Ultrasound-Induced Reactive Oxygen Species Mediated Therapy and

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Ultrasound-Induced Reactive Oxygen Species-Mediated Therapy and Imaging Using a Fenton Reaction Activable Polymersome Wei-Peng Li, Chia-Hao Su, Yi-Ching Chang, Yu-Jiung Lin, and Chen-Sheng Yeh ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b06175 • Publication Date (Web): 31 Dec 2015 Downloaded from http://pubs.acs.org on January 12, 2016

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Ultrasound-Induced Reactive Oxygen Species-Mediated Therapy and Imaging Using a Fenton Reaction Activable Polymersome Wei-Peng Li,†,♯ Chia-Hao Su,§,♯ Yi-Ching Chang,† Yu-Jiung Lin,† and Chen-Sheng Yeh†,* †

Department of Chemistry and Advanced Optoelectronic Technology Center, National Cheng Kung University, Tainan 701, Taiwan §

Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung

Memorial Hospital, Kaohsiung 833, Taiwan ♯

W. P. Li and C. H. Su contributed equally.

*Correspondence author E-mail: [email protected] KEYWORDS: hydrogen peroxide · polymersome ·

ultrasound ·

magnetic

resonance imaging ·sonodynamic therapy ABSTRACT Ultrasound technique has been extensively employed in diagnostic purpose. Because of its features of low-cost, easy access, and noninvasive real-time imaging, toward clinical practice it is highly anticipated to simply use diagnostic ultrasound to concurrently perform imaging and therapy. We report a H2O2-filled polymersome to display echogenic reflectivity and reactive oxygen species-mediated cancer therapy simply triggered by the micro-ultrasound diagnostic system accompanied with MR imaging. Instead of filling common perfluorocarbons, the encapsulation of H2O2 in H2O2/Fe3O4-PLGA polymersome provides O2 as echogenic source and •OH as 1

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therapeutic element. On exposure to ultrasound, the polymersome can be easily disrupted to yield •OH through the Fenton reaction by reaction of H2O2 and Fe3O4. We showed that the malignant tumors can be completely remove in a non-thermal process.

Because of its safety, non-invasive real time visualization, painless operation to patients, and easy accessibility, ultrasound (US) has been extensively employed for disease diagnosis. Beside diagnostic purpose, the focused US is often employed to destruct carriers for drug release or to ablate malignant tumor through the hyperthermia. Thus, a careful manipulation in focused US is necessary to avoid the damage of the surrounding healthy tissues around the targeted site. Using US diagnostic system provides the advantages of the harmless to the healthy tissues and the painless course of treatment to the recipients. Micro-US has been an important tool at the stage of preclinical imaging to study models of human disease in small animals for the facilitation of biomedical discovery. In this context, we are motivated to begin with micro-US diagnostic system to concurrently perform imaging and therapy in a non-thermal condition.

US imaging is generated based on the acoustic impedance difference between the body of the structures to reflect US waves. The US imaging can be enhanced by the 2


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employment of the gas-filled capsules (gas: like perfluorocarbon, nitrogen) made of lipid or polymer compositions.1-5 Encapsulation of low boiling point perfluorocarbon that is thermosensitive liquid to vaporize as gas has also been reported recently. 6-9 Instead of filling gaseous or low boiling point perfluorocarbons, we present a cost-effective liquid H2O2 loaded US contrast agent in a form of H2O2/Fe3O4-PLGA (PLGA: poly(lactic-co-glycolic acid)) polymersome encapsulating H2O2 in the hydrophilic core with Fe3O4 nanoparticles (NPs) packed in the shell of the polymersome (Figure 1a). The liquid H2O2 can be readily encapsulated by the hydrophilic core of the polymersome. Importantly, the encapsulation of H2O2 presents a crucial role to concurrently provide O2 for echogenic reflectivity and •OH as therapeutic reactive oxygen species (ROS). Upon the exposure of the micro-US diagnostic system, the encapsulated H2O2 in the core was liberated and ran through the disruption of PLGA polymersome to react with Fe3O4 packed inside the polymersome membrane, thus yielding •OH following a Fenton reaction. Because of the presence of Fe3O4, the H2O2/Fe3O4-PLGA polymersome was able to give additional MR imaging capability.

RESULTS

Synthesis and Characterization of Polymersomes. H2O2 and trisodium diphosphate (TSPP, stabilizer) were loaded into the hydrophilic core of PLGA polymersome 3


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during the first emulsion process (w/o). Subsequently, Fe3O4 NPs (10.6 ± 0.8 nm) (Figure S1) were embedded into the hydrophobic shell of the PLGA polymersome during the second emulsion process (w/o/w). The TEM images indicated that the synthesized Fe3O4-PLGA (412 ± 86 nm, TEM measurements) exhibited a spherical shape (Figure 1b,c), and the 10-nm Fe3O4 NPs were successfully embedded in these vesicles. During the first emulsion process (w/o), the concentrations of H2O2 could be adjusted to determine the amount of H2O2 that can be loaded in the polymersomes. Three different concentrations of H2O2 (200, 400, and 600 µL) were loaded into the H2O2/Fe3O4-PLGA, and the effect of H2O2 concentrations did not show visible change in the structural morphology and size (Figure 1d,e and Figure S2). The dynamic light scattering (DLS) analysis indicated that the hydrodynamic diameter slightly increased when loaded with H2O2 (Table S1). A colorimetric test involving the formation of yellowish I3- was employed to determine the concentration of the encapsulated H2O2, giving the H2O2 concentrations of H2O2(200 µL)/Fe3O4-PLGA, H2O2 (400 µL)/Fe3O4-PLGA, and H2O2 (600 µL)/Fe3O4-PLGA solutions as 3.5, 8.6, and 14.3 mM, respectively (Figures S3 and S4). The scanning electron microscope (SEM) images showed the spherical morphology for the H2O2(600 µL)/Fe3O4-PLGA polymersomes (Figure 1f). Mapping and line scan modes indicated that the Fe signal was concentrated in the shell region of the PLGA polymersome (Figure S5). A 4


