Novel alginate-stabilized doxorubicin-loaded nanodroplets for ultrasounic theranosis of breast cancer

Accepted Manuscript Title: Novel alginate-stabilized doxorubicin-loaded nanodroplets for ultrasounic theranosis of breast cancer Author: Fatemeh Baghbani Fathollah Moztarzadeh Jamshid Aghazadeh Mohandesi Fatemeh Yazdian Manijhe Mokhtari-Dizaji PII: DOI: Reference:

S0141-8130(16)31493-3 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.09.008 BIOMAC 6474

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

2-5-2016 1-9-2016 2-9-2016

Please cite this article as: Fatemeh Baghbani, Fathollah Moztarzadeh, Jamshid Aghazadeh Mohandesi, Fatemeh Yazdian, Manijhe Mokhtari-Dizaji, Novel alginate-stabilized doxorubicin-loaded nanodroplets for ultrasounic theranosis of breast cancer, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.09.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Novel alginate-stabilized doxorubicin-loaded nanodroplets for ultrasounic theranosis of breast cancer

Fatemeh Baghbania, Fathollah Moztarzadeha,*, Jamshid Aghazadeh Mohandesib, Fatemeh Yazdianc, Manijhe Mokhtari-Dizajid, a

Biomaterials Group, Faculty of Biomedical Engineering (Center of Excellence), Amirkabir University of Technology, Tehran, Iran b

Department of Metallurgy, Faculty of Mining and Metallurgical Engineering, Amirkabir University of Technology, Tehran, Iran c

Faculty of New Science and Technologies, University of Tehran, Tehran, Iran

d

Department of Medical Physics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

*

Corresponding author: Tel: (+98-21)6454-2393

E-mail address: [email protected] (F.Moztarzadeh)

Graphicalabstract

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Abstract Perfluorocarbon nanoemulsions are a new class of multifunctional stimuli-responsive nanocarriers which combine the properties of passive-targeted drug carriers, ultrasound imaging contrast agents, and ultrasound-responsive drug delivery systems. Doxorubicin-loaded alginate stabilized perflourohexane (PFH) nanodroplets were synthesized via nanoemulsion preparation method and their ultrasound responsivity, imaging, and therapeutic properties were studied. Doxorubicin was loaded into the nanodroplets (39.2 nm) with encapsulation efficiency of 92.2%. In vitro release profile of doxorubicin from nanodroplets was an apparently biphasic release process and 12.6% of drug released from nanodroplets after 24 hours incubation in PBS, pH=7.4. Sonication with 28 kHz therapeutic ultrasound for 10 min triggered droplet-to-bubble transition in PFH nanodroplets which resulted in the release of 85.95% of doxorubicin from nanodroplets. Microbubbles formed by acoustic vaporization of the nanodroplets underwent inertial cavitation. In the breast cancer mice models, ultrasound-mediated therapy with doxorubicin-loaded PFH nanodroplets showed excellent anti-cancer effects characterized by tumor regression. Complete tumor regression was observed for the group in which doxorubicin-loaded nanodroplets were combined with ultrasound, whereas the tumor growth inhibition of doxorubicin –loaded nanodroplets was 89.6%. These multifunctional nanodroplets, with excellent therapeutic and ultrasound properties, could be promising drug delivery systems for chemotherapeutic application.

Keywords: Alginate-stabilized nanodroplets; Ultrasound; Breast cancer;

