Intracellularly Acid-Switchable Multifunctional ... - ACS Publications

Subscriber access provided by University of Pennsylvania Libraries

Article

Intracellularly Acid-Switchable Multifunctional Micelles for Combinational Photo/Chemotherapy of the Drug Resistant Tumor Tingting Wang , Dangge Wang , Haijun Yu, Mingwei Wang, Jianping Liu , Bing Feng , Fangyuan Zhou , Qi Yin, Zhiwen Zhang, Yongzhou Huang, and Yaping Li ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b07706 • Publication Date (Web): 11 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Nano is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Intracellularly Acid-Switchable Multifunctional Micelles for Combinational Photo/Chemotherapy of the Drug Resistant Tumor Tingting Wang+,#, Dangge Wang+,#, Haijun Yu+,*, Mingwei Wang╟, Jianping Liu+, Bing Feng+, Fangyuan Zhou+, Qi Yin+, Zhiwen Zhang+, Yongzhuo Huang+, Yaping Li+,*

+

State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. ╟

Department of Nuclear Medicine, Fudan University Shanghai Cancer Center,

Shanghai 200032, China.

#

Equal contribution to this study.

*Corresponding authors: Dr. Haijun Yu ([email protected]); Prof. Yaping Li ([email protected]), Tel/Fax: +86-21-2023-1979.

1 ACS Paragon Plus Environment

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 33

Abstract: The intrinsic or acquired drug resistance is the main challenge for cancer chemotherapy today. So far, lots of nanosized drug delivery systems (NDDS) have been exploited to combat cancer drug resistance. However, the therapy efficacy of current NDDS is severely impaired by the limited tumor penetration of the nanoparticles due to the existence of physiological and pathological barriers in the solid tumor. In this study, we report on the design and fabrication of intracellularly acid-switchable multifunctional micelles for combinational photo and chemotherapy of the drug resistant tumor. The micelles were composed of a pH-responsive diblock copolymer, a photosensitizer and a polymeric prodrug of doxorubicin. The micelle displayed silenced fluorescence and photoactivity during the blood circulation, while switched to active state at weakly acid condition (i.e., pH ≤ 6.2) in the endocytic vesicles to dramatically induce 7.5-fold increase of the fluorescence signal for fluorescence imaging. Upon near infrared (NIR) laser irradiation, the micelle induced notable reactive oxygen species (ROS) generation to trigger cytosol release of the chemotherapeutics and perform photodynamic therapy (PDT). Moreover, the micelle efficiently converted the NIR light to local heat for enhancing tumor penetration of the anticancer drug, tumor specific photothermal therapy and photoacoustic (PA) imaging. Furthermore, the micelles could generate amplified magnetic resonance (MR) signal at acidic microenvironment to perform magnetic resonance (MR) imaging. Collectively, this study presents a robust nanoplatform for multimodal imaging and combinational therapy of the drug resistant tumor, which might provide an insight for developing polymer-based NDDS for cancer therapy.

Keywords:

Acid-switchable

micelles,

Drug

resistance,

Combinational therapy, Multimodal imaging.


Tissue

penetration,

Page 3 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Nanosized drug delivery systems (NDDS) has been investigated to improve the therapeutic performance and biosafety of chemotherapy since nanoparticle (NP) encapsulation notably elongates the blood circulation and increases the bioavailability of the anticancer drugs.1 NDDS can also integrate different therapeutic modalities for combinational cancer therapy.2,3 Despite all these advantages, poor tumor penetration of the nanosized drugs remains a critical challenge for NDDS-mediated cancer chemotherapy due to the existence of the physiological and pathological tumor barriers.4 The tumor microenvironment exhibits an elevated interstitial fluid pressure (IFP) caused by the leaky vasculature, defective lymphatic drainage, and dense extracellular matrix (ECM).5,6 The NPs administrated through the systemic injection are thus restricted predominately in the perivascular area, which can induce acquired drug-resistance due to continued exposure of the cancer cells to a sublethal dose of chemotherapeutics.7-9 To address this problem, a large variety of theranostic NPs combining the therapeutic functions with the imaging modalities including fluorescence imaging,10 magnetic resonance (MR) imaging,11,12 photoacoustic (PA) imaging,13,14 computed tomography (CT),15 or positron emission tomography (PET)16-18 had been developed for cancer treatment. These multifunctional NPs can be used for precise tumor imaging, real-time monitoring of the nanoparticle distribution, evaluation of therapeutic outcomes and individualized optimization of the treatment regimens.19 Bioresponsive theranostic NPs with signal amplification were also exploited by targeting the redox,20 hypoxic,21,22 acidic,23-25 or enzymatic26 microenvironment of the tumor or cancer cells. Among all these stimuli, the low pH in the tumor or endocytic vesicles compared to the normal tissue or cytoplasm is most extensively investigated. It is well documented that the solid tumor has an acid extracellular microenvironment with pH of 6.5~6.8, and the endocytic vesicles are even more acidic (i.e., pH=5.5~6.5 in the early endosome, and pH=5.0~5.5 in the late endosome/lysosome).7,13 A few theranostic NPs with acid-induced hydrophobic to hydrophilic transition,24,27,28 3 ACS Paragon Plus Environment