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cryo-transmission electron microscope further analyzed the cross-sectional area of the H2O2(600 µL)/Fe3O4-PLGA with the iron oxides mainly distributed in the shell of the PLGA polymersome accompanied with the scattered Fe3O4 NPs in the core of the vesicle (Figure 1g). The structure, surface identity, and magnetic property were characterized using XRD, FT-IR, and SQUID measurements (Figures S6 and S10). To provide a beneficial surface to prevent the particles from being engulfed by macrophages in the blood,10 the positively charged BSA engaged in a favorable electrostatic interaction with negatively charged PLGA, thus modifying the surface of PLGA polymersomes. In this study, the concentration of BSA adsorbed on the H2O2/Fe3O4-PLGA was 1.63 µg BSA in 100 ppm H2O2/Fe3O4-PLGAs (Figure S11). To assess the safety of the synthesized polymersomes, we fed different concentrations of the H2O2(600 µL)/Fe3O4-PLGA and Fe3O4-PLGA to the HeLa cells. After 1 day of incubation, the polymersomes were non-cytotoxic to HeLa cells (Figure S12). The stability evaluation indicated that the H2O2(600 µL)/Fe3O4-PLGA can be dispersed in phosphate buffered saline (PBS, pH 7), PBS (pH 5), Dulbecco’s modified Eagle medium (DMEM), and serum (10% fetal bovine serum) without forming precipitation for 7 days storage at 37 oC. However, the small pores were generated on the surface of the polymersomes in the PBS (pH 5), suggesting that an acidic environment induced the degradation of PLGA polymersomes (Figures S13 and S14). H2O2/Fe3O4-PLGA 5


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can thus be treated as the pH-sensitive drug delivery nanocarriers for slowly releasing therapeutic substances in the acidic environments.

Behavior of the Polymersomes upon US Trigger and MR Imaging Relaxivity. Following micro-US diagnostic probe (VisualSonics, 40 MHz) irradiation, the polymersomes exhibited US-triggered disruption behavior. The sonication process facilitated progressively destruction of polymersomes as exposure time prolonged (Figure 2a). A complete collapse of the polymersome structure was seen upon 30 min of US irradiation. It is noted that the destruction of the polymersomes was seen as well under the sonication of 43 KHz at 37 oC (DELTA, ultrasonic cleaner D200H). To evidence the presence of O2 in the resulting polymersomes, an indicator Ru(dpp)3Cl2 was sensitive to react with O2, leading to the reduction of its fluorescence intensity. The H2O2 encapsulated PLGA (H2O2-PLGA) polymersome without Fe3O4 was prepared and subjected to react with Ru(dpp)3Cl2. The fluorescence of Ru(dpp)3Cl2 showed apparent drop when H2O2(600 µL)-PLGA exposed to US irradiation for 30 min resulting in the destruction of the polymersomes to release O2, while lack of US trigger gave no reduction of florescence of Ru(dpp)3Cl2 after 30 min because of the absence of O2 appearance (Figure 2b). We have performed O2 generation with and without US as a function of the storage period. We separately prepared two groups of H2O2-PLGA and H2O2(600 µL)/Fe3O4-PLGA polymersomes to monitor the O2 6


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generation. TSPP has been added to stabilize H2O2 in our formulation. Without US exposure, no O2 generation was detected for 2 days of storage. Once triggered by US, the fluorescence reduction was observed and remained the same in the course of 2 days, indicating that O2 generation was independent of the storage period. (Figure S15). Without the addition of TSPP resulted in the excessive O2 to drastically swelling of polymersome leading to the collapse of the vesicle structure. The polymersomes were not stable showing 20% of the collapsed structures following the immediate preparation and no complete spherical morphology was seen after 3 day incubation in PBS(pH 7). Subsequently, we examined the in vitro echogenic performance of the H2O2(600 µL)/Fe3O4-PLGA polymersomes in the agar gel phantom (Figure 2c). Fe3O4-PLGA in the absence of H2O2 was transparent to US under US exposure. In contrast, considerable echo signals, captured in conventional B-mode, were observed immediately after H2O2(600 µL)/Fe3O4-PLGA injection into gel, indicating that the generation of O2 was responsible for US resonation. The contrast enhancement can last for at least 30 min. Because Fe3O4 NPs could be used as T2 contrast agents for magnetic resonance imaging (MRI), Fe3O4-PLGA and H2O2(600 µL)/Fe3O4-PLGA polymersomes as well as Fe3O4@PLGA NPs were prepared in agarose gel at various iron ion concentrations. For Fe3O4@PLGA NPs, the surface of the Fe3O4 NPs was coated with PLGA and transferred the coated NPs into a water phase solution (Figure 7


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S16). A 1.4 T contrast agent analyzer mq60 system (Bruker Optik GmbH, Germany) measured r2 values of the Fe3O4-PLGA, H2O2(600 µL)/Fe3O4-PLGA, and Fe3O4@PLGA as 524.7, 528.9, and 73.8 mM−1s−1, respectively (Figure 2d). Significantly, many Fe3O4 NPs clustered inside the polymersome shell resulted in large relaxivity value as opposed to the dispersed Fe3O4@PLGA NPs. No noticeable effect in relaxivity was found to encapsulating H2O2 in polymersome between Fe3O4-PLGA and H2O2(600 µL)/Fe3O4-PLGA.

The Evidence of •OH Generation and cytotoxicity under different US frequency. It is known that H2O2 reacts with Fe2+ in the presence of iron as a catalyst to produce highly reactive •OH and hydroxide (OH−), called Fenton reaction.14-16 Because of the deformation of polymersome upon US trigger, the encapsulated H2O2 can infiltrate and run through the degraded PLGA to react with Fe3O4 packed inside the polymersome membrane yielding •OH. To ensure that the Fe3O4 NPs can react with H2O2 and generated hydroxyl radicals, a parallel experiment was conducted using Fe3O4@PLGA (Figure 3a). A 3’-(p-aminophenyl) fluorescein (APF) was used as the reagent for effectively detecting •OH by reaction of APF with •OH to emit intense fluorescent light of 520 nm, which can then be measured to determine the formation of •OH. Once again, TSPP provided the certain degree of stabilization of H2O2. The solution containing Fe3O4@PLGA NPs and H2O2 rapidly generated hydroxyl radicals, 8


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and the fluorescence of the solution was increased 2.5 times after 15 min of reaction time, verifying that a mixture of Fe3O4 NPs and H2O2 can undergo a Fenton reaction. Adding TSPP to the solution containing Fe3O4@PLGA NPs and H2O2 increased the fluorescence of the solution by 1.8 times. Before further experiments were performed, two experiments were conducted to evidence that H2O2 (core) was successfully separated from the Fe3O4 nanoparticles by PLGA shell in polymersomes and the enhanced •OH was generated when exposure of US due to the Fenton reaction. We have prepared H2O2/Fe3O4-PLGA polymersomes incubated in H2O containing APF without exposure of US for the observation of 2 days. To evidence the •OH generation, H2O2/Fe3O4-PLGA polymersome solutions were centrifuged and the supernatants were subjected to observe the APF fluorescence. Following this process, the fluorescence of APF was indeed observed without US irradiation, but the intensity remained constant for 2 days of observation. However, the significantly enhanced fluorescence was seen when the polymersome solutions experienced US irradiation at the end of 2 day incubation (Figure S17). The initial constant fluorescence is anticipated from the presence of the scattered Fe3O4 NPs in the core of the polymersomes, as seen in Figure 1g. To strengthen our observation, we have conducted additional experiment by encapsulation of APF in the core of H2O2/Fe3O4-PLGA polymersomes. With slight centrifugation to remove excess 9