Introduction Developing stimuli-responsive nanocarriers which release their drug cargo only in response to environmental or physical stimuli, such as pH, hyperthermia, light, or ultrasound is an interesting approach to tumor-targeting [1]. Ultrasound as a drug delivery modality has a number of attractive features and could be applied with a variety of drug carriers. Ultrasound is especially attractive because it is non-invasive, accessible, cost effective, and is possible to direct ultrasound toward deeply located body sites in precise energy deposition patterns and sonicate tumors with millimeter precision [2-9]. Ultrasonic irradiation of tumor triggers drug release from the accumulated particles and transiently alters the permeability of cell membrane, which results in effective intracellular drug uptake by tumor cells [2, 10-11]. Ultrasound- mediated drug delivery like all energy-based tumor treatment modalities requires tumor imaging prior to treatment. Combining tumor imaging and treatment by ultrasound is especially attractive because it is cost-effective and makes the procedure more simple. Microbubbles have been used as ultrasound contrast agents in ultrasound imaging for many decades. Nowadays, their therapeutic application as drug carriers and enhancers of drug and gene delivery are being widely investigated [2-5, 12–14]. However, these systems present some difficulties which make them hard to utilize. They have very short circulation time (minutes) and

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large size (within micron range) which hinder their effective extravasation into tumor tissue, which is essential for effective drug targeting. Furthermore, injection of microbubbles may cause some side effects, such as high osmotic pressure and blood vessel dilation. The way to overcome these limitations may consist in developing acoustic phase shift nanodroplets that would effectively accumulate in tumor tissue by passive or active targeting and then convert into microbubbles in situ by ultrasound [15, 16]. These droplets are usually composed of perfluorocarbon (PFC) and coated materials which are stable in the blood stream. By ultrasound irradiation, PFC nanodroplets undergo an instant phase transition into gas bubbles called acoustic droplet vaporization or ADV. ADV promotes vascular permeability and ultrasound ablation for tumor tissue [17, 18], and also triggers the release of encapsulated drugs from nanodroplets [19,20]. The droplet to bubble transition process occurs with inertial cavitation (IC) when the applied acoustic pressure is higher than a particular threshold [21]. IC is the major effect of ADV that cause cell damage via inducing heat and mechanical force in therapeutic ultrasound applications [22-28]. These features of PFC nanodroplets make them promising candidates for overcoming the limitations of contrast bubbles and suggest that acoustic phase shift nanodroplets could be ideal theranostic agents for tumor targeted drug delivery. Doxorubicin (DOX) is one of the most commonly used chemotherapeutic drugs in current oncological chemotherapy. DOX has been approved for treatment of a number of solid tumors, such as leukemia, aggressive lymphoma, breast, lung, ovarian, and broad types of cancers [29]. However, DOX causes severe side effects when administered at high dose systematically [30, 31]. The encapsulation of DOX into micro and nanocarriers would protect normal tissues from exposure and toxicity. Therefore, several studies have been done on delivering DOX to targeted tissue by microspheres [32- 34], micelles [31, 35-37], nanoparticles [38, 39] or synthetic conjugates [40, 41] while minimizing the systemic exposure of the drug. Among various methods, polymeric nanoparticles with charges can load ionic drugs such as DOX easily by a simple absorption method with the highest entrapment efficiency [33]. In this work, the aim was to develop DOX-loaded multi-functional alginate-coated perfluorohexane (PFH) nanodroplets which provide efficient local treatment of tumor by a hybrid strategy involving both controlled triggered drug delivery and ADV-mediated destruction of cancer cells and, also serve as contrast-enhanced ultrasound imaging agents. The development and characterization of alginate-coated PFH acoustic nanodroplets that fits these functional demands have been described.

2. Experimental 2.1. Materials Doxorubicin Hydrochloride (2 mg/ml) was obtained from EBEWE Pharma (Unterach, Austria). Sodium Alginate, Perfluorohexane and Tween 20, phosphate-buffered saline (PBS, pH 7.4) and dialysis membranes (molecular weight cutoff, 12000) were supplied by Sigma-Aldrich (St. Louis, MO, USA). The agar used for in-vitro phantom experiments was purchased from Invitrogen (CA, USA). 3

All other chemicals and solvents were of analytical grade and used as received without further purification or treatment.