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 33

surface charge reversing,29 or size tunable property30 were proposed to improve the imaging sensitivity or therapeutic performance against tumor. Nanoparticles modified with cell penetrable peptide were also developed for enhancing tumor penetration.31 However, bioresponsive theranostic NPs with satisfied tumor penetration capability have rarely been reported.32 Ultra

pH-responsive

diblock

copolymer

poly(ethylene

glycol)-block-poly(diisopropanol amino ethyl methacrylate co-hydroxyl methacrylate) (PEG-b-PDPA, referred to as PDPA) can self-assemble into core-shell micelles at neutral pH condition (i.e., 7.4), but dissociates sharply at weakly acidic condition (≤ 6.2) upon protonation of the tertiary amines.28 We had previously developed PDPA-based dual pH-responsive micelles for co-delivery of siRNA and a membrane poration reagent amphotericin B to overcome the endosome barrier.33 Alternatively, we had also conjugated a NIR absorbent cypate to PDPA micelles for photothermal and chemotherapy of drug resistant tumor.34 In current study, to develop smart nanoplatform for cancer theranostic, we presented a PDPA-based versatile micelles with intracellularly acid-switchable properties for multimodal tumor imaging and combinational treatment of drug resistant tumor. The micelles were composed of three independent functional units. The first unit is a PDPA-based polymeric matrix for acid-induced dissociation of the micelle nanostructure. The second component is a photosensitizer chlorin e6 (Ce6), which is a widely used photosensitizer for PDT,35 and has the intrinsic ability to chelate the MR reagent gadolinium (Ⅲ) (Gd3+) and fluorescence emission in the NIR region (e.g., 680 nm). Ce6-conjugated PDPA micelles (PDPC) display silent fluorescence and MR signals at neutral pH, therefore with low background noise and quenched photoactivity in the blood. The third component is a pluronic prodrug of doxorubicin (PDOX), which can increase accumulation and suppress DOX efflux in drug resistance cancer cells by depleting ATP-production in the mitochondrion.36 The PDOX prodrug is loaded into the micelles through hydrophobic interaction with the micelle core (Scheme 1A). 4 ACS Paragon Plus Environment

Page 5 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Moreover, the micelles administrated through systemic injection can passively accumulate in the tumor via the enhanced penetration and retention (EPR) effect. After internalized via endocytosis, PDPC micelles are quickly dissociated in the early endosome upon protonation and generate strong fluorescence and T1-weighted MR signals for fluorescence and MR imaging, respectively. The activated micelles can induce notable reactive oxygen species (ROS) generation upon NIR laser irradiation for photodynamic therapy (PDT) and promoting tumor penetration of the chemotherapeutics. Furthermore, the PDPC micelles can cause significant temperature elevation via NIR laser illumination for PA imaging and photothermal therapy (PTT) (Scheme 1B). The acid-switchable versatile micelles could thus serve as a robust nanoplatform for combinational treatment of various drug resistance tumors. RESULTS AND DISCUSSION Synthesis and Characterization of Photosensitizer-Conjugated Acid-Switchable Micelles. The pH-responsive diblock copolymer PDPA bearing pendant hydroxyl groups was synthesized by atom transfer radical polymerization (ATRP) method.37 PDPC was then prepared by coupling Ce6 on the pendent hydroxyl groups of PDPA via formation of ester bonds (Figure S1&2). The Ce6 weight percentage in PDPC was ~ 8.0 wt.% as determined by using UV-Vis spectroscopic measurement, roughly about three Ce6 molecules on each PDPC chain. To demonstrate the acid-responsive property of the hybrid micelles, we firstly prepared the PDOX-free PDPC micelles with PDPC and PDPA (1:1 weight ratio) by following a nanoprecipitation procedure described previously.34 Paramagnetic T1-weighted MR agent Gd3+ was loaded into the hydrophobic core of the micelles through coordinating with Ce6.38,39 Diblock copolymer poly(ethylene glycol)-block-poly(tertbutyl methyl acrylate-co-hydroxyl methacrylate) (PTBA) conjugated with Ce6 (referred as PTBC) without pH-responsive property was synthesized by ATRP method (Figure S3). PTBC can self-assemble into spherical micelles with similar core-shell structure as PDPC in 5 ACS Paragon Plus Environment