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solution, the APF-H2O2/Fe3O4-PLGA polymersomes in 96-well were subjected to the laser confocal spectrometer for the direct fluorescence observation. Once again, APF-H2O2/Fe3O4-PLGA polymersomes were stored in H2O for 2 days of observation. Because

of

the

APF

fluorescence,

we

have

seen

that

the

intact

APF-H2O2/Fe3O4-PLGA polymersomes revealed as the spots manner with weak intensity without US irradiation (Figure S18). Those fluorescence remained the same intensity in the course of 2 days that was consistent with the aforementioned results due to the reaction of the scattered Fe3O4 NPs in the core with H2O2. Upon the exposure of US, we can observe the larger spots with intense fluorescence, which is expected from the destruction of polymersomes in an aggregated form. Accordingly, our formulation can successfully perform enhanced •OH generation upon US irradiation. To further understand how H2O2/Fe3O4-PLGA polymersomes generate hydroxyl radicals affected under a US field (40 MHz), we irradiated H2O2/Fe3O4-PLGA as a function of exposure time to observe the formation of hydroxyl radicals (Figure 3b). Longer US irradiation received by H2O2 (600 µL)/Fe3O4-PLGA polymersomes led to more •OH production because of the increased destruction in structure resulting in more intensive Fenton reaction. In addition, more H2O2 loaded in polymersome was responsible for greater •OH production.

10


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We have further conducted the acoustic frequency dependent studies. The influence of generation of •OH and therapeutic outcome of H2O2/Fe3O4-PLGA polymersomes were compared with frequencies of 40 and 55 MHz under 100% power output using micro-US diagnostic system. Based on the APF fluorescence enhanced to evidence •OH, 40 MHz showed showed better performance in generation of •OH compared to 55 MHz (Figure 4a). For the therapeutic comparison, H2O2/Fe3O4-PLGA polymersomes were fed with HeLa cells to receive US exposure. MTT assays indicated that 40 MHz resulted in 13% cell viability, while 55 MHz displayed less capability of killing cancer cells with higher ~20% viability (Figure 4b).

In Vivo US Imaging upon US Irradiation and MR Imaging. Next, an intratumoral injection was arranged to demonstrate potential of the H2O2 (600 µL)/Fe3O4-PLGA polymersomes for US imaging of the tumor (Figure 5a). The HeLa cell carcinoma tumor-bearing nude mice received 5 mg [Fe]/kg dosage. The contrast under B-mode (VisualSonics, 40 MHz) was recorded at different time intervals (5, 15, and 30 min) that showed apparent contrast enhancement and the quantitation of the gray-scale increased to maximum intensity within 5 min under US field. Although the echo signal displayed a descendent trend as exposure time prolong, the continuous imaging could be up to 30 min that was consistent with the in vitro agar gel phantom. A further evaluation of the US echogenic performance of H2O2 (600 µL)/Fe3O4-PLGA was 11


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carried out for intravenous administration through tail vein injection. Because H2O2(600 µL)/Fe3O4-PLGAs featured a favorable magnetic property, with and without magnet attractions were individually conducted for comparison of contrast enhancement. The tumor-bearing mice received two subsequent intravenous injections with each dosage of 5 mg [Fe]/kg. For magnetic attraction, the magnet was placed on top of the tumor for 20 min after each injection. As shown in Figure 5b, immediately after 2nd injection of polymersomes, the US contrast of the tumor tissue (size: 405 mm3) brightened and the gray-scale intensity increased. The enhancement gradually reached a peak at 15 min, and then a slight drop after 30 min exposure. Contrarily, we could not observe any contrast enhancement at tumor tissue in the group of nil magnet in the period of 30 min for US irradiation (Figure S19). The other group of mice carrying smaller tumor (size: 4 mm3) also exhibited similar contrast enhancement in the period of 30 min, except the optimal echo signal appeared at 5 min (Figure S20). Figure 5c shows the MR imaging visibility to monitor polymersomes accumulation following the same operation for intravenous injection. The tumor (size: 405 mm3) was subjected to the measurements using a 9.4 T animal micro MRI system. After 2nd-post injection, the tumor contrast became darkened and the signal continuously reduced to 77 % after exposure of US for 30 min. Although the significant polymersomes were disrupted based on the observation of Figure 2a after 30 min of 12


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US exposure, the released Fe3O4 NPs remained inside the tumor to contribute MR imaging. For the group with smaller tumor (size: 4 mm3), the contrast change behaved similarly (Figure S21). No significant contrast negative effect was seen at tumor site in the absence of magnetic attraction (Figure S22).

Antitumor Efficacy upon US Irradiation and Toxicity Evaluation. The overproduction of ROS can result in oxidative damage of cell functions.17-19 In addition, cancer cells are more vulnerable than normal cells to the damage caused by the additional ROS. Thus, the generation of •OH upon US trigger provides an advantageous benefit to possibly suppress tumor growth for cancer therapy. The regression efficacy in tumor growth was monitored in terms of tumor volume change (Figure 5d). HeLa cells were transplanted hypodermically in the thighs of the nude mice. Fifteen nude mice bearing HeLa tumors were divided into five groups. Following the same administration procedure as used in US and MR imaging, the mice received two sequential injections through tail vein at a dosage of 5 mg [Fe]/kg. Taking together the information acquired from TEM, US, and MR imaging, the groups with mice treated with PBS + US, polymersomes + US and polymersomes/magnet + US all experienced 30 min irradiation of US after each intravenous injection. Hence, the tumor-bearing mice totally received 1 h exposure of US after following two sequential injections. Because we extended MR imaging 13


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tumor tissues at post-24 h in, it was found that the contrast continued to improve the negative contrast effect that might suggest the possibly continuous accumulation of polymersomes to tumor (Figure 5c). The US-treated mice received additional US exposure for 1 h on 2nd day. On the whole, the groups under magnetic attraction exhibited statistically significant in antitumor efficacy, indicating the important therapeutic factor for the amount of the material localized in the tumor. When the magnetic attraction was applied without US irradiation, tumor growth was inhibited in the initial 8 days, but they relapsed slowly as days prolonged. This initial suppression was likely induced from the degradation of the polymersomes to release •OH in the tumor acidic environment, as seen in Figures S13 and S14. Contrarily, the focus of the magnetic target for polymersomes at the tumor along with US irradiation led to complete suppression of tumor growth. Both US triggering and polymersomes degradation in acidic condition concurrently contributed this effective antitumor efficacy. Thermographic analysis of tumor temperature using a Thermo Tracer H2640 (NEC, Japan) camera was performed to monitor tumor in the course of US exposure (30 min) and indicated the temperature to remain at ~22 oC showing no sign of increase, suggesting a non-thermal process under US field (Figure S23). For the toxic evaluation, H2O2(600 µL)/Fe3O4-PLGA on healthy mice without tumor was evaluated through tail vein injection at the dosage of 5 mg [Fe]/kg. All the mice stayed alive for 14


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the

experimental

period

of

30

days.

