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Ultrasound Triggered Conversion of Porphyrin/CamptothecinFluoroxyuridine Triad Microbubbles into Nanoparticles Overcomes Multidrug Resistance in Colorectal Cancer Min Chen, xiaolong liang, Chuang Gao, Ranran Zhao, Nisi Zhang, shumin wang, Wen Chen, Bo Zhao, Jinrui Wang, and Zhifei Dai ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b03674 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 14, 2018

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Ultrasound Triggered Conversion of Porphyrin/Camptothecin-Fluoroxyuridine Triad Microbubbles into Nanoparticles Overcomes Multidrug Resistance in Colorectal Cancer Min Chen†,#, Xiaolong Liang‡,#, Chuang Gao†, Ranran Zhao‡, Nisi Zhang†, Shumin Wang‡, Wen Chen‡, Bo Zhao‡, Jinrui Wang‡, Zhifei Dai*,†

†

Department of Biomedical Engineering, College of Engineering, Peking University,

Beijing 100871, People’s Republic of China ‡

Department of Ultrasound, Peking University Third Hospital, Beijing 100191, China

Corresponding Authors *E-mail: [email protected] Homepage: http://mimit-pku.org # These authors contributed equally to this work.

Keywords: photodynamic therapy, chemotherapy, microbubble, drug resistance, colorectal cancer Abstract Multidrug resistance remains one of the main obstacles to efficient chemotherapy of colorectal cancer. Herein, an efficient combination therapeutic strategy is proposed based on porphyrin/camptothecin-floxuridine triad microbubbles (PCF-MBs) with high drug loading contents, which own highly stable co-delivery drug combinations and no premature release. The triad PCF-MBs can act not only as a contrast agent for ultrasound (US) /fluorescence bi-modal imaging but also a multi-modal therapeutic agent for synergistic chemo-photodynamic combination therapy. Upon local ultrasound exposure under the guidance of ultrasound imaging, in situ conversion of PCF-MBs into porphyrin/camptothecin-floxuridine nanoparticles (PCF-NPs) leads to high accumulation of chemo-drugs and photosensitizer in tumor due to the induced high permeability of the capillary wall and cell membrane temporarily via sonoporation effect, greatly reducing the risk of systemic exposure. Most importantly, it was found that the PCF-MBs mediated photodynamic therapy could significantly

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reduce the expression of adenosine-triphosphate (ATP)-binding cassette sub-family G member 2 (ABCG2), which is responsible for the drug resistance in chemotherapy, resulting in a prominent intracellular camptothecin increase. In vivo experiments revealed that the PCF-MBs in combination of ultrasound and laser irradiation could achieve a 90% tumor inhibition rate of HT-29 cancer with no recurrence. Therefore, such triad PCF-MBs based combination therapeutic strategy shows great promise for overcoming drug resistance of colorectal cancer and other cancers.

Patients with metastatic colorectal cancer (CRC) usually receive chemotherapy drugs as their primary treatment. Irinotecan and fluoropyrimidines combination treatment is a standard of care for inoperable colorectal cancer. 1-3 A rational molar ratio of 1:1 was suggested to achieve the best synergistic therapeutic effect.4 However, most of the above clinical trials are based on free drugs, which are very toxic to normal cells and have limited half-life in vivo.5 Although recent advances in liposomal technology help to improve the drug delivery to some extent, problems such as premature drug release6-8 and limited cell uptake, are still main obstacles for efficient cancer treatment. To overcome the drug premature release, an amphiphilic camptothecin-floxuridine conjugate (CF) was developed recently for spontaneous self-assembly into liposomal nanoparticles (NPs) in water.9 Irinotecan is an active chemo drug against colorectal cancer in patients, whose disease is refractory to fluorouracil.10 However, one of the major impediment to efficient chemotherapy of colon cancer remains to be the multidrug resistance (MDR).11,12 Known as a half-transporter of the G subfamily of ATP-binding cassette (ABC) transporter genes, ABCG2 plays a vital role in the cause of MDR,13 which greatly limits the chemotherapeutic efficacy. Although, several ABC transporter inhibitors had been under investigation, their clinical usage was limited due to some drug-drug interactions with other chemo-drugs.14 Therefore, another blocking mechanism apart from chemical agents should be employed to overcome such dilemma. Photodynamic therapy (PDT) is a clinical cancer therapeutic procedure, with minimally invasiveness that utilizes the photosensitive intravenous drug in


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combination with a low power, long duration laser to achieve a selective malignant cells killing effect.15 Nevertheless, PDT treatment alone often results in poor prognosis and recurrence. The deeply located cancer cells can be beyond the accessibility of light irradiation thus escape from PDT.16 PDT can usually cause the strong reactive oxygen species (ROS) generation,17 which consumes a great deal of oxygen and blocks down most vessels of the tumor, resulting in tumor hypoxia.18 Subsequently, hypoxia can stimulate the cancer resistance and drive the tumor metastasis by activating multiple pathways, such as hypoxia induced factor-1-α (HIF-1α).19 Hence, to normalize the tumor vasculatures instead of shutting them during PDT treatment would be beneficial to improve the therapeutic efficacy. Robert et al reported that, irinotecan, can improve vascular function while inhibit cancer progress.20,21 Therefore, a combination of PDT and chemotherapy can be a potential strategy for the thorough clearance of cancer cells. Over the past decades, nanotechnology has been contributing to the development of cancer drug delivery.22,23 Nevertheless, full potential of nanocarriers for cancer treatment is hindered by limited intratumoural drug perfusion.24 A series of biological barriers have been reported to hinder the drugs accumulation in tumors,25-27 reducing the efficacy of conventional and nanoparticle-based chemotherapeutics, especially in the treatment of metastatic disease.28 In order to surmount these barriers and reduce the unwanted phototoxicity in healthy tissue, much effort has been devoted to designing combination nanomedicine rationally to accommodate the multiple drugs or therapeutic modalities for increasing the antitumor effect.29-32 Recently, microbubbles (MBs) have attracted broad research interests because they can act as not only a contrast agent to enhance ultrasound imaging, but also a delivery system to load various therapeutics.33 Focused ultrasound (FUS) has been shown to disrupt the biological barriers in a non-invasive, safe, and targeted manner.34 By using the technique of ultrasound-targeted microbubble destruction (UTMD),35 the cellular uptake and therapeutic efficacy can be significantly improved due to the enhanced permeation of cell membrane and vasculature via sonoporation effect.34 However, limited drug-loading capacity of MBs remains a big obstacle in cancer


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theranostics. In present study, stable triad MBs (PCF-MBs) were constructed by mixing porphyrin grafted lipid (PGL) and amphiphilic CF drug-drug conjugate with the solvent to obtain a mixture and then sonicating to generate MBs by cavitation from perfluoropropane (C3F8) in the mixture (Scheme 1). The CF conjugate is synthesized by coupling two hydrophobic camptothecin (CPT) molecules and two hydrophilic floxuridine (FUDR) molecules to multivalent pentaerythritol through a hydrolyzable ester linkage according to the reported method.9 CF conjugate is used for combined chemotherapy of colorectal cancer while PGL functions as a photosensitizer for fluorescence imaging and photodynamic therapy. Both PGL and CF can self-assemble into microbubbles for contrast-enhanced ultrasound imaging. The self-assembling nature of PGL and CF offers a strategy to optimize the drug loading contents (DLC) by manipulating lipid molar ratios. By using both PGL and CF themselves as MBs, the obtained PCF-MBs show remarkably high DLC, highly stable co-delivery drug combinations and no premature release.9 Compared

with

conventional

ultrasound-mediated

microbubble

burst

therapy,36-39 sonodynamic therapy40,41 and nanoparticles based drug delivery and therapy,37,

42,43

PCF-MBs can be converted into PCF-NPs upon local ultrasound

exposure under the guidance of ultrasound imaging by using UTMD technique. Moreover, the induced high permeability of the capillary wall temporarily facilitates escape of converted PCF-NPs into the tumor interstitium.44,45 After internalization into tumor cells, both CPT and FUDR can be coordinately released for effective tumor growth inhibition due to the hydrolysis of the ester bond. In addition, PGL induced PDT can deplete ABCG2 by which chemo drugs can be pumped out of cancer cells (Scheme 1). CPT can normalize the PDT-generated vessel disruption which may exacerbate the hypoxia effect causing tumor recurrence.