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

aqueous solution. To demonstrate the acid-activatable property of the PDPC micelles, we thus prepared the PTBC micelle with 4.0 wt.% Ce6 loading as a pH-insensitive control of PDPC ones. The pH-responsive property of the PDPC micelles was firstly investigated by using transmission electron microscopic (TEM) examination. PDPC formed spherical micelles ca. 30 nm at pH 7.4 (Figure 1A), but the micelles dissociated completely at pH 6.0 and form amorphous aggregates due to protonation of PDPA segments.40 In contrast, the PTBC micelles did not respond to the pH change from 7.4 to 6.0, and remained constant morphology. Acid-triggered dissociation of the PDPC micelles was further confirmed by examining their hydrodynamic diameter using dynamic light scattering (DLS) measurement (Figure S4A&B). The acid-activatable fluorescence property of the PDPC micelles was examined by measuring fluorescence emission of Ce6 at different pH values. The fluorescence emission of PDPC and PTBC micelles was quenched at pH ≥ 6.6 due to fluorescence resonance energy transfer (FRET) effect between the Ce6 molecules and photo-induced electron transfer (PET) from the nitrogen atom of PDPA to the porphyrin ring of Ce6 inside the hydrophobic core.33 Upon protonation at pH 6.2, the fluorescence intensity of the (4.0 wt.%) Ce6-loaded PDPC micelles was dramatically increased by 7.5-fold over that examined at pH 7.4 (Figure 1B and Figure S4C-H), implying the FRET and PET effects were stopped because micelle dissociation and protonation of the tertiary amine groups, respectively. The PDPC micelles were stable in phosphate buffer solution (PBS) and mouse blood after 24 h incubation at 37 °C as indicated by the negligible fluorescence change (Figure S5). By contrast, the fluorescence emission of the pH-insensitive PTBC micelles was not recovered at acidic pH (e.g., ≤ 6.0) since Ce6 always aggregated inside the micelle core. The T1-weighted MR phantom of Gd3+-loaded PDPC micelles was examined at 7.0-T small animal magnetic field. Figure 1C showed a significant intensity variation of PDPC micelles in response to the pH change. A high longitudinal relativity (r1) of 6 ACS Paragon Plus Environment

Page 6 of 33

Page 7 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

10.2 mM-1·s-1 was detected at pH 6.0, displaying 6-fold higher than that examined at pH 7.4. The r1 value of PDPC micelles at pH 6.0 was 27-fold higher than that of Gd3+-loaded PTBC micelles examined at the same pH condition (Figure S6A). This could be explained by acid-triggered dissociation of the micelles, and access of water molecules to the Gd3+ ions since the r1 value was majorly attributed to the direct water coordination with Gd3+.41 The r1 value of the PDPC micelles examined at pH 6.0 was 2.4-fold higher than that of the commercialized T1-weighted MRI reagent DTPA-Gd (Magnevisit®, r1 = 4.29 Mm-1s-1).12 The acid-activatable fluorescence and MR properties of PDPC micelles can thus realize tumor specific imaging with signal amplification while minimized background noise in the blood circulation. The influence of buffer pH on the photoactivity of the PDPC micelles was examined by using 9,10-anthracenediyl- bis(methylene) dimalonic acid (ABDA) as a reactive oxygen species (ROS) indicator. PTBC produced negligible ROS when exposed to 655 nm laser at pH values ranging from 7.4 to 5.8. Similarly, PDPC generated insignificant ROS at pH ≥ 6.6 due to quenched photoactivity of Ce6 inside the hydrophobic core. By sharp contrast, the PDPC micelles induced significant ROS at pH ≤ 6.2, implying acid-induced recovery of the photoactivity (Figure 1D). The photothermal property of PDPC micelles was verified by determining the temperature elevation upon irradiation with 655 nm laser. PDPC micelles caused tunable temperature elevation, depending on either the micelle concentration or the power density (Figure 1E and Figure S6B&C), in good consistence with the literature reports by Zheng and Lam et al. that the porphyrin-incorporated nanovehicles could efficiently convert laser light energy into local heat for PTT of solid tumor.20,42 The photothermal conversing efficacy of the PDPC micelles was not affected by pH change. PDPC induced comparable temperature elevation ca. 30 °C at pH ranging from 7.4 to 6.0 as shown in Figure 1E. We attributed the consistent photothermal property of the PDPC micelles to their stable absorption at different pH conditions (Figure S6D). Subsequently, we examined the capability of PDPC micelles for PA 7 ACS Paragon Plus Environment

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 33

imaging by using multispectral optoacoustic tomography (MSOT) with an excitation wavelength of 680 nm. Figure 1F showed that the PDPC micelles produced notable PA signal in a Ce6 concentration-dependent manner at either neutral or acidic pH. Acid-Triggered Activation of the Photosensitizer-Conjugated Micelles in Vitro. We next investigated the acid-triggered intracellular activation of the PDPC micelles in vitro in DOX-resistant MCF-7/ADR breast cancer cells since drug resistance is a major obstacle for cancer chemotherapy.17,18 Strong fluorescence signal assigned to Ce6 was found in the MCF-7/ADR cells post 6 h micelle incubation (Figure 2A). The fluorescence signal of Ce6 was abolished when the cells were pretreated with an ATP vascular inhibitor, Bafilomycin A1 (Baf-A1) for 1 h, confirming the intracellular protonation of the PDPA moiety accounts for fluorescence recovery of the PDPC micelles. The intracellular photoactivity of the PDPC micelles was

examined

by

confocal

laser

scanning

microscopy

(CLSM)