2.2. Preparation of doxorubicin-loaded alginate stabilized PFH nanodroplets Doxorubicin-loaded nanodroplets were obtained via nano-emulsion process. Briefly, perfluorohexane (PFH), doxorubicin and Tween 20 (surfactant) were homogenized in distilled deionized water for 2 min at 24,000 rpm using Ultra-Turrax SG215 homogenizer. Then, polymer solution (alginate 1.5%w/v) was added drop-wise whilst the mixture was homogenized at 13,000 rpm for 3 min. Finally, CaCl2 solution (0.2 w/v) was added dropwise to the emulsion under homogenization at 3000 rpm for 3 min. Nanodroplets were characterized for: morphology, by transmission electron microscopy (TEM); size distribution, and polydispersity index by dynamic light scattering; and drug entrapment and release by UV-vis spectroscopy. 2.3. Nanodroplets introduction into gel To introduce nanoemulsion into agarose gel, 1% agarose solution in phosphate buffered saline (PBS) was prepared and 200 μL of a nanodroplet emulsion was purred into it. 2.4. Cell culture 4T1 human breast cancer cells were obtained from Pasteur Institute (Tehran, IR). The cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells were cultured at 37 °C in humidified air containing 5% CO2. 2.5. Animal procedures Animals study was performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health. All experiments were conducted in accordance the Guiding Principles for the Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA). Six to 8 weeks old female BALB-c mice were obtained from Pasteur Institute (Tehran, IR). For inoculation, 4T1 human breast cancer cells were suspended in serum-free RPMI-1640 medium and injected subcutaneously to the flank of unanaesthetized mice (7×105 cells/100 μL/mouse). One week after transplantation, tumor gelosis formed at the injection site. Mice with similar tumor areas (0.5-0.6 mm length and width of the tumor) were selected as the model animals for the pharmacodynamic study.

2.6. Characterization of Doxorubicin-loaded nanodroplets 2.6.1. Particle size analysis

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The average size distribution and polydispersity index of nanodroplets was determined by DLS using a Zeta-sizer 3000HS (Malvern Instruments, Malvern, UK).). The analysis was performed at a scattering angle of 90° after the nanodroplet solution diluted adequately with double-distilled water. The measurement yielded the mean size and polydispersity index (PI).

2.6.2. TEM The morphology of drug-loaded nanodroplets was observed using a transmission electron microscopy (H-7650; Hitachi, Tokyo, Japan) operating at an acceleration voltage of 80 kV. To prepare sample for imaging, one drop of the solution was placed onto a 400-mesh carbon-coated copper grid and dried at room temperature.

2.6.3 Stability of the nanodroplets Nanodroplets were suspended in phosphate-buffered saline (PBS) at pH 7.4 and incubated at 4 °C for predetermined time intervals. The stability of nanodroplets was determined by changes in their size and drug entrapment efficiency. 2.6.4. Determination of entrapment efficiency To determine the entrapment efficiency, drug-loaded nanodroplets were separated from the solution by centrifugation (Centrifuges 5424 R, Eppendorf, Canada) at 11000 rpm for 30 min. Supernatants recovered from centrifugation were decanted. Doxorubicin content in the supernatant was analyzed by a UV-Vis spectrophotometer at 480 nm (U.V-1601; Shimadzu, Japan). Samples were prepared and measured in triplicate. Drug entrapment efficiency (EE) was calculated using the following equation: Entrapment Efficiency (%) =

(𝑇𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑎𝑑𝑑𝑒𝑑)− (𝐹𝑟𝑒𝑒 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔) 𝑇𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑎𝑑𝑑𝑒𝑑