Results and Discussion Synthesis and characterization of PCF-MBs The triad microbubbles were fabricated by stabilizing the PFC gas core with the


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shell composed of CF, PGL, 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidyl-ethanolamine-poly(ethylene glycol)2000 (DSPE-PEG2000) and cholesterol with different molar ratios. Concerning the design of PCF-MBs, cholesterol serve as the small molecule to fill in the space gap between the rigid structure of porphyrin rings, thus stabilizing the shell. DSPE-PEG2000 is incorporated into the microbubble shell as a steric stabilizer due to the bulky hydrophilic PEG headgroup pointing into the continuous media, which prevents the coalescence to stabilize the microbubbles.46 In order for modulation of the blood stability of PCF-MBs, three formulations of PCF-MBs were fabricated by varying the CF molar percentage from 10% to 50% (Table 1). For comparative studies, the PGL-MBs with no CF were prepared from PGL, cholesterol and phospholipids. It was found that the CF molar percentage has impact on the stability of PCF-MBs. The PCF-MB formulation #1 (30% CF) owns higher microbubble production yield with a concentration of 1.90 × 109 MBs/ml but its drug loading content is relatively low. For formulation PCF-MB#3(50% CF), higher content of CF results in lower microbubble production yield with a concentration of 0.70 × 109 MBs/ml. Nevertheless, the PCF-MB formulation #2 (40% CF) shows relatively high microbubble production yield with concentration of 1.02 × 109 MBs/ml as well as high drug loading contents (4.5% porphyrin, 14.3% FUDR and 20.2% CPT). CF is capable to form a very condensed monolayer to impede extremely gas escape from the core into the aqueous medium while the introduction of DSPE-PEG2000 into the microbubble shell prevents PCF-MBs from the aggregation.47 Therefore, the PCF-MB formulation #2 were selected for all the following studies. The stability of PCF-MBs was further investigated by observing the morphology by a fluorescence microscope and measuring the concentration by a Coulter cell counter (Fig S1). For microscope observation, the PCF-MBs were diluted with glycol to avoid shaking. The concentration of the PCF-MBs was reduced to half over the period of 3 hours. Table 1 Compositions of PCF-MB formulations with different molar ratios. Formulations

PGL

CF

DSPC

Cholesterol


DSPEPEG2000

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PCF-MB #1 PCF-MB #2 PCF-MB #3 PGL-MB

10% 10% 10% 10%

30% 40% 50% 0

40% 30% 20% 70%

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10% 10% 10% 10%

10% 10% 10% 10%

The ultraviolet-visible (UV-Vis) absorption spectra of PCF-MBs and PCF-NPs aqueous solutions were examined with PGL and CF as controls (Figure 1a). PCF-MBs and PCF-NPs exhibited the characteristic absorption peaks from both free PGL and CF. No significant change in UV-vis absorption was seen after converting PCF-MBs into PCF-NPs. Transmission electron microscope (TEM) image further demonstrated that the converted PCF-NPs were well dispersed after 3 min ultrasound (1.0MHz, 50% duty, 1W/cm2) exposure to PCF-MBs (Figure 1c). These PCF-NPs had diameters ranging from 30 nm to 100 nm which corresponded with size distribution measured by dynamic laser scattering (DLS) measurements (Figure S2). The fluorescence spectrum (Figure 1b) of PCF-MBs was recorded with an excitation wavelength of 365 nm. The blue fluorescence was emitted by CPT in CF, while the red region was attributed to porphyrin in PGL. These two fluorescence signals were very well isolated, beneficial to intracellular observation. The PCF-MBs were observed under a fluorescence microscope (Figure 1d-f). Well dispersed microbubbles were presented with clear boundary between the non-fluorescent core and the fluorescent shell emerging with red PGL fluorescence (Figure 1d) and blue CF fluorescence (Figure 1e). The overlaid channel (Figure 1f) showed co-localization purple signals on the shells of most PCF-MBs, which indicated the hybrid composition of PGL and CF on the surface of PCF-MBs. Further Image-J based analysis was carried out to measure the diameters of the as-prepared PCF-MBs. Enlarged images (Figure 1g-i) from the representative sight in Figure 1d-f were analyzed by intensity profiles (bottom of Figure 1g-i) of corresponding lines. Two peaks can be seen for each microbubble, and the diameter of a microbubble can be measured by drawing the line right across the center of the microbubble. The diameter was measured to be approximately 1 μm, which was in consistent with the size distribution (Figure 1j) of PCF-MBs measured by a Coulter cell counter.


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Cellular internalization of PCF-NPs and PCF-MBs Cellular internationalization is the upstream behavior of the interaction between precisely defined particles and cells. After incubation of PCF-MBs with HT -29 colon cancer cells for 4 hours, confocal imaging was carried out to map the distribution of PGL and CF within cancer cells (Figure 2A). Acridine orange (AO) with green fluorescence was chosen to label the nucleus.48 The blue fluorescence was infiltrated into the whole cell including the nucleus, upon which co-localization with AO can be observed, indicating the nucleus penetration of the chemo-drugs. Such phenomenon revealed the breakdown of the ester bonds between camptothecin and floxuridine, allowing chemo-drug molecules to penetrate into the nuclear pores.4 Yet, the red fluorescence from PGL can only be detected within the cytoplasm, and no co-localization with AO can be seen, demonstrating the localized accumulation of photosensitizers within cytoplasm. Such remarkably different subcellular distributions of photosensitizers and chemo-drugs proclaimed the necessity to combine therapeutic agents with different subcellular targets and blocking mechanisms in order to completely inhibit cancer cells and overcome drug resistance.4 Human umbilical vein endothelial cells (HUVECs) were treated with phospholipid microbubbles combined with ultrasound (1.0 MHz, 50% duty, 1W/cm2, 3min). The cell viability was evaluated by using 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. As shown in Figure S3, no obvious cytotoxicity to HUVECs demonstrated the good safety of the ultrasound parameters employed in the MBs+US treatment.