using

2’,7’-dichlorfluorescein-diacetate (DCFH-DA) as a fluorescence probe of ROS. Red fluorescence spots of Ce6 was found surrounding the nuclei in the absence of laser irradiation (Figure 2B), implying the majority of the micelles were entrapped in the lysosome (Figure S7). Strong green fluorescence appeared in the micelle-treated cells upon 5 min irradiation with 655 nm laser, indicating notable ROS generation, along with the cytosol diffusion of Ce6. The photoactivity of the PDPC micelles was completely silenced by Baf-A1 treatment (Figure 2C). The influence of laser dose on intracellular ROS generation of PDPC micelles was quantitatively examined using flow cytometry. Figure 2D&E demonstrated that the intracellular ROS concentration was positively correlated with the power density, and reached a plateau at photo density of 2.0 W/cm2. Baf-A1 treatment reduced the ROS generation efficiency of PDPC micelles by 15.3-fold. To evaluate the phototoxicity of PDPC and PTBC micelles, MCF-7/ADR cells were pre-incubated with PDPC micelles for 12h, and then illuminated with 655 nm laser for 3 min. As shown in Figure 2F, PDPC micelles caused tunable phototoxicity 8 ACS Paragon Plus Environment

Page 9 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

in a power density-dependent manner. The PTBC micelles induced negligible cell death when illuminated at power density ≤ 1.5 W/cm2. Notably, over 40% of the MCF-7/ADR cells treated with PTBC micelles were killed when illuminated at a power density of 2.0 W/cm2. The phototoxicity of PDPC micelles decreased significantly with Baf-A1 pre-incubation. All these information suggested that the phototoxicity of PDPC micelles is positively correlated with the extent of ROS generation. However, the phototoxicity of PTBC was not affected by Baf-A1 treatment, suggesting that the phototoxicity of PTBC micelles was most likely caused by PTBC-induced hyperthermia effect (Figure S6B&C). To elucidate the contribution of PDT and PTT on the phototoxicity of PDPC micelles, we examined the cell apoptosis by flow cytometric analysis. Figure 2G revealed a positive correlation between the laser-induced cell apoptosis and the power density. For instance, the apoptotic cell population changed from 6.2% to 33.3% along with the increase of photo density from 0.5 to 2.0 W/cm2. Baf-A1 treatment reduced the apoptotic cell percentage to 18.6%, although Baf-A1 notably inhibited the PDT effect of the PDPC micelles by suppressing ROS generation (Figure 2D&E). Given the obvious photothermal effect of PDPC micelles at high power density (e.g., 2.0 W/cm2), the phototoxicity of PDPC micelles at high photo density can be attributed to a cumulative effect between laser irradiation-induced PTT and PDT. Fluorescence, MR and PA Multimodal Imaging In Vivo. The ability of the acid-activatable PDPC micelles to perform multimodal tumor imaging was assessed in the MCF-7/ADR tumor bearing nude mice. The mice were randomly grouped when the tumor volume reached 200 mm3 (n = 3 for each group). PDPC or PTBC micelles were then intravenously (i.v.) injected at an identical Ce6 dose of 5.0 mg/kg. Whole animal fluorescence imaging showed the PTBC micelles generated weak fluorescence signal at 4h postinjection (p.i.). By contrast, strong fluorescence emission was observed in the tumor of the PDPC group at 2 h p.i. (Figure 3A, top panel). The passive tumor accumulation and activation of the PDPC micelles was further 9 ACS Paragon Plus Environment

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

confirmed by fluorescence imaging of the excised tissues (Figure 3A, bottom panel), which can most likely be attributed to the enhanced penetration and retention (EPR) effect of the micelles. The maximal tumoural signal to background (S/B) ratio appeared at 2 h p.i., 2.2-fold higher than that of the PTBC group (Figure 3B). The fluorescence intensity decreased gradually over time due to the blood clearance of the micelles from the tumor. The T1-weighted MR property of PDPC micelles was examined in the nude mouse model bearing MCF-7/ADR tumors. Figure 3C displayed that the PDPC micelles generated much higher T1 contrast in the MCF-7/ADR tumor than PTBC. To elucidate the influence of Gd3+ concentration on the T1-weighted MR signal, the in vivo T1 MR signal of PDPC and PTBC micelles was then normalized with the intratumoral Gd3+ concentration. The normalized T1 MR signal of the PDPC group was 3.9-fold higher than that of the PTBC group examined at 2h p.i. (Figure 3D), which verified the acid-activatable MR property of the PDPC micelles since PTBC micelles delivered more Gd3+ to the xenograft tumors than PDPC (Figure S8). The T1 MR signal increased over time p.i., most likely attributed to Gd3+ ion deposition in the tumor. The ability of the PDPC micelles for PA imaging in vivo was demonstrated in the MCF-7/ADR tumor bearing mice by MSOT examination. The PA signal of the PDPC micelles increased over time due to the intratumoral accumulation of the micelles (Figure 3E). The quantifications of the PDPC signal intensity and signal distribution area in tumors showed that the maximal tumor uptake of PDPC occurred at 2h p.i., in good agreement with the fluorescence imaging data. The distribution area at 4h p.i. was 1.5-fold larger than that examined at 1h due to the diffusion into the deep tumor (Figure 3F). It was worth noting that the majority of the micelles were entrapped in the perivascular area without notable tumor penetration. Intracellular Uptake and Cytotoxicity of PDOX-Loaded Micelles In Vitro. To exploit the potential of PDPC micelles for combinational cancer therapy, DOX prodrug (PDOX) formed self-assemble PDOX/PDPC micelles with PDPC. PDOX 10 ACS Paragon Plus Environment