× 100

2.6.5. In-vitro drug release study 2.6.5.1. Passive Drug release The release of doxorubicin from doxorubicin-loaded PFH/Alginate nanodroplets was evaluated using phosphate buffer (pH 7.4) and citrate buffer (pH 5.5) at 37 °C. Two milliliter of the nanodroplet solution was poured into a dialysis bag (Spectrapor, MW cutoff 12000 g/mol) and placed into 10 mL of phosphate buffer (pH 7.4) or citrate buffer (pH 5.5) severally. The release study was carried out in a shaker incubator (MS MP8 Wise Stir Wertheim, Germany) with 5

shaking rate of 100 rpm at 37 °C for 24 h. At predetermined time intervals (0, 1, 2, 3, 4, 5, 6 and 24) the sampling was performed. In each time point, 2 ml of the buffer was elicited, and then replaced with an equivalent volume of fresh buffer. The amount of released doxorubicin in the buffer was analyzed by a UV-Vis spectrophotometer (U.V-1601; Shimadzu, Japan) at a wavelength of 480 nm. The accumulated release was calculated utilizing the following equation: R= [V Σ n - i (Ci + V0 Cn)] / mdrug Where, R is the accumulated release (%), V is the sampling volume, V0 is the initial volume, Ci and Cn are the doxorubicin concentrations, i and n are the sampling times, and mdrug is the mass of doxorubicin in nanoparticles.

2.6.5.2 Ultrasound-induced release from drug-loaded nanodroplets To determine the influence of 28 kHz ultrasound on doxorubicin release from nanodroplets, the nanodroplet solution was purred into Plexiglas scaffold with a latex cover and placed into a water bath at 37 °C, and then was sonicated for 0.5, 1, 4 and 10 min with 0.34 W/cm2. After processing, the nanodroplet solution was withdrawn, and centrifuged at 11000 rpm, then the freedoxorubicin concentration was determined as described above. Each experimental condition was repeated 3 times. The ultrasound system was designed in our co-worker’s lab (Ultrasound Lab, Tarbiat Modares University, Tehran, Iran) to operate in the low kHz range with a center frequency of 27.7 kHz (nominal frequency of 28 kHz), a bandwidth of 421 Hz and a probe diameter of 60 mm.

2.6.6. Ultrasound imaging Ultrasound imaging was performed using a 12-MHz linear transducer (Acuson Sequoia 512, Siemens, Mountain View, CA).

2.6.7. Pharmacodynamic study Mice with the desired tumor size were divided into five groups of five. In all groups, injection was performed via peritoneum: The mice in group I (control) underwent injection of 0.2mL of PBS solution. The mice in group II underwent injection of doxorubicin. The mice in group III underwent injection of doxorubicin-loaded nanodroplets. The mice in group IV underwent injection of 0.2 mL of PBS solution and ultrasound exposure. The mice in group V underwent injection of doxorubicin-loaded nanodroplets and ultrasound exposure (nanodroplets with ultrasound). One and a half hours after the injection, group IV and V were sonicated with the ultrasound diagnostic apparatus for 4 min (28 kHz CW ultrasound; nominal output power density 0.34 W/cm2). Mice were treated by three systemic injections of PBS, doxorubicin or 6

nanodroplet-encapsulated doxorubicin (2 mg/kg of body weight as Dox) given every 4 days. Ultrasound was applied directly to the mouse skin by Aquasonic coupling gel. Tumor sizes were measured three times a week for 2 weeks after treatment with a caliper. The first administration day was recorded as day 0. Tumor volume (V) was calculated as follows: 𝑉 = 0.5𝐿 × 𝑊 2 where L and W are the length and width of the tumor, respectively. Two weeks after administration, mice were killed and tumors were removed. The normalized tumor size (Dn) was calculated according to the following formula: 3

𝑉

𝐷𝑛 = √𝑉 , 0

where V and Vo are current and initial tumor volumes (Vo is a tumor volume at the start of the treatment). The tumor growth inhibition rate (IR) was calculated as follows:

(𝑉 )

𝐼𝑅 = 1 − (𝑉𝑡 ) × 100 𝑐

Where Vc is the tumor volume in control group at day x and Vt is the tumor volume in treated group at day x.