Singlet oxygen generation capability of PCF-MBs The production of ROS, especially singlet oxygen (1O2), plays a vital role in porphyrin mediated PDT. Next, the singlet oxygen generation capability of PCF-MBs was investigated using singlet oxygen sensor green (SOSG) reagent by incubating HT-29 cancer cells with 5 μM PCF-MBs for 4 h at 37 °C, followed by irradiation with ultrasound or laser.49 The 1O2 generated by PDT can mediate the oxidation of SOSG and result in green fluorescence increase, allowing for the monitoring of 1O2


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production with confocal scanning laser microscopy. The control group treated with phosphate buffer solution (PBS) treated group had no fluorescence (Figure 2b①), while the PCF-MBs group showed weak PGL and CF fluorescence (Figure 2b②). Upon ultrasound irradiation (1.0 MHz, 1 W/cm2) for 1 min, the PCF-MBs+US group exhibited significant increase in PGL and CF fluorescence due to the higher uptake of PCF-NPs converted from PCF-MBs via sonoporation effect (Figure 2b④). More PCF-NPs entered the tumor cells due to the induced high cell membrane permeability. Upon laser irradiation (650 nm, 0.2 W/cm2) for 3 min, the PCF-MBs+Light group had moderate SOSG fluorescence enhancement (Figure 2b③) while both the PCF-MBs group and the PCF-MBs+US group showed negligible SOSG fluorescence. It indicated no 1O2 generation without light exposure. Compared with the groups of PCF-MBs+US and PCF-MBs+Light, the HT-29 cancer cells treated with PCF-MBs+US+Light exhibited greatly enhanced 1O2 generation not only in the cytoplasm but also inside the nucleus and more dramatic morphology change (Figure 2b⑤). For the PGL-MBs+US+light group, strong PGL fluorescence was observed while the SOSG fluorescence was seen within the cytoplasm instead of the nucleus (Figure 2b⑥). Therefore, these in vitro experiments showed that the CF incorporation didn't abate the capability of porphyrin induced 1O2 production under light irradiation. Furthermore, the combination of ultrasound and microbubble led to a great enhancement of PGL and CF internalization, causing sever cytotoxicity to cancer cells through synergistic chemo-photodynamic effects.

Ultrasound enhanced penetration and cancer killing capabilities of PCF-MBs To further investigate the penetration depth of PCF-MBs with or without ultrasound, a multicellular 3-dimensional (3D) tumor spheroid composed of HT-29 cancer cells was established.50 HT-29 3D tumor spheroids were harvested and observed by using confocal microscopy when the diameter reached 300 μm in mimicking a solid tumor in vitro (Figure 2c and Figure S4). The penetration of PCF-MBs without ultrasound was strictly limited to the outer few cell layers of the spheroids after incubation with 5 μM PCF-MBs for 4 h at 37 °C. On the contrary,


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PCF-MBs penetrated throughout the entire spheroid upon ultrasound irradiation. As seen in the fluorescence images (Figure S4), the US(-) group showed very weak CF fluorescence inside the collagen. On the contrary, strong CF fluorescence was observed inside the collagen for the US (+) group, revealing deep penetration into the 3D spheroid. Such penetration enhancement in tumor spheroid model can be ascribed to PCF-MBs induced inertial cavitation51 and sonoporation34 accompanied with ultrasound irradiation. Thus, when PCF-MBs and ultrasound are joined together, the chemo-drug and PDT induced cancer cell death would be more effective than PCF-MBs alone owing to the higher PCF-NPs uptake. The cancer cell killing capabilities of PCF-MBs were investigated by incubating HT-29 cancer cells with PCF-MBs of various concentrations, followed by different treatments. The cell viability was evaluated by MTT assay 72 hours post treatment. As seen in Figure 2d, the cell killing capability of PCF-MBs showed a remarkable concentration-dependent ranging from 0 ~ 12 μM. The cell viabilities of HT-29 cancer cells significantly decreased with the concentration increasing. At the same concentration, the viabilities of HT-29 cancer cells decreased in the order: PCF-MBs > PCF-MBs+US > PCF-MBs+Light > PCF-MBs+US+Light. At a concentration of 12 μM of PCF-MBs, the viabilities of HT-29 cancer cells were 24.1 ± 1.3%, 16.1 ± 1.2%, 10.3 ± 1.1% and 9.4 ± 0.4% for PCF-MBs, PCF-MBs+US, PCF-MBs+Light and PCF-MBs+US+Light, respectively. Compared with the PCF-MBs group, the PCF-MBs+US group exhibited significant higher killing effect upon exposure to ultrasound (1.0 MHz, 1 W/cm2, 50% duty cycle, 1 min) due to ultrasound triggered conversion of PCF-MBs into PCF-NPs and sonoporation induced higher PCF-NPs uptake. Interestingly, the PCF-MBs+US+Light group exhibited lower cell viabilities than the PCF-MBs+US group. It further confirmed the photodynamic effect of PCF-MBs for killing tumor cells upon additional laser irradiation

(650

nm

and

0.2

W·cm-2

for

10

min).

As

expected,

the

PCF-MBs+US+Light group showed the most effective cancer killing capability due to the synergistic anticancer activity. All these experimental findings demonstrated that PCF-MBs based PDT and chemotherapy combination can be a potential strategy for


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the thorough clearance of cancer cells.

ABCG2 depletion induced by the PCF-MBs mediated PDT The PCF-MBs induced PDT treatments were found to deplete greatly the expression of drug efflux transporter ABCG2, which was responsible for cancer drug resistance. In order to investigate the modulation of ABCG2 and apoptosis related caspase-3 by PDT treatments, HT-29 cancer cells were first treated with 5μM PGL-MBs, followed by irradiation with ultrasound (1.0 MHz, 1 W/cm2, 50% duty cycle) for 1 min, and laser (650 nm, 200 mW/cm2) for 0, 2 and 4 min, respectively. As shown in Figure 3a-c, the immunofluorescence (IF) intensity of ABCG2 was gradually decreased with increasing PDT exposure duration. On the contrary, the level of apoptosis related protein caspase 3 was gradually promoted with morphological change in cytoskeleton. The quantified measurement of IF intensity showed the efficacy of PDT induced ABCG2 depletion was 75% (Figure 3c), and the PDT induced caspase 3 expression was promoted more than 7 folds (Figure 3d). After treatments of HT-29 cancer cells with PCF-MBs in combination of ultrasound and laser, western blot was carried out to the cell lysates (Figure 3e). Different treatments showed a dramatic PDT dose-dependent ABCG2 depletion (Figure 3f) and caspase-3 up-regulation (Figure 3g). HT-29 is a human colon cancer cell line, which express excess ABCG 2. And ABCG2 is the mostly described as one of the three major multidrug-resistance pumps, whose substrates include mitoxantrone, irinotecan, flavopiridol, 5-Fluorouracil, and etc.52 In order to investigate whether the PDT mediated ABCG2 depletion could affect the intracellular accumulation of chemo-drugs in HT-29 colon cancer cells. Different PDT treatments were furthermore carried out and intracellular CPT levels were analyzed by the fluorescence of CPT from the cell lysates using a multi-plate reader.53 We found that HT-29 cells treated with PCF-MBs+Light and PCF-MBs+US+Light showed 2 and 4 folds increases in intracellular CPT concentrations at 1 hour post-PDT, compared with those treated with PCF-MBs alone (Figure 3g). While the intracellular CPT in PCF-MBs+Light group increased was only 1.2 folds compared with PCF-MB


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alone after another 5 hours incubation although with average intracellular CPT increase. Such phenomenon can be owing to the time dependent cell uptake of PCF-NPs when the PCF-MBs were maintained during and after PDT. So, in order to exclude the additional contribution from uptake of CPT after PDT at the present of PCF-MBs, the media that contained PCF-MBs were removed after PDT in another separated study, whose results were surprisingly different (Figure 3i). The intracellular CPT accumulation was reduced after another 5.5 h incubation. Such phenomenon could be attributed to the ABCG2 mediated CPT efflux. Although all groups showed the tendency of CPT flowing out 6 h post PDT compared with 0.5 h post PDT, the PCF-MBs+US+Light group maintained the highest CPT retention 6 h post PDT. Such phenomenon should be ascribed to the influence of ABCG2 drug efflux transporter, which drove the chemo-drugs out of cells when it was intact. While, upon light irradiation, the drug efflux could be greatly inhibited caused by PDT induced ABCG2 depletion, resulting in higher drug retention intracellularly. In addition, the PCF-MBs+US+Light group had more than 2 folds the drug accumulation intracellularly compared with PCF-MBs group. This was well consistence with the confocal images shown in Figure 2b, further validated the outstanding drug delivery performance of PCF-MBs+US+PDT platform. Compared with free CPT and CPT+FUDR groups, the PCF-MBs+US+Light group showed more than 1.5 folds of intracellular CPT levels (Figure S5).