Page 10 of 33

Page 11 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

prodrug was synthesized by conjugating a molecular of DOX on the end of P85 via a lysosome cleavable Gly-Pro-Leu-Gly (GFLG) tetrapeptide (Figure S10&11). DOX percentage in the prodrug was 8.0 wt.% as determined by using fluorescence spectroscopy. The prodrug strategy has several obvious advantages over non-covalent encapsulation of small molecular free drug. Firstly, pluronic copolymer P85 can significantly increase the accumulation and cytotoxicity of DOX in the MCF-7/ADR cells by inhibiting drug efflux from the cancer cells (Figure S9). Secondly, the encapsulation of a polymeric prodrug into micelle nanoparticles through co-assembling allows tunable drug loading and prevents premature drug leakage in the blood circulation. Moreover, PDOX/PDPC micelles shows diminished nonspecific cytotoxicity since PDOX prodrug can only be released from the PDOX/PDPC micelles in the acidic endocytic vesicles of the cancer cells, where DOX was liberated from the PDOX prodrug via degradation of the GFLG spacer.43 The PDOX/PDPC hybrid micelles were prepared by self-assemble of PODX and PDPC at 1:1 weight ratio, with an identical DOX and Ce6 loading percentage of 4.0 wt.%. The resulting PDOX/PDPC micelles displayed spherical morphology with hydrodynamic particle size ca. 30 nm at pH 7.4 as determined by TEM and DLS, respectively. The hybrid micelles swelled up to 100 nm at pH 6.0 due to the dissociation of PDPC diblock copolymer. The hybrid micelles showed comparable acid-activatable fluorescence and photoactivity as PDPC, implying the pH-responsive property of PDPC was not interfered by loading PDOX (Figure S12). The cellular uptake, distribution and cytotoxicity of PDOX/PDPC micelles were examined in parental MCF-7 and drug resistant MCF-7/ADR cells by flow cytometry, CLSM and MTT examination, respectively. The hybrid micelles induced 2-fold higher DOX accumulation in the MCF-7/ADR cells than free DOX (Figure 4A&B). Meanwhile, PDOX/PDPC micelles showed a DOX resistance index of 1.3 (calculated by dividing the IC50 of DOX in the MCF-7/ADR cells with that in DOX-sensitive MCF-7 cells), 100-fold lower than that of free DOX (Figure 4C and Table S1), due to 11 ACS Paragon Plus Environment

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 33

the P85-medaited inhibition of DOX efflux from MCF-7/ADR cells . Pharmacokinetics and Combinational Treatment of the Drug Resistant Breast Tumor. Improved blood circulation, efficient tumor accumulation and deep tumor penetration of NPs are crucial factors for highly efficient cancer therapy using nanomedicine.1,44 The pharmacokinetic of the PDOX/PDPC micelles was investigated in the SD rat by examining the blood concentration of DOX using fluorescence spectroscopy. Compared to free DOX, the PDOX/PDPC micelles significantly improved the maximal blood concentration (Cmax) and area under the curve of DOX (AUC(0-t)) by 35 and 17-order of magnitude, respectively (Figure 5A and Table S2). The tumoural DOX concentration of the PDOX/PDPC group was 3.3 and 1.9-fold higher than that of the free DOX group, respectively, at 2 and 24 h p.i., due to tumoural accumulation of the PDOX/PDPC micelles (Figure 5B). To verify the potential of the PDOX/PDPC hybrid micelles to induce the hyperthermia effect in vivo, the PDOX/PDPC micelles were i.v. injected into the MCF-7/ADR tumor bearing nude mice at two Ce6 doses of 2.5 and 5.0 mg/kg. In a Ce6 dose-dependent manner, the hybrid micelles caused notable tumor temperature elevation upon laser irradiation at power density of 2.0 W/cm2 (Figure 5C&D). Hyperthermia triggered intratumoral penetration of the PDOX/PDPC micelles was verified by CLSM examination of the cryo-sections of the tumor. Without laser irradiation, the majority of the hybrid micelles were entrapped in the perivascular area of the tumor. In contrast, highly diffused PDOX distribution was observed post laser irradiation, which indicated laser irradiation induced hyperthermia effect could trigger deep tumor penetration of the PDOX/PDPC micelles since the tumoural neurovasculature

was

ablated

upon

laser

illumination

as

revealed

by

immunohistofluorescence (IHF) staining of CD31 (Figure 5E).33 To demonstrate the PDT effect in vivo, the nude mice bearing the MCF-7/ADR tumor were i.v. injected with the PDOX/PDPC micelles at a Ce6 dose of 5.0 mg/kg. The tumors were then illuminated with 655 nm laser at 2h p.i. under the guidance of 12 ACS Paragon Plus Environment

