1:e53. Kantarci G, Cavalli R. Non-viral systems for gene delivery: Up to date. In: Nanotechnology in progress: Pharmaceutical applications. Kerala: Research SignPost; 2012. Kwan JJ, Myers R, Coviello CM, Graham SM, Shah AR, Stride E, Carlisle RC, Coussios CC. Ultrasound-propelled nanocups for drug delivery. Small 2015;11:5305–5314. Lin CY, Javadi M, Belnap DM, Barrow JR, Pitt WG. Ultrasound sensitive eLiposomes containing doxorubicin for drug targeting therapy. Nanomedicine 2014;10:67–76. Lindgren M, Langel U. Classes and prediction of cell-penetrating peptides. Methods Mol Biol 2011;683:3–19. Lundberg P, El-Andaloussi S, Sutlu T, Johansson H, Langel U. Delivery of short interfering RNA using endosomolytic cell-penetrating peptides. FASEB J 2007;21:2664–2671.

Volume 42, Number 6, 2016 Mahalingam S, Raimi-Abraham BT, Craig DQ, Edirisinghe M. Formation of protein and protein–gold nanoparticle stabilized microbubbles by pressurized gyration. Langmuir 2015;31:659–666. Muratovska A, Eccles MR. Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells. FEBS Lett 2004; 558:63–68. Parhizkar M, Stride E, Edirisinghe M. Preparation of monodisperse microbubbles using an integrated embedded capillary T-junction with electrohydrodynamic focusing. Lab Chip 2014;14:2437–2446. Ren J, Xu C, Zhou Z, Zhang Y, Li X, Zheng Y, Ran H, Wang Z. A novel ultrasound microbubble carrying gene and Tat peptide: Preparation and characterization. Acad Radiol 2009;16:1457–1465. Ren J, Zhang P, Tian J, Zhou Z, Liu X, Wang D, Wang Z. A targeted ultrasound contrast agent carrying gene and cell-penetrating peptide: preparation and gene transfection in vitro. Colloids Surf B Biointerfaces 2014;121:362–370. Rizzitelli S, Giustetto P, Cutrin JC, Delli Castelli D, Boffa C, Ruzza M, Menchise V, Molinari F, Aime S, Terreno E. Sonosensitive theranostic liposomes for preclinical in vivo MRI-guided visualization of doxorubicin release stimulated by pulsed low intensity nonfocused ultrasound. J Control Release 2015;28:21–30. Schroeder A, Avnir Y, Weisman S, Najajreh Y, Gabizon A, Talmon Y, Kost J, Barenholz Y. Controlling liposomal drug release with low frequency ultrasound: Mechanism and feasibility. Langmuir 2007; 23:4019–4025. Simeoni F, Morris MC, Heitz F, Divita G. Insight into the mechanism of the peptide-based gene delivery system MPG: Implications for delivery of siRNA into mammalian cells. Nucleic Acids Res 2003;31:2717–2724. Son S, Min HS, You DG, Kim BS, Chan KI. Echogenic nanoparticles for ultrasound technologies: Evolution from diagnostic imaging modality to multimodal theranostic agent. Nano Today 2014;9:525–540. Stride E. Physical principles of microbubbles for ultrasound imaging and therapy. Front Neurol Neurosci 2015;36:11–22. Suzuki R, Oda Y, Utoguchi N, Maruyama K. Progress in the development of ultrasound-mediated gene delivery systems utilizing nanoand microbubbles. J Control Release 2011;149:36–41. Tsu-Yin W, Wilson KE, Machtaler S, Willmann JK. Ultrasound and microbubble guided drug delivery: Mechanistic understanding and clinical application. Curr Pharm Biotechnol 2013;14:743–752. Vives E. Present and future of cell-penetrating peptide mediated delivery systems: ‘‘Is the Trojan horse too wild to go only to Troy?’’ J Control Release 2005;109:77–85. Xiang B, Dong DW, Shi NQ, Gao W, Yang ZZ, Cui Y, Cao DY, Qi XR. PSA-responsive and PSMA-mediated multifunctional liposomes for targeted therapy of prostate cancer. Biomaterials 2013;34:6976–6991. Yang Y, Yang YF, Xie XY, Cai XS, Mei XG. Preparation and characterization of photo-responsive cell-penetrating peptide-mediated nanostructured lipid carrier. J Drug Target 2014;22:891–900. Yang Y, Yang Y, Xie X, Wang Z, Gong W, Zhang H, Li Y, Yu F, Li Z, Mei X. Dual-modified liposomes with a two-photon-sensitive cell penetrating peptide and NGR ligand for siRNA targeting delivery. Biomaterials 2015;48:84–96. Zhang Q, Tang J, Fu L, Ran R, Liu YY, Yuan MQ, He Q. A pHresponsive a-helical cell penetrating peptide-mediated liposomal delivery system. Biomaterials 2013a;34:7980–7993. Zhang Y, Peng L, Mumper RJ, Huang L. Combinational delivery of c-myc siRNA and nucleoside analogs in a single, synthetic nanocarrier for targeted cancer therapy. Biomaterials 2013b;34:8459–8468. Zhao ZX, Gao SY, Wang JC, Chen CJ, Zhao EY, Hou WJ, Feng Q, Gao LY, Liu XY, Zhang LR, Zhang Q. Self-assembly nanomicelles based on cationic mPEG–PLA–b-polyarginine (R15) triblock copolymer for siRNA delivery. Biomaterials 2012;33:6793–6807. Zhou ZY, Zhang P, Ren JL, Ran H, Zheng YY, Li P, Zhang Q, Zhang MH, Wang ZG. Synergistic effects of ultrasound-targeted microbubble destruction and TAT peptide on gene transfection: An experimental study in vitro and in vivo. J Control Release 2013; 170:437–444. Zhu F, Jiang Y, Luo F, Li P. Effectiveness of localized ultrasound-targeted microbubble destruction with doxorubicin liposomes in H22 mouse hepatocellular carcinoma model. J Drug Target 2015;23:323–334. Zorko M, Langel U. Cell-penetrating peptides: Mechanism and kinetics of cargo delivery. Adv Drug Deliv Rev 2005;57:529–545.