Ultrasound/fluorescence bimodal imaging capability of PCF-MBs In order to evaluate the contrast enhancement capability of the prepared PCF-MBs in vitro. A suspension of the above PCF-MBs was injected into a latex tube containing circulating saline. As shown in Figure S6, the B mode images showed no significant change, while the harmonic mode showed greatly contrast enhancement, which indicated the promising application of PCF-MBs in ultrasound imaging. To investigate the capability of the PCF-MBs to enhance ultrasound imaging in vivo, 1mg/ml PCF-MBs were intravenously administrated into HT-29 cancer bearing Balb/c nude mice, and the xenograft tumor was monitored using a 7.0 MHz ultrasound


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transducer (Figure 4a). Before injection, there was almost no signals inside tumor, while, 20s post injection, the US imaging signal reached the maximum, and the contrast enhancement could last for more than 3min (Figure S7). A therapeutic ultrasound transducer (NapaGene, 4000) was then used to locally disrupt the microbubbles. The ultrasound signal was seen diminished immediately upon ultrasound irradiation (1.0 MHz, 1 W/cm2, 50% duty, 1 min) due to ultrasound triggered conversion of PCF-MBs into nanoparticles. The converted PCF-NPs could be efficiently up-taken by cancer cells because of the enhance membrane permeability via sonoporation effect. The recovery ultrasound signal indicated the PCF-MBs second in-flow from other part of the body. More sequence of ultrasound was further exposed to trigger the micro-to-nano conversion for producing higher PCF-NPs accumulation in the tumor. After a total 3 min ultrasound irradiation, no significant ultrasound signal can be detected, which indicated the full conversion from PCF-MBs to PCF-NPs. The ultrasound triggered PCF-NPs local accumulation process was further validated by in vivo fluorescence imaging technique (Figure 4b). After PCF-MBs were administrated to HT-29 tumor bearing mice, and 15 min after US irradiation, the fluorescence of porphyrin was detected by using an excitation wavelength of 640 nm and an emission wavelength of 720 nm. The maximum accumulation of PCF-MBs appeared at about 2 hours after injection (Figure 4d). The fluorescence signal was still detectable more than 48h post-injection despite of 75% decrease in intensity. In order to study the deposition pathway in organs, the organs were dismembered and detected in living animal fluorescence imaging system 24 hours post injection with or without ultrasound, respectively (Figure 4c,e). In the PCF-MBs group, a maximum integrated intensity of 55×107 was located in liver, which should be owing to the macrophage clearance of PCF-MBs.54 Meanwhile, the spleen had an intensity of 15×107, and the tumor had only 10×107 integrated intensity. Dramatically, 50×107 integrated fluorescence intensity was detected within tumor in the PCF-MBs+US group, the intensity of liver reduced to only 30×107, and the spleen of less than 5×107. The sharp fluorescence increase (a.c. 5 folds) in tumor in assistance of ultrasound indicated the


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great successful PCF-MBs to PCF-NPs conversion and local PCF-NPs accumulation in the tumor. The decrease intensity of other organs, such as liver (45% off) and spleen (50% off), further demonstrated the UTMD induced reduction of PCF-MBs accumulation in un-targeted organs, reducing the risks of systematic exposure. The blood circulation dynamics of PCF-MBs was investigated by determining the blood fluorescence intensity of PGL. The blood PCF-MBs signals decreased to 37% after 5 h and only 10% was observed after 24 h, exhibiting rapid clearance kinetics (Figure S8). Values of elimination half-life (t1/2) and area under the plasma concentration-time curve from 0 to 24 h (AUC0-24) for PCF-MBs were calculated to be 1.95 h and 512 mghmL-1, respectively. The half-life of PCF-MBs is comparable to previous reports. 54 The rapid clearance of PCF-MBs insures the safety issue without ultrasound irradiation.

In vivo anticancer efficacy of PCF-MBs To evaluate the in vivo therapeutic efficacy of PCF-MBs, the HT-29 colon cancer bearing nude mice were randomly divided into 6 groups and the treatment began when the volume reached to about 100 mm3. The mice were injected with PBS, PCF-MBs and PGL-MBs, respectively, followed by treatments with or without ultrasound / light irradiation. In order to examine the ROS generated levels in vivo, SOSG was first intratumorally injected 30 min before light irradiation. The resulting tumor tissue was resected and cryo-sliced for fluorescence analysis. Both the PGL-MBs+US+Light and PCF-MBs+US+Light group emitted noticeable SOSG green

fluorescence.

Nevertheless,

the

fluorescence

intensity

of

the

PCF-MBs+US+Light group was much higher than that of the PGL-MBs+US+Light group. This demonstrated higher singlet oxygen generation capability of the treatment using the triad PCF-MBs in combination with ultrasound and light irradiation (Figure S9). Figure 5a showed the tumor volume growing curve versus time. 28 days after


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treatment (Figure S10), the average tumor volume of the PBS and PCF-MBs group grew to approximately 2000 mm3 and 1237 mm3, respectively. A tumor inhibition rate of 38% revealed that the fundamentally synergistic CPT and FUDR chemotherapy had significant colon cancer inhibition. Upon exposure of CF conjugate to esterase and acidic microenvironment of the tumor, both CPT and FUDR were released due to the hydrolysis of the ester bond and work collaboratively.9 After the addition of 650 nm laser irradiation, the PCF-MBs+Light group exhibited a tumor inhibition rate of more than 50%, which was attributed to the additional PGL mediated PDT on the basis of CPT and FUDR. When the PCF-MBs were associated with ultrasound exposure on the tumor site, the average tumor volume of the PCF-MBs+US group reduced to 615.63 mm3. The increased tumor inhibition rate of 69% was ascribed to the UTMD induced higher PCF-NPs accumulation at tumor sites. In order to acquire the efficacy of PDT monotherapy, PGL-MBs were injected intravenously, followed by 3 min ultrasound irradiation and 10 min laser irradiation of 650 nm at the tumor site. Within the first 10 days, the PGL-MBs+US+Light group showed almost no tumor volume growth. However, 10 days after treatment, the apparent tumor recurrence was observed, reaching a final average tumor volume of 769.88 mm3. A tumor inhibition rate of 61% was achieved, lower than that of the PCF-MBs+US group. Interestingly, the tumor volume of the PCF-MBs+US+Light group dramatically retained at 191.44±66 mm3, achieving a 90% tumor inhibition rate. Cell permeability played an important role for the nanomedicine across the membrane barrier. Different endothelial cells showed different permeability, which can be affected by the properties of nanoparticle,55 and be also regulated by different stimulus such as ultrasound. Most studies have suggested that low-intensity ultrasound can enhance the cell membrane permeability,56-58 and the cavitation effect produced by ultrasonic destruction of microbubbles can cause an increase in microvascular permeability, microvascular rupture, and increased membrane permeability to enhance drug uptake in related tissues.59,60 In our study, ultrasound irradiation was confined in the tumor tissue, and was able to enhance effectively the tumor vascular and tumor cell membrane permeability when combined with the MBs circulating through the tumor,