The

biodistribution

of

H2O2(600

µL)/Fe3O4-PLGA was monitored and compared with the results obtained by injections of H2O2(600 µL)-PLGA and PBS which reflect the native iron distributed in mice. It indicated that the H2O2(600 µL)/Fe3O4-PLGA can be gradually eliminated within a month (Figure S24). The histological and blood biochemical analysis showed no acute toxicity as a cancer medicine (Figures S25 and S26).

DISCUSSION

In this study, we have developed a theranostic polymersome sensitive to US irradiation. Different from the previous reports filling gaseous or low boiling point perfluorocarbons, a liquid H2O2 can be an effective US source. Encapsulation of H2O2 presents a crucial role to concurrently provide O2 for echogenic reflectivity and •OH as therapeutic ROS species. Importantly, the generation of long echo signal and disruption of polymersome yielding ROS through the Fenton reaction were simply triggered by the micro-US diagnostic system via a non-thermal process. Usually, the focused US is employed to destruct carriers for drug release or to ablate malignant tumor through the hyperthermia. Thus, a careful manipulation in US is necessary to avoid the damage of the surrounding healthy tissues around the targeted site. Using the micro-US diagnostic system as demonstrated in this study provides the advantages

15


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of the harmless to the healthy tissues and the painless course of treatment to the recipients.

At the current stage, understanding the polymersome destruction mechanisms remains to be resolved and requires extensive further work. However, the behavior of the H2O2/Fe3O4-PLGA polymersomes rupture may be associated with the inclusion of Fe3O4 NPs in the polymersome shell. Frinking and co-workers nicely demonstrated that the acoustic properties of microbubble US contrast agents strongly depend on the shell stiffness and thickness as well as microbubble sizes.ref When insonated at resonance frequency, microbubbles compress and oscillate to undergo large variation inducing microbubbles destruction. Importantly, the shell properties, i.e. stiffness and thickness, strongly affect resonance frequency. The thicker and more rigid shell notably increased resonance frequency. We propose that the inclusion of Fe3O4 resulting in the change of the mechanic property of the PLGA polymersome leading to the increased rigidity in the shell of PLGA polymersomes might create the resonance frequency upon US exposure under VisualSonics (40 MHz). Accordingly, we have performed the parallel experiments to monitor the rupture of PLGA (PLGA polymersome without H2O2 and Fe3O4), H2O2-PLGA (H2O2 encapsulated PLGA polymersomes without Fe3O4 in the shell) and Fe3O4-PLGA (Fe3O4 in the PLGA polymersomes shell without H2O2), which were compared with H2O2/Fe3O4-PLGA 16


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polymersomes under US exposure at 40 MHz for 0, 5, 15 and 30 min (Figure 6). The TEM images showed that Fe3O4-PLGA revealed the same destruction behavior as those of H2O2/Fe3O4-PLGA polymersomes. On the other hand, the structures of both PLGA and H2O2-PLGA polymersomes in the absence of Fe3O4 in the shell displayed intact throughout the period of US irradiation. Fe3O4 NPs embedded creating the higher hardness PLGA shell could have varied the oscillation frequency upon US exposure.

Considering the therapeutic treatment, the sonodynamic therapy is a cure that is recognized to rely on the generation of ROS by the combination of US exposure and the existence of the sensitizer.20-22 Unfortunately, the exact mechanisms remain to be dubious and have been proposed that the cavitation effect upon US trigger leads to either sonoluminescence or pyrolysis process resulting in the ROS production from sensitizer. Instead of using sensitizer following the uncontrolled mechanisms, our ROS-mediated therapeutic strategy derived from our designed polymersome can be viewed as a new type of the sensitizer-free sonodynamic approach to completely supress tumor growth in mice model with the capability of showing controllable ROS (•OH) generation, by the adjustment of the encapsulation amount of H2O2, from the well-known Fenton reaction mechanism.

In addition, the degradation property in

acidic condition presents a synergistic effect for H2O2/Fe3O4-PLGA to act as the 17


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pH-sensitive carrier giving a great facilitation against malignant tumor following the external US stimulus because the un-destruction polymersomes would slowly degrade to release •OH as time prolonged.

Finally, the presence of O2 has been a limitation for radiotherapy, such as X-ray and gamma ray, and photodynamic treatment. For example, in radiotherapy, radiation is used to produce radicals on deoxyribonucleic acid (DNA) that cause further oxidation under aerobic conditions, leading to DNA double strands breaking, while the photodynamic therapy requires a photosensitizer to generate singlet oxygen upon light irradiation. The need of O2 has critically limited their applications in the hypoxia region in solid tumors. Thus, the presence of O2 in H2O2/Fe3O4-PLGA has an important implication in cancer therapy to deliver these polymersomes against hypoxia environment to raise antitumor efficacy.

CONCLUSION

Toward the practical practice, we report a promising polymersome, which is a form of H2O2/Fe3O4-PLGA polymersome encapsulating H2O2 in the hydrophilic core with Fe3O4 nanoparticles packed in the shell of the polymersome, to simply use the diagnostic micro-US system for concurrently conducting imaging and therapy. Instead of filling gaseous or low boiling point perfluorocarbons, a cost-effective liquid H2O2 loaded polymersome was designed to display echogenic reflectivity and reactive 18


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oxygen species (ROS)-mediated cancer therapy triggered by the micro-US diagnostic system accompanied with MR imaging. The encapsulation of H2O2 presents a crucial role to concurrently provide O2 for echogenic reflectivity and •OH as therapeutic ROS species. The disruption of polymersome yielding •OH through the Fenton reaction simply initiated by the micro-US diagnostic system via a non-thermal process presents a newly developed sensitizer-free sonodynamic therapy to completely supress tumor growth in mice model.