Page 13 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

fluorescence imaging at power density of 2.0 W/cm2. The fluorescence ROS probe DCFD-DA was intratumorally injected 10 min before laser irradiation. Figure 6 demonstrated that the green fluorescence diffused throughout the tumor section of the laser-treated PDOX/PDPC group, indicating that laser irradiation caused significant ROS generation. Negligible green fluorescence was detected in the tumor section of the PDOX/PDPC group without laser illumination and the PBS group with laser treatment, confirming laser-induced ROS generation was specific to the PDOX/PDPC micelles. Moreover, notable cytosol release of the red fluorescence was found in the tumor section of the laser-treated PDOX/PDPC group. This might be caused by the ROS-induced lysosome damage and successive cytosol release of DOX after it was liberated from PDOX in the endocytic vesicles.34,45 With the satisfied photothermal and photodynamic effect of the PDOX/PDPC micelles in hand, we next evaluated the anti-tumor efficacy of the hybrid micelles by using an orthotopically implanted MCF-7/ADR drug resistant tumor model. The tumor bearing mice were randomly grouped when the tumor volume reached 100 mm3 (n = 6). The tumor bearing mice were then i.v. injected with 100 µL of micelle or free DOX solution. Laser irradiation was performed in the PDPC and PDOX/PDPC groups at 2h p.i.. The tumor volume was monitored during the animal studies and the mice were photographed at the end of the anti-tumor study. PDPC micelle alone showed negligible influence on the growth of the MCF-7/ADR tumor compared to PBS control (Figure 7A&B). Although PDOX/PDPC micelles showed highly improved cytotoxicity in vitro and much more tumor accumulation in vivo than DOX (Figure 4C), they displayed slightly improved anti-tumor efficiency over DOX. This paradox can most likely be explained by the entrapment of the micelles in the perivascular area as demonstrated by CLSM examination of the tumor sections (Figure 4B&5E). PDOX/PDPC-mediated chemotherapy inhibited around half of the tumor growth, slightly more efficient than free DOX or PDOX, which could be explained by the 13 ACS Paragon Plus Environment

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

increased tumor distribution of PDOX due to the elongated blood circulation and EPR effect of PDOX/PDPC micelles. Upon localized laser illumination, PDPC micelles notably inhibited the tumor growth due to the synergistic effect of PDT and PTT. However, tumor recurred quickly when the treatment was finished. PDOX/PDPC + Laser displayed the highest antitumor efficiency among the animal groups and completely eradicated two of the tumors (Figure 7A). These results clearly suggested a cumulative anti-tumor effect between PDOX-mediated chemotherapy and laser irradiation induced PDT and PTT (Figure 7B). PDPC + Laser treatment caused negligible body weight change, implying a good biocompatibility of the PDPC micelles even under the condition that the mice were handled without shielding from room light (Figure 7C). Hematoxylin-eosin (H&E) staining revealed obvious DNA damage and membrane lysis in the PDOX/PDPC + Laser group (Figure 7D). This result implied that laser irradiation caused notable apoptosis of the tumor cells as confirmed by the TUNEL staining (Figure 7E). Histological examination of the major organs (i.e., heart, liver, spleen, lung, and kidney) confirmed the good biosafety of the PDOX/PDPC micelles since they caused negligible organ damage (Figure S13). Although the hybrid micelles also distribute to the health organs (e.g., liver and spleen), the results we presented herein demonstrated that the phototherapy and laser-induced micelle penetration can be limited to the tumor under the guidance of the tumor-specific fluorescence, MR and PA imaging. This can maximize the benefits of the combinational therapy while reduce the side effects. We believe the therapeutic potential of our acid-switchable micelles can be further improved by functionalizing with a targeting ligand,46 which will be exploited in our future study. CONCLUSION In summary, we successfully developed a micelle-based robust nanoplatform with laser-triggered tumor penetration capability and acid-activatable fluorescence and photoactivity. The micelles displayed silent fluorescence, MR and PDT activity in blood, which can be dramatically activated upon cellular uptake and intracellular 14 ACS Paragon Plus Environment