3. Results and discussion 3.1. Preparation of doxorubicin-loaded alginate stabilized PFH nanodroplets Alginate stabilized PFH nanodroplets were obtained by nanoemulsion process. The encapsulation efficiency of doxorubicin in both nanodroplet formulations was over 90% (Table 1). The absorption of cationic doxorubicin molecules by anionic alginate molecules may have resulted in the high entrapment of doxorubicin in alginate shell. Both nanodroplet formulations possessed significantly higher doxorubicin release at acidic pH (5.5, the pH of endosomal/lysosomal environment) compared to normal pH (7.4) (Fig 1 a,b). The cumulative amount of drug release from formulations A and B at normal pH after 24 h was 12.6% and 9.4% respectively, while at acidic pH, about 38.2% and 29.5% drug was released from formulations A and B respectively. The relatively higher drug release from formulation A could be because of the smaller size of the particles. Smaller nanodroplets have a larger surface area which can lead to a greater drug release. This phenomenon has been discussed in detail in our previous paper [42]. 7

3.2. Size and morphology of nanodroplets Upon increasing the PFH volume fraction in nanodroplet formulation, the mean size of particles increased from 39.2±3 nm (formulation A) to 102.3±11 nm (formulation B) Table 1. Increasing PFH volume fraction enhances collision frequency among PFH droplets and subsequently the rate of flocculation which results in formation of larger nanodroplets. The morphology of both formulation of doxorubicin-loaded PFH nanodroplets was observed using TEM (Fig. 2). The difference between TEM particle size of each formulation with the size from DLS analysis could be because TEM gives the size of nanoparticles in dried form while DLS tells the hydrodynamic diameter that includes the inorganic core along with any molecule and the solvent layer attached to the particle as it moves under the influence of brownian motion. The nanodroplets had a spherical shape. The volume fraction of PFH in formulation A was low (5%v/v), so the particles had a micelle like structure. Since PFH serves as the template core, the amount of PFH affects the nanoparticle size and morphology. Nanodroplets from formulation B with higher PFH content showed a well-defined shell–core structure -a light core with a dark ring around it. The light core could because of liquid–gas conversion of PFH under high vacuum during TEM [43].

3.3. Stability of nanodroplets The nanodroplets showed good stability over three months at 4 °C. Nearly no changes in size and entrapment efficiency of nanodroplets were observed (Fig 3). The nanodroplets could remain sufficiently stable so as not to break or fuse together because of their high negative zeta-potential and well-constructed shell.

3.4. Ultrasound-induced release from drug-loaded nanodroplets In controlled drug delivery, the ideal delivery systems are the ones that do not release their drug cargo in the circulation system, and after reaching the target tissue, release their drug payload in a controlled manner. The in vitro release profiles of doxorubicin from PFH/alginate nanodroplets at pH, 7.4 indicated that doxorubicin was tightly retained by the nanodroplets (Fig. 4 a) but it was rapidly released by ultrasound exposure. The passive and ultrasound induced release profiles of doxorubicin from the nanodroplets (formulation A and B) within 10 min are presented in Fig. 4 a. A possible drug release mechanism for the PFH nanodroplets, may be acoustic droplet vaporization and bubble formation under ultrasound exposure [2]. Doxorubicin is localized in the stabilizing alginate shell of synthesized nanodroplets. By ultrasound exposure at sufficiently high rarefactional pressures [15], the PFH nanodroplets vaporize into gas microbubbles, and undergo 8