METHODS Materials. All reagents were of analytical purity and used without further purification. iron(III) acetylacetonate (Fe(C5H7O2)3 , 99.9%), trioctylamine (TOA, [CH3(CH2)7]3 N, 98%), oleic acid (OA, CH3(CH2)7CHCH-(CH2)7COOH, 90%), nitric acid (HNO3, 65%), tetrasodium pyrophosphate (TSPP, Na4P2O7, 95%), poly(vinyl alcohol) (PVA, M.W.= 47000, 98%), hydrogen peroxide solution (H2O2, 30%), potassium iodide (KI, 99.5%),

albumin

from

bovine

serum

(BSA,

98%),

and

3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, C18H16BrN5S, 97.5%) were used as purchased from Sigma-Aldrich. Poly(lactic-co-glycolic acid) (PLGA, 5050 DLG 4.5A, M.W.= 66 kDa) were bought from Evonik Industries. Hydrochloric acid (HCl, 36%) was obtained from BASF. Aminophenyl fluorescein solution (APF, C26H17NO5, 98%) was purchased from Life Technologies. Dichloromethane (CH2Cl2, 99.5%), and hexanes (C6H14, 99.9%) were bought from MACRON. Chloroform (CHCl3, 99.8%) was obtained from MERCK. Ethanol (C2H5OH,

99.9%)

was

purchased 19


from

J.T.Baker.

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Tris(4,7-diphenyl-1,10-phenanthroline)

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ruthenium(II)

dichloride

(Ru(dpp)3Cl2,

C72H48Cl2N6Ru) was obtained from Alfa Aesar. Water was obtained by using a Millipore direct-Q deionized water system throughout all studies. Characterization. Morphology and characterization of the nanoparticles and polymersomes were monitored by the transmission electron microscopy (TEM, Hitachi H-7500) and high-resolution transmission electron microscopy (HR-TEM, JEOL JEM-2100F). Surface analysis of the polymersomes was observed by the high-resolution scanning electron microscope (HR-SEM, JEOL JSM-7001F). UV-Vis spectra (protein assay, XO and I3-) were recorded on a UV-Vis absorption spectrometer (Hewlett-Packard Model 8453). X-ray diffraction signal of the materials was obtained by the X-ray diffractometer (XRD, Shimasz Cu Kα radiation (λ = 1.54060 Å), 30 kV, 30 mA). Concentrations of the materials were measured by atomic spectrometer (Thermo Scientific, iCE 3000 Series AA spectrometer). Magnetic properties of the polymersomes were measured by superconducting quantum interference device vibrating sample magnetometer (SQUID, Quantum Design MPMS). FT-IR spectra of the polymersomes was measured by Fourier transform

infrared

spectrometer

(FT-IR,

JASCO

200E).

A

fluorescence

spectrophotometer (HORIBA, Fluoromax-4) was used to measure the emission intensity of APF. The quantification of cell viability was performed using an enzyme-linked immune-sorbent assay reader (ELISA reader, Thermo Scientific Multiskan EX). The dynamic light scattering spectrometer (DLS, MALVERN Nano-ZS90) was used to measure the zeta potentials and the hydrodynamic diameters of polymersomes. Thermogravimetric analysis (TGA, Seiko SSC 5000) was used to measure the weight loss of polymersomes. Preparation of 10 nm and 22 nm Fe3O4 NPs. The 10 nm Fe3O4 nanoparticles were prepared by the thermal decomposition method. The 0.5 g iron(III) acetylacetonate, 20


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0.52 ml oleic acid and 20 ml trioctylamine were mixed in a round-bottomed flask, and then heated at 150 °C for 30 min to dewater, following a decrease of temperature to 120 °C. Using the suction pump to degas for 30 min was followed by an increase of the temperature to 305 °C (rise rate: 1.7°C/per min) under full argon condition. Keeping the reaction in 305 °C for 30 min and then cooled to room temperature. The iron oxide nanoparticles were collected by the centrifugation (8000 rpm, 10 min) and then washed using toluene at least 3 times. For preparation of 22 nm Fe3O4 nanoparticles, the amount of iron(III) acetylacetonate was increased to 1.4 g from 0.5 g. Following the same process, the uniform 22 nm Fe3O4 nanoparticles were obtained and then dried to further characterization. Preparation of Fe3O4@PLGA Nanoparticles. The 1000 ppm of 10 nm Fe3O4 NPs was washed by alcohol. After centrifugation, the precipitates were dispersed in 100 µL chloroform containing 1 mg PLGA. Add the Fe3O4 NPs solution dropwise to 10 mg/ml polyvinyl alcohol (PVA) and then the mixture was sonicated for 2 h. Subsequently, the as-prepared Fe3O4@PLGA nanoparticles were dispersed in water phase solution after a wash process conducted at least 3 times. Preparation of Fe3O4-PLGA Polymersomes. The PLGA polymersomes can be prepared through a double emulsion (water/oil/water) method. The core area of the polymersome is hydrophilic, whereas the space between the core and the outer shell is hydrophobic. Therefore, the oiled phase Fe3O4 nanoparticles can embed in the space between the PLGA double-shell. First, we prepared the oil phase solution containing 4 mL dichloromethane with 10 mg/mL of PLGA. The 8 mg PVA was dissolved in 0.8 mL diH2O (deionized water) as water phase solution. For the first emulsion process, the oil phase solution was slowly added to PVA solution under sonication accompanied with ice-water bath condition for 2 h. This process can generate w/o PLGA polymersomes. Next step, the 1400 ppm Fe3O4 NPs were dispersed in 0.5 mL 21


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dichloromethane and then added to the first emulsion solution. To embed the Fe3O4 NPs into PLGA shell, we first let emulsion solution containing Fe3O4 NPs dropwise in 12 mL (10 mg/ml) PVA solution for the second emulsion conducted using a homogenizer in ice bath for 20 min to form w/o/w Fe3O4 -PLGA polymersomes. The products were purified by centrifugation (1000 rpm, 1 min) to remove micro-sized PLGA (the precipitate). Subsequently, the supernatants were centrifuged (3000 rpm, 1 min) again to collect precipitates, Fe3O4 -PLGA polymersomes, which were subjected to wash using an ultrapure water. Finally, the bovine serum albumin (BSA) was chosen to conjugate on the surface of polymersomes. Added 60 µg BSA in 8 mL solution containing 10 ppm Fe3O4-PLGA and then shaked the solution for 2 h. The less amount of Fe3O4 NPs in Fe3O4-PLGA was prepared in the same emulsion manner, but the Fe3O4 NPs concentrations was changed from 1400 ppm to 600, 140 or 30 ppm. The number of the embedded-Fe3O4 NPs in the Fe3O4-PLGA polymersomes was obtained from the average number of Fe3O4 NPs by calculation of five respective Fe3O4-PLGA polymersomes shown in the TEM images. The results indicated that the amount of Fe3O4 NPs can be effectively controlled. Preparation of H2O2 (200, 400, or 600 µL)/Fe3O4-PLGA Polymersomes. First, we prepared the oil phase solution containing 4 mL dichloromethane with 10 mg/mL PLGA. The water phase solution containing 8 mg PVA was dissolved in x mL diH2O (x = 0.6 for 200 µL, 0.4 for 400 µL, and 0.2 for 600 µL) with y µL 30% H2O2 (y = 200 for 200 µL, 400 for 400 µL, and 600 for 600 µL) and 10 µL 0.1M TSPP. The first emulsion process, the oil phase solution was slowly added in PVA solution under sonication accompanied with ice-water bath condition for 2 h. This process can generate w/o PLGA polymersomes. Next step, the 1400 ppm Fe3O4 NPs were dispersed in 0.5 mL dichloromethane and then added to the first emulsion solution. To embed the Fe3O4 NPs into PLGA shell, we first let emulsion solution containing 22