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thus greatly improved the drugs uptake and retention at the tumor area. Apart from UTMD induced PCF-NPs accumulation in tumor tissues, the ROS generated by PGL induced PDT can increase the microvascular permeability near tumor tissues, allowing more PCF-NPs penetration into tumor interstium.61 This further proved the synergistic effect of PDT and chemotherapy with the aid of UTMD, thus maximized the therapeutic activity of PCF-MBs. Simultaneously, no significant change in animal body weight (Figure 5b) or organ damage can be detected in all PCF-MBs treated groups after 28 days post-treatment (Figure 6). It demonstrated that all treatments were tolerated well by the tumor-bearing animals and the combined chemo-PDT treatment showed no unacceptable toxicity. The reported liposome-like nanocapsules of Janus camptothecin-floxuridine conjugate aimed at delivering two drugs efficiently at a precise molar ratio of 1:1.9 While, the current PCF-MBs was develop for enhancing the therapeutic efficacy of the combined chemo-PDT treatment under the assistance of ultrasound targeted microbubble destruction to overcome multidrug resistance in colorectal cancer. Ex vivo prognosis evaluation Immunohistochemistry and Immunofluorescence analysis of tumor tissues were carried out to evaluate the prognosis after different treatments. Different treatments on drug resistance, proliferation ability, tumor vasculature, and hypoxia level were evaluated by immunohistochemistry staining of ABCG2, Ki67, CD31 and HIF-1α.62 As can be seen in Figure 7a-b, without no laser or ultrasound irradiation, the PCF-MBs treated group showed 90% Ki67 positive area compared with PBS group, indicating the limited cancer inhibition ability of chemotherapy alone. Nevertheless, the PCF-MBs+Light treated group showed better inhibition ability of cancer cell proliferation (50% Ki67 positive area) compared with the group treated with PCF-MBs alone since PDT could exacerbate the hypoxia and resulted in an increase in the expression of HIF-1 (27% positive). Meanwhile, the microvessel density dropped down to 60%, further verifying the PDT induced hypoxia mechanism. The 20% Ki67 positive rate of the PCF-MBs+US treated group demonstrated that the UTMD assisted CPT and FUDR synergistic chemotherapy caused the efficient cancer


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cell death. Yet, the high-level ABCG2 expression (approximately 75%) could increase risks of drug resistance which can be detrimental to prognosis. As expected, the high PDT efficacy of the PGL-MBs+US+Light treated group remarkably down-regulated the ABCG2 expression (30% positive rate), intensively blocked microvesseles (10% CD31 positive rate), and gave rise to the sharp increase of HIF-1 (7 folds higher than the PBS treated group). The high-level HIF-1 expression induced recurrence. A Ki67 positive rate of 30% revealed that the final tumor inhibition rate of the PGL-MBs+US+Light treated group was inferior to the PCF-MBs+US group. In stark contrast, the PCF-MBs+US+Light treated group showed the lowest ABCG2 expression because the high PCF-NPs accumulation mediated highly efficient PDT which could induce ABCG2 depletion. The reason why the strong PDT did not cause severe hypoxia effect could be ascribed to the CPT induced tumor vasculature normalization. The final microvessel density remained half that of the PBS group, resulting in lower HIF-1 expression. Therefore, the PCF-MBs+US+Light group achieved the highest therapeutic efficacy with the lowest Ki67 positive rate (10%). As shown in Figure 7c, the ABCG2 fluorescence (green) was completely depleted in the PCF-MBs+US+PDT group, while the PGL-MBs+US+Light group with no CF still showed very high ABCG2 expression in those areas deep inside the tissue. Such phenomenon can be attributed to the limited penetration of PDT in deeper tissues. A moderate ABCG2 expression was seen in the PCF-MBs+US group. The abnormal vasculature (A.V.) was seen in the PBS group (Figure 5c), but it was converted into normalized vasculature (N.V.) both in the PCF-MBs+US and PCF-MBs+US+PDT groups (Figure 5c, CD31 channel), which allowed the chemotherapeutics penetrated deeply into the tumor. In a word, the synergistic chemo-photodynamic therapy can be achieved with PCF-MBs in combination of ultrasound and laser irradiation by depleting ABCG2 with PDT and normalizing tumor vasculature with CPT, which provides a strategy to circumvent hypoxia-induced drug resistance. Conclusion In summary, we have successfully constructed porphyrin/camptothecin-fluxoridine


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triad microbubbles, which show remarkably high drug loading content, highly stable co-delivery drug combinations and no premature release. The obtained PCF-MBs can serve not only as a contrast agent to enhance ultrasound imaging but also a multi-modal therapeutic agent for synergetic chemo-photodynamic therapy. Upon local ultrasound exposure under the guidance of ultrasound imaging, PCF-MBs can be converted into PCF NPs. The induced high permeability of the capillary wall temporarily resulted in high accumulation of PCF-NPs in tumor, greatly reducing the risk of systemic exposure. By combining ultrasound with photodynamic therapy, the use of PCF-MBs can significantly reduce the ABCG2 expression, leading to a prominent intracellular CPT increase. In vivo anticancer activity showed the PCF-MBs with the assist of ultrasound exposure and laser irradiation can greatly inhibit the HT-29 cancer. Furthermore, ex vivo immunohistochemistry and immunofluorescence of the resected tumor verified the ability of CPT for vasculature normalization, reducing the hypoxia responsive HIF-1α expression. Therefore, such combination therapeutic strategy based on triad PCF-MBs is having great promise for overcoming drug resistance of colorectal cancer and other cancers by addressing the mechanisms of tumor recurrence and treatment escape. Further studies using targeting ligands for modification of PCF-MBs to further increase the selectivity are merited.

Experimental Section Materials Camptothecin-Floxuridine (CF) was synthesized according to the reported method9 and porphyrin-grafted lipid (PGL) was synthesized according to the previous method.63 DSPC, DSPE-PEG2000, cholesterol were purchased from Xi'an Ruixi Biological Technology Co., Ltd. SOSG, AO and 4’,6-diamidino-2-phenylindole (DAPI) were purchased from Thermo Fisher Scientific. Primary and second antibodies were obtained from Cell Signaling Technology. All other reagents and solvents were purchased from the domestic suppliers and used as received.


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Preparation and optimization of PCF-MBs CF was first dissolved in Dimethyl sulfoxide (DMSO), PGL was dissolved in Tetrahydrofuran (THF), and DSPC, DSPE-PEG2000 and cholesterol were dissolved in ethanol. Then 1mg of the above mixture with different molar ratio was dropwise added to deionized water at a 37 oC water bath sonication accompanied with gentle shaking. The resulting aqueous suspension were transferred to dialysis bag (8000-14000 Da) for 3 h to remove the excess organic solvents. The obtained PCF-NPs solution was transferred to a small vial by mixing with glycol and 1,2-propyleneglycol at a molar ratio of 8 : 1 : 1. Then C3F8 was introduced to the vial for 45s to place the air within the vial and sealed carefully. A Vial-mix mechanical agitator was employed to generate microbubbles by violent vibration for 45s. The as prepared microbubbles can be handled by small injectors.