stable or inertial cavitation which results in triggered release of doxorubicin from these droplets. In fact, due to a 125-fold density difference between liquid and gaseous form PFH, by complete vaporization of the droplet under ultrasound exposure, its volume increase 125-fold which results in a 25-fold increase of surface area followed by 25-fold decrease of the initial thickness of the droplet shell. This significantly increases the drug release from stabilizing shell, especially because the drug can be “ripped off” by ultrasound. This mechanism is schematically illustrated in Fig. 4 b. The higher rate of ultrasound-aided drug release in formulation B showed that ultrasound active drug release was directly proportional to the content of PFH in the nanodroplets. As it was observed in TEM images, nanodroplets in formulation B had bigger PFH core and thinner alginate shell compared to formulation A, which could result in stronger droplet expansion and eventually more efficient drug release from the stabilizing shell. 3.5. Echogenic properties 3.5.1. droplet-to-bubble transition via injection The echogenic behavior of the PFH nanodroplets was evaluated in vitro using ultrasonic imaging. There are three factors that induce droplet-to-bubble transition in polymer stabilized perfluorocarbon nanodroplets: injection (mechanical factor) [44], heating (thermal factor) [2,3] and sonication (thermal and/or mechanical factor) [42]; which among of those, ultrasound is the most powerful [44]. Microbubbles have much higher echogenicity than nanodroplets which allows discrimination between droplets and bubbles using ultrasound images. We studied the ADV effect for formulation B via injection through a thin needle and 1-MHz ultrasound exposure at room temperature, in PBS solution and gel. However, the efficiency of droplet-to-bubble transition upon injection is not as much as ADV, but it may have clinical relevance [45, 46]. Fig. 5.a shows the control image with no nanodroplet injection. The image appeared black which demonstrates a lack of bubbles. Droplet to babble transition of alginate stabilized PFH nanodroplets upon injection into PBS solution through 26 G and 18 G needles is shown in Fig. 5.b and c respectively. It can be seen that injection through a thinner needle (26 G) is more potent in inducing droplet-to-bubble transition compared to a thicker needle (18 G). Observation of the echoes upon injection of nanodroplets into PBS solution, suggested that the injected nanobubbles coalesced into highly echogenic microbubbles. In fact, the nanodroplets first form nanobubbles, and then aggregate into microbubbles. PFH microbubbles are highly echogenic and generate relatively strong ultrasound contrast which is due to the large difference in acoustic impedances of perfluorocarbons and water. Formation of large microbubbles in tumor tissue upon direct intratumoral injection of nanoemulsion has been reported before [2,44].

3.5.2. Ultrasound-triggered droplet-to-bubble transition in gel matrices Nanodroplets in tumor tissue are surrounded by a viscous extracellular matrix which may affect their behavior including response to ultrasound irradiation. To simulate the increased viscosity of 9

tumor tissue, the nanodroplets were introduced to or agarose gel. Nanodroplet vaporization under the action of ultrasound of various frequencies was monitored. Fig. 6 presents ultrasoundinduced droplet-to-bubble transition of the nanodroplets by 28 kHz ultrasound inserted in agarose gel.

Formation of contrast microbubbles -bright specks in the ultrasound images- from PFH nanodroplets embedded in a gel matrix under the action of ultrasound is presented in Fig 6. Despite the relatively high boiling temperature of PFH (58-60 °C), nanodroplets converted into microbubbles under the action of ultrasound. The formation of microbubbles from nanodroplets by ultrasound exposure indicated that the mechanical component of ultrasound played a predominant role in inducing droplet vaporization because the temperature of sample medium under ultrasound only changed 0.5-1 °C in the experimental setting, which is not enough for purely thermal vaporization of PFH. Ultrasoundinduced droplet-to-bubble transition is called acoustic droplet vaporization or ADV. Acoustic droplet vaporization and microbubble formation in tumor tissue could be extremely beneficial for contrast-enhanced ultrasound imaging and controlled drug delivery.

3.6. Therapeutic effects of doxorubicin-loaded nanodroplets/ultrasound in cancer models In pharmacodynamic study, to demonstrate the enhanced anti-cancer effect of nanodroplets under ultrasound, tumor volume was measured 3 times a week (Fig. 7). Considerable differences were observed between the nanodroplet-ultasound treated group and other groups. Ultrasound waves can disrupt the accumulated nanodroplets in tumor tissue and release a significant amount of drug in a short time which results in considerable tumor regression (Fig 8). Moreover, ultrasound can facilitate the entry of drugs into cells by increasing the permeability of the plasma membrane and reducing the thickness of the unstirred layer at the cell surface through its nonthermal effects such as cavitation, radiation pressure and acoustic microstreaming. The tumor size of mice after 2 weeks in groups I, II, III, IV and V was 750.7±21 mm2, 314.2±14 mm2, 77.5±8 mm2, 475±23 mm2, and 1.96±1.7 mm2, respectively (Fig 8). Significant differences were shown for tumor size between control group and the other groups. Complete tumor regression was observed for the group in which doxorubicin-loaded nanodroplets were combined with ultrasound, whereas without sonication, doxorubicin–loaded nanodroplets did not cause tumor regression but they effectively inhibited the growth of tumor (IR: 89.6%). Therefore, the combination of ultrasound and the ultrasound sensitive core was the keys to obtain a highly enhanced anti-cancer effect.