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Fe3O4 NPs dropwise in 12 mL (10 mg/ml) PVA solution for the second emulsion performed using a homogenizer in ice bath for 20 min to form w/o/w H2O2/Fe3O4-PLGA polymersomes. The products were purified by centrifugation (1000 rpm, 1min) to remove micro-sized PLGA (the precipitate). Subsequently, the supernatants were centrifuged (3000 rpm, 1 min) again to collect precipitates, H2O2/Fe3O4-PLGA polymersomes that were subjected to wash using an ultrapure water. Finally, BSA was modified on the surface of the polymersomes following the aforementioned process. Quantitation of BSA on Polymersomes from the Protein Kit. The calibration curve was obtained from the absorbance of protein kit (600 nm) after a mix with different concentrations of BSA solution. The 60 µg BSA was dissolved in 8 mL diH2O as a control group. Replace 8 mL diH2O to 8 mL of 10 ppm H2O2(600µL)/Fe3O4-PLGA for BSA-modification. After 2 h conjugation, the supernatants were obtained by centrifugation (3000 rpm, 1min). The supernatants with BSA content was analyzed by the addition of 25 µM protein kit for 5 min reaction and then the absorbance of supernatants was measured by UV-Vis spectrometer. Finally, the amount of modified-BSA was determined through a subtraction between the control group and the supernatant solution. Ultrasection of Polymersomes. Sample were prefixed in 2.5% glutaraldehyde for 1.5 h and then washed twice with diH2O. Dehydration was then performed in an ascending series of ethanol concentrations. Sample was polymerized using Spurr resin at 68℃ for 15 h. Ultrathin sections were carried out with Leica UC7 ultramicrotome and observed with a FEI Tecnai F20 transmission electron microscope (TEM) at 120 kV accelerating voltage. Sample handling and images taken were assisted by the Bio MA-TEK, Taiwan.

23


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Analysis of Fenton Reaction for Fe3O4@PLGA NPs. The APF dye was used as an indicator for the generation of hydroxyl radicals that were produced from Fenton reaction. To prepare the solution contained 10 µM APF + 1 mL diH2O as a control group. The 1 mL diH2O was then replaced to 10 mM H2O2, 10 mM H2O2 + 1 mM TSPP (stabilizer), 100 ppm Fe3O4@PLGA NPs + 10 mM H2O2, and 100 ppm Fe3O4@PLGA NPs + 10 mM H2O2 + 1 mM TSPP as experiment groups. After reaction of 0 min, 1 min, 3 min, 5 min, 10 min and 15 min under dark condition, we centrifuged (10000 rpm, 1 min) the solutions to obtain APF supernatants. The 520 nm fluorescence intensity of supernatants was measured to reflect the quantity of hydroxyl radicals. The relative ratio of fluorescence intensity was obtained from APF dye (ex/em: 490/520 nm) before reaction (I0) and after reaction (In) at pH 7 condition. All of the date was made in triplicate. Analysis of Oxygen Produced from H2O2 Redox Reaction in Polymersomes. The inorganic complex of Ru(dpp)3Cl2 was used as an indicator for the generation of oxygen that were produced from the H2O2 redox reaction. The solution containing 40 µM Ru(dpp)3Cl2 + 1 mL H2O was prepared as a control group. 40 µM Ru(dpp)3Cl2 was individually added to 14 mM H2O2, 14 mM H2O2 + 1 mM TSPP solution, and 100 ppm H2O2(600 µL)-PLGA polymersomes for the experimental groups. All samples were placed in 37 ºC bath. For the purpose of the structure destruction, H2O2(600 µL)-PLGA polymersomes were exposed to US (DELTA, ultrasonic cleaner D200H, 43 KHz) for 30 min. After US exposure, solutions were centrifuged (10000 rpm, 1 min) to obtain Ru(dpp)3Cl2 supernatants. The relative ratio of fluorescence intensity was obtained from Ru(dpp)3Cl2 (ex/em: 455/610 nm) to reflect the presence of oxygen. All of the date was made in triplicate. In Vitro Echogenic Performance of the Polymersomes in the Agar Gel Phantom. The 3% agarose gel was prepared in 96-wells. 5 µL of 100 ppm Fe3O4-PLGA and of 24


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ACS Nano

100 ppm H2O2(600 µL)/Fe3O4-PLGA were respectively injected to gel. The contrast was monitored by the Vevo 770 micro-US imaging system (VisualSonics RMV-704, 40 MHz, B-mode). The US enhancement of H2O2(600 µL)/Fe3O4-PLGA were obtained at 0 min, 5 min, 15 min, and 30 min under a continuous US treatment. Quantitation of H2O2 Loaded in Polymersomes. The H2O2 solutions reacted with KI to generate I3- quantified by UV-Vis measurements. The reaction processes are shown below: KI + H2O2 → I2 + 2 KOH KI → K+ + II2 + I- → I3The 1 mL solutions containing 0.5 M KI with different concentrations of H2O2 for 10 min reaction produced I3-. Subsequently, the calibration curve was obtained from 350 nm absorbance of I3- vs corresponding concentration of H2O2. On the other hand, the 1 mL solution containing 1.2 mg polymersomes (without Fe3O4 NPs) with 0.1M KI (excess) was heated 50°C for 15 min resulting in solution color changed to yellow due to H2O2 release. After centrifugation (14000 rpm, 3min), the yellow supernatants were collected to measure the absorbance of I3- for quantitation of H2O2. Fluorescence Intensity Derived from Fenton Reaction for the Polymersomes Containing Different H2O2 Concentration. The APF dye was used as an indicator for the generation of hydroxyl radicals that were produced from Fenton reaction. Prepared the solutions containing 10 µM APF + 100 ppm Fe3O4-PLGA or H2O2/Fe3O4-PLGA polymersomes at different H2O2 concentrations. The solutions were added to 96-well, and then covered with a plastic wrap. The Vevo 770 micro-US diagnostic probe (VisualSonics RMV-704, 40 MHz) was used to irradiate solutions for triggering Fenton reaction. The APF supernatants were obtained by centrifugation (10000 rpm, 1min). The relative ratio of fluorescence intensity was obtained from 25