Characterization of PCF-MBs UV-vis absorption spectra were measured by a UV-vis spectrophotometer (Thermo Fisher, Evolution 220). Fluorescence determined by a

spectra of

the

fluorescence spectrophotometer

different

samples

were

(Thermo Fisher, Lumina).

The morphology analysis of ultrasound induced PCF-MBs to PCF-NPs conversion was carried out by TEM (FEI Tecnai G2 T20) by negative stained samples on cubic grid (200 mesh). Briefly, the as-prepared PCF-MBs solution was irradiated by 1.0 MHz ultrasound (1.0 W/cm2, 50% duty) transducer for 5 min, then 10 μL of the resulting sample was dropped onto the grid, air-dried and negatively stained with 10


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μL, 3.0% phosphotungstic acid) followed by ddH2O washing and air-drying. Imaging was performed at an accelerating voltage of 200 kV. The concentration of the PCF-MBs was determined with a Coulter Counter Multisizer (Coulter Electronics Ltd., Luton, Bedfordshire, UK), in which 20 µL samples were diluted in 20 mL Isoton-II electrolyte solution. The ultrasound imaging (Linear and Nonlinear) of the PCF-MBs in solution was performed in a latex tube using L12-3E ultrasound imaging transducer (3-12 MHz) (Mindray resona 7).

Cell culture studies Monolayer cultures of HT-29 and HUVEC cells were cultured in a complete medium containing Dulbecco's Modified Eagle Medium (DMEM, Gibco), 10% fetal bovine serum (Biological Industries, USA), 100 units/mL penicillin and 100 μg/mL streptomycin. Cellular uptake of PCF-MBs with ultrasound was tested in 12-well plates with coverglass bottoms, where HT-29 cells were allowed to attach and grow overnight. PCF-MBs were added to the wells at certain concentrations and treated with or without ultrasound/light irradiation. Imaging was carried out using a confocal microscope (TCS SP8 STED 3X, Leica). Porphyrin and CPT fluorescence were excited by 405 nm xenon lamp with an emission from 650 nm to 720 nm and 410-490 nm, while AO was excited by a 488 nm diode laser, and collected from 500 nm to 550 nm. SOSG, as an indicator to monitor the generation of 1O2, could react irreversibly with 1O2 to cause an enhancement in its fluorescence was employed to map singlet


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oxygen distribution in porphyrin-PDT. Briefly, PCF-MBs or PGL-MBs were incubated with cells for 4h and replaced by SOSG containing media (10 μM) for 30 min before laser irradiation (650 nm, 0.2 W/cm2) and then washed immediately after PDT. After fixing by 4% paraformaldehyde, the fluorescence from oxidized SOSG, CPT, and porphyrin were observed by the confocal microscope. For in vitro MTT assay, 1×104 HT-29 colon cancer cells were seeded into 96-well plates. 24 h post cell seeding, cells were incubated with various amounts of PCF-MBs and treated with/without ultrasound (1.0 MHz, 1 W/cm2, 50% duty, 3 min) and laser irradiation (650 nm, 0.2 W/cm2, 10 min), and incubated for additional 72 h. Then, 20 μL MTT stock solution (5 mg/mL) was added into each well and incubated for another 4 h, after which the media were removed and dissolved in DMSO, the absorbance at 490 nm was recorded using a microplate reader (BioTek Synergy HT). For the MB destruction investigation, different concentration of phospholipids MBs were incubated with HT-29 cancer cells and treated with/without ultrasound (1.0 MHz, 1 W/cm2, 50% duty, 3 min) followed by additional 24 hours growth before adding MTT. 3D spheroids model The 3D tumor spheroids were established64 by co-culture of 4.5×103 CCD-18Co normal colon cells and 3×103 HT-29 colon cancer cells (3:2) for 8 days in a low adherence 6-well plate (Lot number: 3262, Corning) to harvest spheroids with diameters about 200 micrometer. 10 μM PCF-MBs were incubated with the spheres with or without US irradiation (1 W/cm2, 3 min, 50% duty cycle). Then, the collagen


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was labeled with Alexa-488 conjugated Collagen-VI mono-clone antibody and observed under confocal microscopy. In vivo animal models and treatments The animal experiments protocols were approved by Peking University Institutional Animal Care and Use Committee (IACUC). In vivo experiments were carried out on 4-week-old male Balb/c nude mice weighing 20–22 g (Beijing Vital River Laboratory Animal Technology Co., Ltd.). For tumor inoculations and US/PDT irradiation, animals were inhalation anesthetized with isoflurane. Subcutaneous tumors were implanted by injection of a volume of 50 μL cell suspension containing 5×106 HT-29 cells implanted on the right hind leg. Ten days after cancer cell implantation, the volumes of the subcutaneous tumors reached about 100 mm3, the treatments were started. HT-29 tumor-bearing mice were divided into six groups (n = 6), then intravenously injected via tail vein with PBS, PCF-MBs, and PGL-MBs at a dose of PCF-MBs (13.5 mg/kg,) once every 3 days for 2 times. Tumor volume and body weight were monitored every 2 days. A digital caliper was employed to measure the three tumor dimensions, and tumor volumes were calculated by the hemi-ellipsoid formula: volume = π/6 · L ·W· H ( L, W and H, are the tumor length, width and height). Different groups were treated according to Supplementary Table 1. A Beckman Coulter cell counter (Coulter Electronics Ltd., Luton, Bedfordshire, UK) was employed to determine the concentration of each type of MB. The concentration of MB that injected for each treatment can be calculated according to the volume and mass concentration injected. For an animal weight 20 g should get 0.02 kg·13.5


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mg/kg = 0.27 mg PCF-MBs, which accounts for a volume of 0.27 mg/2 mg·mL-1 = 0.13ml injection dose. And since Concerning the concentration of PCF-MBs injected was 1.02×109 MBs/mL, the MB injected should be 1.02×109 MBs/mL × 0.13 mL = 1.326 ×108 MBs each treatment. In the literature, the CPT and FUDR dosages were commonly reported to be 4-8mg/kg65 and 2.5-100 mg/kg,66 respectively. The CPT and FUDR dosages used in this study were calculated to 4.04mg/kg and 2.86mg/kg, respectively.