Page 14 of 33

Page 15 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

dissociation in the cancer cells. Upon NIR laser irradiation, the activated micelles induced the significant ROS generation and hyperthermia effect for combinational photo/chemotherapy of drug resistant tumor. Meanwhile, the hybrid micelles displayed good potential for fluorescence, MR and PA triplemodal tumor imaging. The versatile micelles reported in this study might provide a novel insight for combating the drug resistant tumor with nanoparticulate drugs. MATERIALS AND METHODS Fabrication and Characterization of the PDOX/PDPC Hybrid Micelles. The PDOX/PDPC hybrid micelles with an identical DOX and Ce6 weight percentage of 4.0 wt.% were prepared by nano-precipitation method as described elsewhere.33 Briefly, 5.0 mg of PDOX and 5 mg of PDPC was dissolved in 150 µL of anhydrous DMAC. The DMAC solution was added to 1.0 mL of DI water under sonication. DMAc was removed by dialyzing against DI water. The size distribution and zeta potential of the PDOX/PDPC micelles were examined by dynamic light scattering (DLS, Nanosizer, Malvern, UK) measurement. The morphology of the hybrid micelles was examined at two different pH values (i.e. 7.4 and 6.0) using transmission electron microscopy (TEM) operated at 200 kV (Joel, Japan). Absorption spectra of the micelles were recorded with an UV-Vis spectrophotometer (Shimadzu UV-2450, Japan). To prepare Gd3+-loaded PDPC micelles, aqueous solution of gadolinium chloride (GdCl3) was added to the DMAc solution of PDPC at a Gd3+ to Ce6 molar ratio of 5:1. The solution was incubated overnight at RT allowing loading Gd3+ to Ce6. The DMAc solution was drop-wise added to 1.0 mL of DI water under sonication. The excess Gd3+ and organic solvent were removed by dialyzing against DI water. The Gd3+ concentration in the resulting micelle solution was determined using inductively coupled plasma mass spectrometry (ICP-MS) measurements. Acid-Activatable Photoactivity and Photothermal Effects of The PDPC micelles. To investigate acid-triggered dissociation of the PDPC micelles, the micelle stock solution was diluted to 0.5 mg/mL with 0.2 M citric-phosphate buffers with different pH values. The fluorescence images of the micellar solutions were collected using a Caliper IVIS Lumina II in


ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

vivo imaging system (Perkin Elmer, USA). Fluorescence spectra was recorded on a fluorescence spectrophotometer (Ex 405 nm, Em 600~750 nm, Hitachi F-4600, Japan). To examine influence of buffer pH on the photoactivity of the PDPC micelles, the micelle solution was diluted to 0.25 mg/mL using 0.2 M citric-phosphate buffer solution with various pH values. A ROS probe ABDA was added at a final concentration of 50 µM. The micelle solution was then illuminated with a 655 nm laser at photo density 1.5 W/cm2 for 30 s (Changchun New Industries Optoelectronics Tech. Co., Ltd, Changchun, China). The ABDA consumption was determined by measuring the 378 nm absorption and normalized with that determined at pH 7.4. The photothermal property of PDPC and PDOX/PDPC micelles was determined by measuring laser irradiation-induced temperature elevation. Typically, 30 µL of micelle solution with desired concentration was placed in a 1.5 mL EP tube. The micelle solution was then irradiated with a 655 nm laser at selected photo density for 2 min (Changchun New Industries Optoelectronics Tech. Co., Ltd, Changchun, China). The laser-induced temperature elevation was recorded using an IR camera (IRTech Co. Ltd., Shanghai, China). Cellular Uptake, Distribution, and Cytotoxicity Assay of the PDOX/PDPC Micelles In Vitro. To examine the intracellular uptake of the PDOX/PDPC micelles, MCF-7 or MCF-7/ADR cells seeded in 24-well plates (2×105 cells/well) were treated with free DOX, PDOX or PDOX/PDPA micelles respectively, for different time durations (i.e. 1, 2, 4, 8 or 12 h) at an identical DOX concentration of 10 µg/ml. The cells were then trypisinized, thoroughly washed with PBS, resuspended in 500 µL of PBS and examined using a FACS Calibur flow cytometric system (BD Biosciences, Oxford, UK). To investigate intracellular distribution of DOX, MCF-7 or MCF7/ADR cells were seeded on 10 mm2 glass coverslips placed in 24-well tissue culture plates at a density of 5×104 cells/well. Cells were incubated with DOX, PDOX or PDOX/PDPA for 12 h at an identical DOX concentration of 10 µg/mL. Afterwards, the cells were stained with Hoechst 33342, washed with PBS, fixed with 4% paraformaldehyde and mounted on glass slides with anti-fade solution. The cells were then examined using CLSM (FluoView FV1000, Olympus, Japan). To determine the chemotoxicity of the PDOX-loaded hybrid micelles, MCF-7 or