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APF dye (ex/em: 490/520 nm) before reaction (I0) and after reaction (In) for 0 min, 5 min, 15 min, and 30 min upon US irradiation at pH 7 condition. All of the date was made in triplicate. Stability Performance of H2O2(600 µL)/Fe3O4-PLGA Polymersomes. The 50 ppm H2O2(600 µL)/Fe3O4-PLGA polymersomes were dispersed in PBS (pH7), PBS (pH5), DMEM medium and serum for 0, 3 and 7 days. The solutions were stored at 37°C. The SEM and DLS were used for analysis of structure stability of polymersomes. Biocompatibility Studies Evaluated by MTT Assay for Polymersomes. The HeLa cells were used as for in vitro experiments. The cells were subcultured in 96-wells plates (8000 HeLa cells/per well) and incubated for 24 h. The DMEM medium was removed and then added fresh medium to culture with different concentrations of Fe3O4-PLGA and H2O2(600 µL)/Fe3O4-PLGA polymersomes. Incubated for another 24 h, the uninternalized polymersomes were removed, followed by a wash with PBS at least 3 times. Finally, the MTT reagent was used and subjected to analyze cell viability by standard ELISA method. All data are obtained in quadruplicate. In Vivo US and MR Imaging. All animal treatments and surgical procedure were performed in accordance with the guidelines of Chang Gung Memorial Hospital Laboratory Animal Center (Kaohsiung, Taiwan). All animals received humane care in compliance with the institution’s guidelines for maintenance and use of laboratory animals in research. All of the experimental protocols involving live animals were reviewed and approved by the Animal Experimentation Committee of Chang Gung Memorial Hospital Laboratory Animal Center. The imaging efficacy of H2O2(600 µL)/Fe3O4-PLGA polymersomes were evaluated using nude mice (Nu/Nu), which was prepared by implanting subcutaneously the suspension of 6 × 106 HeLa cancer cells in medium (100 µ L) into the right thigh of mice (6 − 8 weeks old; 25 − 30 g). After 8 days (tumor: 4 mm3) and 24 days (tumor: 405 mm3) of tumor xenografts, the 26


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tumor-bearing mice were ready for studies. The tumor-bearing mice were anesthetized using 2% isoflurane (Abbott Laboratories, Abbott Park, IL) mixed with 100% O2 delivered using a veterinary anesthesia delivery system (ADS 1000; Engler). For the echo images of the intratumoral injection, H2O2(600 µL)/Fe3O4-PLGA polymersomes (5 mg [Fe]/kg) was injected to tumor region directly. The images were obtained by Vevo 2100 micro-US imaging system (VisualSonics MS-550D, 40 MHz, B-mode) on pre-injection, immediately after post-injection and in the course of 5 min, 15 min and 30 min of US irradiation. For the echo images of the intravenous injection, the tumor-bearing mice were administrated with H2O2(600 µL)/Fe3O4-PLGA polymersomes (5 mg [Fe]/kg) via tail vein. For magnetic attraction, the external magnetic field was placed on the top of tumor for 20 min after injection. The 2nd dose was administrated following the same procedure. The tumors experienced US (VisualSonics MS-550D, 40 MHz, B-mode) after 2nd dose injection. The echo images were obtained on pre-injection, immediately after 2nd post-injection and in the course of 5 min, 15 min and 30 min of US irradiation. The signal intensities of echo imaging were measured using ImageJ 1.49u software. For the MRI images, the intravenous injection procedure followed the treatment processes using for echo image. A sequential MRI acquisition was performed under isoflurane anesthesia in a 9.4T horizontal-bore animal MR scanning system (Biospec 94/20, Bruker, Ettingen, Germany) equipped with a high-performance transmitter-receiver RF volume coil to evaluate the contrast enhancement of polymersomes. As a routine, we first employed multislice turbo rapid acquisition with refocusing echoes (Turbo-RARE) sequence to record high resolution T2-weighted coronal anatomical reference images at pre-injection, 2nd post-injection, exposure of US for 30 min, and post 24 h with the following parameters: field of view= 45.0 mm × 45.0 mm; matrix dimension= 256 × 256 pixels; spatial resolution/pixel= 176 µm × 176 µm; slice thickness= 1.0 mm; 27


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interslice distance= 1.0 mm; echo time= 9.3 ms; effective echo time= 28 ms; repetition time= 3500 ms; rare factor= 8; number of averages= 5; acquisition time= 9 min 20 sec. Based on the orientation of landmark structures from the coronal images, T2-weighted axial anatomical reference imaging was performed under Turbo-RARE sequence acquisition: field of view= 35.0 mm × 35.0 mm; matrix dimension= 256 × 256 pixels; spatial resolution/pixel= 137 µm × 137 µm; slice thickness= 1.0 mm; interslice distance= 1.0 mm; echo time= 9.3 ms; effective echo time= 28 ms; repetition time= 3500 ms; rare factor= 8; number of averages= 5; acquisition time= 9 min 20 sec. The MR imaging signal intensities were measured using ImageJ 1.49u software. In Vivo Antitumor Studies. All animal treatments and surgical procedure were performed in accordance with the guidelines of National Cheng Kung University (NCKU) Laboratory Animal Center (Tainan, Taiwan). The antitumor efficacy was evaluated using nude mice (BALB/cAnN), which was prepared by implanting subcutaneously the suspension of 1 × 107 HeLa cancer cells in medium (100 µL) into the right thigh of mice (4 weeks old; 18 − 22 g, three mice per group). After 7 days of tumor xenografts, the tumor volumes were approximately 50 − 100 mm3, and the tumor-bearing mice were ready for studies. The tumor size was measured along the longest width and the corresponding perpendicular length. The tumor volume was calculated using the volume of an ellipsoid, where volume = 4 π /3 (length/2 × width/2 × depth/2). This study assumed that depth = width and π = 3, resulting in volume = 1/2 × length × (width)2. The Vevo 770 micro-US imaging system (VisualSonics RMV-704, 40 MHz) was used for US irradiation of tumors. Two sequential injections of H2O2(600 µL)/Fe3O4-PLGA polymersomes (5 mg [Fe]/kg) were administrated by the intravenous injection via tail vein. After each injection, the magnet was placed on the top of tumor for 20 min followed by the US irradiation for 28


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30 min. On the 2nd day, the US-treated groups experienced additional US irradiation for 1 h. For the groups with the injection of PBS solution or H2O2(600 µL)/Fe3O4-PLGA

polymersomes without magnet,

the

materials

were

also

administrated through tail vein. Once again, the mice received H2O2(600 µL)/Fe3O4-PLGA polymersomes without magnet for two sequential injections. Mice tumor sizes were observed for every 2 days. In Vivo Studies of Biodistribution, H&E Stain and Blood Analysis. The healthy mice were sacrificed on 1st, 7th and 30th days after injection of H2O2(600 µL)/Fe3O4-PLGA polymersomes at a dose of 5 mg [Fe]/kg. The tissues (heart, lung, liver, spleen, and kidneys) were collected, washed twice with normal saline solution, and stored in 4% para-formaldehyde solution. The H&E results were stained from tissue bank, National Cheng Kung University Hospital (Tainan, Taiwan). For biodistribution, the tissues were disrupted into powders by TissueRuptor (QIAGEN), and the powders were acid-digested in aqua regia for 1 weeks. The iron content of the samples was measured by atomic absorption spectrometer (AA). For blood analysis, the mice blood was obtained from the heart, and then heparin sodium was added immediately. The clotted blood samples were centrifuged at 1200 rpm for 15 min to obtain serum. The blood biochemistry analysis (AST, ALT, T-Bil, BUN, UA, and CREA) was determined by biochemical analyzer (FUJI DRI-CHEM 4000i). All of the results are triplicate.