In vivo ultrasound imaging HT-29 colon cancer cells (2×106 in 80 μL PBS) were subcutaneously injected into the right hind leg of male Balb/C mice which were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. After about 10 days, the mice had developed tumors with volumes of about 100 mm3. After the tumor-bearing mouse was injected a 200 μL bolus of 2×108/mL PCF-MBs or PGL-MBs intravenously, the tumor was imaged using the Mindray Resona 7 ultrasound system with the L12-3E probe (3 ~ 12 MHz) at a mechanical index (MI) of 0.044. About 20 s after the injection, the MBs in the tumor were triggered destruction with a sudden increase of mechanical index in order to observe the reperfusion of the MBs in the tumor tissue. The differences between parameters used in ultrasound imaging and ultrasonic burst mainly include the ultrasonic frequency and acoustic intensity. The center frequency used for imaging was 7 MHz, and mechanic index less than 0.06. While the frequency of ultrasonic burst applied in the UTMD was 1.0 MHz, which is reported to produce more


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ultrasonic cavitation effect than higher frequency and deeper penetration depth. The acoustic intensity for UTMD was 1.0 W/cm2 with a duty cycle of 50%. In vivo fluorescence imaging and distribution analysis For in vivo fluorescence imaging, 200 μL PCF-MBs or PGL-MBs with a 100 μM equivalent concentration of PGL and CF were injected through the tail vein. The tumor issues were irradiated by a US probe (1.0 MHz, 50% duty, 1 W/cm2, 3 min). Then IVIS Spectrum In Vivo Imaging System (Perkin-Elmer) was utilized to collect the fluorescence signals of porphyrin at 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 6 h, 12 h, 24 h and 48 h, respectively. The mice after 24 h fluorescence imaging were sacrificed and the hearts, livers, spleens, lungs, kidneys, tumors were collected for analysis of fluorescence based semi-quantitative bio-distribution. Pharmacokinetics study of PCF-MBs For determining the blood clearance of PCF-MBs, male Balb/c mice (body weight 20-22 g, 6 weeks old) were maintained in standard housing. The porphyrin levels of plasma in mice were determined by a multi-plate microreader. PCF-MBs were administered into the mice via the tail vein, with an injection dose of 15 mg/kg PGL. At different time intervals, blood samples were collected with heparin coated syringe. Subsequently, plasma was separated by centrifugation (3000 g, 10 min) to be applied to the analysis of porphyrin concentration. The supernatant was subjected to the measurement of fluorescence intensity of porphyrin at 650 nm using a multi-plate microreader. A pharmacokinetic analysis using Origin (9.1) was performed to determine the key parameters including elimination half-life (t1/2), area under the


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plasma concentration–time curve from 0 to 24 h (AUC0–24) by fitting the curve with an ExpDec1 exponential function.

Histological analysis 28 days post-injection, the mice were dissected and main organs (liver, heart, spleen, lung and kidney) were excised for haematoxylin and eosin (H&E) staining at the end of the treatment to evaluate the biocompatibility and toxicity of various formations. And the tumors were sliced for immunohistochemistry assay to analyze the tumor hypoxia,

vasculature,

and

proliferation

biomarker

expressions.

Then,

the

immunohistochemistry and H&E staining images were captured with microscope (Leica DMI3000B, Germany). Antibodies used for immunostaining were summarized in Supplementary Table 2.

Statistical Analysis Student’s t-test was applied to test the significance of the difference between two groups, while for variance in multiple (>3) groups, one-way analysis of variance (ANOVA) was taken, where was considered to be significance when *p < 0.05, **p < 0.01 and ***p < 0.001. Data were expressed as mean ±standard deviation (SD). ASSOCIATED CONTENT Supporting Information The Supporting Information (SI) is available free of charge on the ACS Publications


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website at DOI: xxxxxxxxxx. Stability, DLS, cytotoxicity, intracellular levels compared with free drugs, in vitro and in vivo US imaging, pharmacokinetics, in vivo 1O2 distribution of PCF-MBs and the photographs taken at the end point can be found in the SI. Conflict of Interest: The authors declare no competing financial interest.

Acknowledgment:

This research was financially supported by National Key Research and Development Program of China (No. 2016YFA0201400), Beijing Natural Science FoundationHaidian original innovation joint fund (No. 17L20170), National project for research and development of major scientific instruments (No. 81727803), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 81421004), the National Natural Science Foundation of China (No. 81771846, No. 81571810) and grant from Peking University Third Hospital (BYSY2015023).

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(20) Neijzen, R.; Wong, M. Q.; Gill, N.; Wang, H.; Karim, T.; Anantha, M.; Strutt, D.; Waterhouse, D.; Bally, M. B.; Tai, I. T.; Ng, S. S. W.; Yapp, D. T. Irinophore C (TM), a Lipid Nanoparticulate Formulation of Irinotecan, Improves Vascular Function, Increases the Delivery of Sequentially Administered 5-FU in HT-29 Tumors, and Controls Tumor Growth in Patient Derived Xenografts of Colon Cancer. J. Control Release 2015, 199, 72-83. (21) Verreault, M.; Wehbe, M.; Strutt, D.; Masin, D.; Anantha, M.; Walker, D.; Chu, F.; Backstrom, I.; Kalra, J.; Waterhouse, D.; Yapp, D. T.; Bally, M. B. Determination of an Optimal Dosing Schedule for Combining Irinophore C (TM) and Temozolomide in an Orthotopic Model of Glioblastoma. J. Control. Release 2015, 220, 348-357. (22) Shi, J. J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Cancer Nanomedicine: Progress, Challenges and Opportunities. Nat. Rev. Cancer 2017, 17, 20-37. (23) Jin, Y. S.; Ma, X. B.; Feng, S. S.; Liang, X.; Dai, Z. F.; Tian, J.; Yue, X. L. Hyaluronic Acid Modified Tantalum Oxide Nanoparticles Conjugating Doxorubicin for Targeted Cancer Theranostics. Bioconjug. Chem. 2015, 26, 2530-2541. (24) Chen, H. M.; Zhang, W. Z.; Zhu, G. Z.; Xie, J.; Chen, X. Y. Rethinking Cancer Nanotheranostics. Nat. Rev. Mater. 2017, 2 ,17024. (25) Blanco, E.; Shen, H.; Ferrari, M. Principles of Nanoparticle Design for Overcoming Biological Barriers to Drug Delivery. Nat. Biotechnol. 2015, 33, 941-951. (26) Holohan, C.; Van Schaeybroeck, S.; Longley, D. B.; Johnston, P. G. Cancer Drug Resistance: An Evolving Paradigm. Nat. Rev. Cancer 2013, 13, 714-726. (27) Fang, J.; Nakamura, H.; Maeda, H. The EPR Effect: Unique Features of Tumor Blood Vessels for Drug Delivery, Factors Involved, and Limitations and Augmentation of the Effect. Adv. Drug Deliver. Rev. 2011, 63, 136-151. (28) He, C. L.; Tang, Z. H.; Tian, H. Y.; Chen, X. S. Co-delivery of Chemotherapeutics and Proteins for Synergistic Therapy. Adv. Drug Deliver. Rev. 2016, 98, 64-76. (29) MacKay, J. A.; Chen, M. N.; McDaniel, J. R.; Liu, W. G.; Simnick, A. J.; Chilkoti, A. Self-assembling Chimeric Polypeptide-doxorubicin Conjugate Nanoparticles that Abolish Tumours after a Single Injection. Nat. Mater. 2009, 8, 993-999. (30) Li, H. J.; Du, J. Z.; Du, X. J.; Xu, C. F.; Sun, C. Y.; Wang, H. X.; Cao, Z. T.; Yang, X. Z.; Zhu, Y. H.; Nie, S. M.; Wang, J. Stimuli-responsive Clustered Nanoparticles for Improved Tumor Penetration and Therapeutic Efficacy. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 4164-4169. (31) Li, W. P.; Su, C. H.; Chang, Y. C.; Lin, Y. J.; Yeh, C. S. Ultrasound-Induced Reactive Oxygen Species Mediated Therapy and Imaging Using a Fenton Reaction Activable Polymersome. ACS Nano 2016, 10, 2017-2027. (32) Yin, Q.; Shen, J. N.; Zhang, Z. W.; Yu, H. J.; Li, Y. P. Reversal of Multidrug Resistance by Stimuli-responsive Drug Delivery Systems for Therapy of Tumor. Adv. Drug Deliver. Rev. 2013, 65, 1699-1715. (33) Guo, C. X.; Jin, Y. S.; Dai, Z. F. Multifunctional Ultrasound Contrast Agents for Imaging Guided Photothermal Therapy. Bioconjug. Chem. 2014, 25, 840-854. (34) Lentacker, I.; De Cock, I.; Deckers, R.; De Smedt, S. C.; Moonen, C. T. W. Understanding Ultrasound Induced Sonoporation: Definitions and Underlying Mechanisms. Adv. Drug Deliver. Rev. 2014, 72, 49-64. (35) Mayer, C. R.; Geis, N. A.; Katus, H. A.; Bekeredjian, R. Ultrasound Targeted Microbubble Destruction for Drug and Gene Delivery. Expert Opin. Drug Deliv. 2008, 5, 1121-1138.