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4. Conclusion Doxorubicin was successfully encapsulated into ultrasound-responsive alginate stabilized perfluorohexane nanodroplets which were able to transform into microbubbles by ultrasound exposure. Drug release study indicated the triggered release of doxorubicin from nanodroplets under the action of ultrasound. Nanodroplets possessed highly enhanced anti-cancer effects under ultrasound, and displayed long-lasting, strong ultrasound contrast. Therefore, ultrasoundresponsive alginate stabilized PFH nanodroplets could be promising carriers of anti-cancer drugs.

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Figure captions: Fig 1. In vitro drug release profiles of doxorubicin-loaded PFH/alginate nanodroplets with different PHF volume fractions at normal (7.4) and acidic pH (5.5) (data represent mean ± standard deviation, n = 3). Fig 2. TEM images of formulation a) E1 and E2 with different PFH volume fractions. Fig 3. Changes in a) particle size and b) entrapment efficiency of the doxorubicin-loaded PFH

nanodroplets incubated at 4 °C for predetermined time intervals. Fig 4. a) In vitro release profiles of doxorubicin from formulations A and B without ultrasound (control) and with ultrasound exposure (28 kHz, 0.34 w/cm2) in pH 7.4, at 37 °C for 10 min, data are the mean ± SD (n = 3); b) Schematic illustration of drug transfer from nanodroplets through microbubbles into cells under the action of ultrasound. Fig 5. Ultrasound images of a) pure PBS (Control), and alginate/PFH nanodroplets (formulation A) injection into PBS through a 26 G needle (b) or 18 G needle (c). Bubbles formed when nanoemulsion was injected through a thin needle are visualized as bright spots; Injection through the thin needle results in immediate formation of very bright bubbles whose size and brightness increase with time; Fig 6. Ultrasound image of PFH nanodroplet-loaded agarose gel; 1 % agarose solution in PBS was mixed with 0.2 ml PFH nanodroplet emulsion at 45 °C. The mixture was cooled to room temperature to solidify.

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Fig 7. Tumor growth/regression curves for breast cancer bearing mice treated with physiological saline (control), doxorubicin, doxorubicin-loaded PFH nanodroplets, ultrasound and doxorubicin-loaded PFH nanodroplets with ultrasound. Doxorubicin dose was 2 mg/kg of body weight. Data are the mean ± SD (n = 5). Fig 8. Photographs of mice bearing breast carcinoma tumors 2 weeks after the treatment; a) control mouse, b) mouse treated with ultrasound, c) mouse treated with doxorubicin-loaded PFH nanodroplets and, d) mouse treated with doxorubicin-loaded PFH nanodroplets and ultrasound exposure.

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20

21

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Table captions:

Formulation code

Alginate (%w/v)

Tween 20 (%v/v)

PFH (%v/v)

Doxorobicin (µg)

Particle Size (nm ± SD)

Mean Polydispersity index

zetapotential

Entrapment Efficiency (% ± SD)

Cumulative Released at 24 H (% ± SD)

A

0.1

0.3

5

250

39.2 ±3

0.19

- 32.72

92.2±2.1

12.6±0.41

B

0.1

0.3

10

250

102.3±11

0.27

- 35.37

90.1±1.4

9.4±0.32

All ingredients were added to DD water with final volume of 3 ml.

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