Conflict of Interest: The authors declare no competing financial interest.

Acknowledgments: We appreciate the financial support from the Ministry of Science and Technology (MOST 103-2113-M-006-008-MY2), and in port by the Headquarters of University Advancement at the National Cheng Kung University, which is 29


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sponsored by the Ministry of Education, Taiwan.

Supporting Information Available: DLS analysis of polymersomes (Table S1). Additional TEM, HRTEM, ED, EDX, colorimetric analysis, quantitative analysis, XED, FTIR, TGA, SQUID, stability analysis, thermographic image, biodistribution analysis, histological analysis, blood biochemical analysis, and MTT assay. This material is vailable free of charge via the Internet at http://pubs.acs.org

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ACS Nano

Figure 1. Formulated H2O2/Fe3O4-PLGA polymersomes and TEM analysis of Fe3O4-PLGA polymersomes and H2O2(600 µL)/Fe3O4-PLGA polymersomes. (a) The H2O2 encapsulated Fe3O4-embedded PLGA polymersomes (H2O2/Fe3O4-PLGA) were prepared via a double emulsion process. (b) The low resolution TEM image of the Fe3O4-PLGA polymersomes and (c) high magnification image of a single Fe3O4-PLGA polymersome with embedded Fe3O4 NPs (black dots). (d) The TEM image of the H2O2(600 µL)/Fe3O4-PLGA polymersomes and (e) high magnification image of a single H2O2(600 µL)/Fe3O4-PLGA containing H2O2. (f) The SEM image of the H2O2(600 µL)/Fe3O4-PLGA polymersomes (inset: the high magnification image of a single H2O2(600 µL)/Fe3O4-PLGA polymersome). (g) The cryo-TEM image of a single H2O2(600 µL)/Fe3O4-PLGA polymersome showing a hollow structure.

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Figure 2. The behavior of the polymersomes upon US trigger and the relaxivity. (a) The deformation of H2O2/Fe3O4-PLGA polymersomes observed by TEM upon micro-US diagnostic probe (VisualSonics, 40 MHz) trigger as a function of the exposure period. (b) Ru(dpp)3Cl2 (ex/em: 455/610 nm) was used as an O2 indicator. The fluorescence intensity decreased when Ru(dpp)3Cl2 reacted with O2. The ratio of fluorescence intensity was obtained from Ru(dpp)3Cl2 before reaction (I0) and after reaction (In). All of the date were obtained in triplicate. (c) In vitro US images in agarose gel upon micro-US diagnostic probe (VisualSonics, B-mode) exposure for Fe3O4-PLGA and as a function of period for H2O2(600 µL)/Fe3O4-PLGA. (d) The r2 values of Fe3O4-PLGA, H2O2(600 µL)/Fe3O4-PLGA, and calculated by T2 relaxation

(T2−1/s−1)

Fe3O4@PLGA were

rate versus iron concentration.

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ACS Nano

Figure 3. The evidence of •OH generation. (a) Fluorescence intensity derived from the process of Fenton reaction as a function of reaction time. The Fenton reaction-induced •OH was processed from H2O, 10 mM H2O2 with 1 mM TSPP stabilizer, 10 mM H2O2, 100 ppm Fe3O4@PLGA NPs + 10 mM H2O2 with 1 mM TSPP stabilizer, and 100 ppm Fe3O4@PLGA NPs + 10 mM H2O2. (b) Fe3O4-PLGA and H2O2/Fe3O4-PLGA containing different amount of H2O2 upon micro-US diagnostic probe (VisualSonics, 40 MHz) exposure as a function of period. The ratio of fluorescence intensity was obtained from 10 µM APF dye (ex/em: 490/520 nm) before reaction (I0) and after reaction (In). All of the date were obtained in triplicate.

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Figure 4. The evidence of •OH generation and therapeutic efficiency of H2O2/Fe3O4-PLGA polymersomes triggering by micro-US diagnostic system with 40 and 55 MHz probes. (a) The APF as the indicator for amount •OH that were generated from H2O2/Fe3O4-PLGA polymersomes after irradiation of US diagnostic probes with 40 and 55 MHz. The ratio of fluorescence intensity was obtained from 10 µM APF dye (ex/em: 490/520 nm) before US treatment (I0) and after US trreatment (In). All of the date were obtained in triplicate. (b) In vitro cytotoxicity analysis evaluated by MTT assay. HeLa cells were used as cell model to examine the therapeutic efficiency of H2O2/Fe3O4-PLGA polymersomes after US irradiated of 40 and 55 MHz.

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ACS Nano

Figure 5. In vivo US imaging and the antitumor efficacy upon micro-US diagnostic probe (VisualSonics, 40 MHz) irradiation and MR imaging monitored using a 9.4 T 39


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animal micro MRI system. (a) US imaging (B-mode) of the tumor by intratumoral injection of H2O2(600 µL)/Fe3O4-PLGA as a function of the exposure period and the corresponding US intensity. (b) US imaging (B-mode) of the tumor by intravenous injection of H2O2(600 µL)/Fe3O4-PLGA as a function of the exposure period and the corresponding US intensity. (c) MR coronal imaging of the tumor by intravenous injection of H2O2(600 µL)/Fe3O4-PLGA as a function of the exposure period and the corresponding MR signal intensity. (d) Tumor growth curves with different treatments (n = 3) after intravenous injection (**p < 0.01). The enclosed area shown in red and the arrow indicate the tumor position. b and c conditions were conducted under magnetic attraction after intravenous injection. Tumor size: 405 mm3.

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ACS Nano

Figure 6. The deformation of a) PLGA, b) H2O2-PLGA and c) Fe3O4-PLGA polymersomes observed by TEM upon micro-US diagnostic probe (VisualSonics, 40 MHz) trigger as a function of the exposure period.

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Ultrasound-Induced Reactive Oxygen Species Mediated Therapy and

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