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(36) Eisenbrey, J. R.; Shraim, R.; Liu, J. B.; Li, J. Z.; Stanczak, M.; Oeffinger, B.; Leeper, D. B.; Keith, S. W.; Jablonowski, L. J.; Forsberg, F.; O'Kane, P.; Wheatley, M. A. Sensitization of Hypoxic Tumors to Radiation Therapy Using Ultrasound-Sensitive Oxygen Microbubbles. Int. J. Radiat. Oncol. 2018, 101, 88-96. (37) Liang, Y.; Chen, J.; Zheng, X.; Chen, Z.; Liu, Y.; Li, S.; Fang, X. Ultrasound-Mediated Kallidinogenase-Loaded Microbubble Targeted Therapy for Acute Cerebral Infarction. J. Stroke Cerebrovasc Dis. 2018, 27, 686-696. (38) Zhu, L. L.; Zhao, H. Y.; Zhou, Z. Y.; Xia, Y. H.; Wang, Z. G.; Ran, H. T.; Li, P.; Ren, J. L. Peptide-Functionalized

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Scheme 1. (a) Schematic illustration of self-assembly of PCF-MBs with porphyrin grafted lipid of PGL and camptothecin-floxuridine conjugate of CF, which can be converted into PCF-NPs by ultrasound targeted microbubble destruction. (b) PCF-MBs mediated chemo-photodynamic combination therapy with the assistance of UTMD technique. The therapeutic efficacy may be enhanced since PDT can deplete ABCG2

by

which

chemo

drugs

can

be

cells.


pumped

out

of

cancer

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Figure 1. Characterization of PCF-MBs. (a) UV-vis absorption spectra of PCF-MBs and PCF-NPs aqueous solutions, while CF and PGL dissolved in organic solvents were used as controls. (b) Fluorescence spectrum of PCF-MBs excited by 365 nm. (c) TEM image of PCF-NPs converted from PCF-MBs upon ultrasound exposure. (d-f) Fluorescence images of PCF-MBs where red fluorescence represented the PGL(d), blue fluorescence indicated the CF (e), and the co-localization signal was displayed as purple (f) (scale bar: 3 μm). (g-i) Representative enlarged images of PCF-MBs collected from d-f (scale bar: 1 μm) and their intensity surface plots. (j) Size distribution of PCF-MBs.


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Figure 2. In vitro studies of PCF-MBs and PCF NPs in HT-29 cells. Confocal laser scanning microscopy images of (a) intracellular distribution of PCF NPs PGL (red for blue for CF, green for AO) (scale bar: 10 μm); (b) cell uptake (CPT, blue; PGL, red) and singlet oxygen generation (green) after different treatments (① control, ② PCF-MBs, ③ PCF-MBs+light, ④ PCF-MBs+US, ⑤ PCF-MBs+US+light, ⑥ PGL-MBs+US+light) (scale bar: 20 μm); (c) confocal images of the morphology (shown in white filed) , PGL fluorescence and surface intensity plots of HT-29 3D tumor spheroid models treated with PCF-MBs (c1-3) or PCF-MBs+US (c4-6). (d) concentration dependent cancer killing ability after different treatments by MTT assay 72 hours post treatment. (*p < 0.05)


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Figure 3. In vitro studies of PDT induced ABCG2 depletion in HT-29 cells. Immunofluorescence imaging of ABCG2 (green) (a) and Caspase-3 (b) before and after PGL-MBs+US+Light treatment for 2 min and 4 min, respectively. Nuclei is stained blue with DAPI. Scale bar, 10 μm. Quantification of the ABCG2 (c) and caspase 3 (d) fluorescence intensity per DAPI area after 2 min and 4 min PDT irradiation. (e) Western blotting of ABCG2, caspase-3 and β-actin in cell lysates collected from PCF-MBs+US+Light treated HT-29 cells after different laser exposure durations (50 mW/cm2, 0-5 min). ABCG2 (f) and Caspase-3 (g) expressions relative to no-treatment (NT) were normalized to β-actin in the western blotting results. (h) Intracellular CPT levels of PCF-MBs post-PDT while PCF-MBs containing media were maintained during and after PDT. HT-29 cells were incubated with PCF-MBs (5 μm) for 4 hours before irradiation. Intracellular CPT levels from cell lysates at 1 and 6 hours post-PDT


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were determined by fluorescence signals of CPT (Ex/Em: 355/460 nm). (i) Intracellular CPT levels of PCF-MBs post-PDT while PCF-MBs were removed after PDT. (***p < 0.001, not significant, ns, p > 0.05)


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Figure 4.

(a) Ultrasound imaging of xenograft tumor before (left) and after (right)

PCF-MBs injection. (b) Whole body in vivo fluorescence images obtained after tail vein injection of PCF-MBs (1 mg mL−1, 100 μL) followed by UTMD and their integrated fluorescence intensity (d). Fluorescence images (c) and integrated fluorescence intensity (e) of major organs and tumors obtained after tail vein injection of PCF-MBs (1 mg mL−1, 100 μL) for 24 h. (*p < 0.05)


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Figure 5. Therapeutic efficacy in HT-29 colon xenograft tumor model. (a) Tumor growth profiles in nude mice treated with PBS, PCF-MBs, PCF-MBs+Light, PGL-MBs+US+Light, CF-MBs+US and PCF-MBs+US+Light on Day 0 and Day 3 (see arrows). Each PCF-MBs injection dose was 13.5 mg/kg and PGL-MBs injection dose was 4.98 mg/kg. (b) Body weight change after different treatments. Results are presented as mean ±SD (n = 6). (***p < 0.001)


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Figure 6. H&E staining of major organs resected from the sacrificed animals after different treatments for 28 days. (Scale bar: 200 μm)


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Figure 7. Immunohistochemistry images of HT-29 cancer cells after different treatments for 28 days. (a) Effects of different treatments on drug resistance, proliferation ability, tumor vasculature, and hypoxia level were evaluated by immunohistochemistry staining of ABCG2, Ki67, CD31 and HIF-1α. (b) Quantification of staining intensity of ABCG2, Ki67, CD31 and HIF-1α using Image J. (c) Immunofluorescence images of different treatments showed tumor vasculature (CD 31, red), drug resistance transporter ABCG2 expression (green) and the nucleus (blue). (Scale bar: 40 μm)


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Ultrasound Triggered Conversion of Porphyrin ... - ACS Publications

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