Page 16 of 33

Page 17 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

MCF-7/ADR cells were seeded in 96-well tissue culture plates (5×104 cells/well) and incubated with DOX, PDPC, PDOX or PDOX/PDPC at the desired DOX concentrations. The cell viability was measured by MTT assay after 48 h micelle incubation. To determine the phototoxicity of the PDPC and PDOX/PPDPC micelles, MCF-7/ADR cells seeded in two 96-well tissue culture plates (5×104 cells/well) were incubated with the PDPC or PDOX/PDPC micelles at a Ce6 concentration of 3.0 µg/mL for 24 h. The cells were then treated with 655 nm laser for 2 min at varied photo density. To elucidate the influence of intracellular protonation on the phototoxicity of the PDPC and PDOX/PDPC micelles, the cells were pre-treated with 1.0 µM Baf-A1 for 1 h and then incubated with the micelles for 24 h. Afterwards, the cells were treated with 655 nm laser for 2 min and continually cultured for additional 24 h. The cell viability was then analyzed by MTT assay. The cells without light illumination were set as negative control. To determine laser irradiation inducing cell apoptosis, MCF-7/ADR cells were seeded in 12-well tissue culture plates at a density of 2×105 cells/well. The cells were then treated with the PDOX/PDPC micelles for 4 h at the identical Ce6 and DOX concentration of 3.0 µg/mL. The cells were harvested, re-dispersed in 30 µL of PBS and treated with 655 nm laser for 5 min. The cells were then stained with Annexin V-FITC and PI for 15 min at 37 °C and analyzed using flow cytometric examination. ROS Generation In Vitro and In Vivo. Laser irradiation induced ROS generation in vitro was detected using Reactive Oxygen Species Assay Kit following the manufacturer’s instruction. MCF-7/ADR cells seeded in 12-well plates (2 × 105 cells/well) were incubated with PDOX/PDPC at an identical Ce6 and DOX concentration of 3 µg/mL. The cell culture medium was removed after 4 h incubation and 10 µM of fluorescent probe DCFH-DA was added. The cells were continually incubated for 20 min, washed with PBS, harvested and resuspended in 30 µL of PBS. The cells were then treated with 655 nm laser for 5 min at photo density of 0.5, 1.0, 1.5 or 2.0 W/cm2, respectively. The intracellular ROS generation was examined by measuring the green fluorescence intensity using flow cytometric examination. To visualize ROS generation in vitro, MCF-7/ADR cells were seeded on 10 mm2 glass


ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

coverslips placed in 24-well tissue culture plates at a density of 5×104 cells/well. The cells were incubated with the PDPC or PDOX/PDPC micelles for 4h at an identical Ce6 or DOX concentration of 3.0 µg/mL-1. Afterwards, DCFH-DA and Hoechst 33342 were added into the cell culture medium following the similar protocol described above. Twenty minutes later, the cells were irradiated with 655 nm laser at photo density of 1.5 W/cm2 for 2 min, and examined using CLSM. To elucidate acid-triggered photoactivity of the PDOX/PDPC micelles, the cells were pre-treated with 1.0 µM Baf-A1 for 1 h and then illuminated with 655 nm laser at the same condition. The efficiency of the hybrid micelle to induce ROS generation in vivo was examined in tumor-bearing nude mice. When the tumor volume reached 200-300 mm3, the mice were intravenously (i.v.) injected with PBS, PDPC or PDOX/PDPC micelles at the same DOX or Ce6 dose of 5.0 mg/kg. Two hours p.i., the mice were anaesthetized with pentobarbital sodium and intratumorally injected with DCFH-DA at a dose of 2.5 mg/kg. Ten minute later, the tumors were irradiated with 655 nm laser for 2 min at photo density of 2.0 W/cm2. The tumors were collected, frozen sectioned at 5 µm thickness, stained with DAPI and observed by using CLSM examination. The tumoural temperature elevation was recorded with the IR camera thermographic system. To investigate the influence of laser irradiation on the blood vessels, the laser-treated tumor organs without DCFH-DA injection were collected, stained with CD31 antibody and observed using CLSM examination. Fluorescence, MRI and PA Imaging In Vitro and In Vivo. For fluorescence imaging in vitro, PDPC micelle solution was diluted into 0.5 mg/mL with 0.2 M citric-phosphate buffers with different pH and the fluorescence imaging was conducted using a Caliper IVIS Lumina II imaging system (Perkin Elmer, USA). For fluorescence imaging in vivo, 100 µL of PDPC or PTBC micelle solution were i.v. injected into MCF-7/ADR tumor-bearing nude mice at an identical Ce6 dose of 5.0 mg/kg. The whole body fluorescence images were collected at 1, 2 and 4 h p.i.. The mice were sacrificed 4 h post micelle administration. All major organs were colleced and examined for fluoresence imaging ex vivo. MRI in vitro was performed by 7.0 Tesla 70/20 BioSpec MR system (Bruker, Germany).


Page 18 of 33

Page 19 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

T1-weighted MR images and T1 map were acquired using RARE sequence. T1-weighted MRI: TE = 6.0 ms, TR = 1500 ms, Average = 4, Rare factor = 4, slice thickness = 1.0 mm, FOV = 41× 32 mm, and Matrix size = 256 × 256. T1 map: TE = 7.02 ms, TR = 550, 800, 1200, 2000 and 5000 ms, Average = 1, Rare factor = 2, slice thickness = 1.0 mm, FOV = 41× 32 mm, and Matrix size = 192 × 192. T1 MR map was generated with a 96-well plate containing 100 µL aliquots of micelle suspension at pH 7.4 or 6.0, ranging in Gd3+ concentration from 0 to 0.25 mM. For MRI in vivo, MCF-7/ADR tumor bearing nude mice were i.v. injected with 200 µL of PDPC or PTBC micelles at an identical Ce6 dose of 5.0 mg/kg. The mice were then imaged at the desired time point p.i.. PA imaging in vitro and in vivo was conducted on a multispectral optoacoustic tomography (MSOT) small animal scanner (InVision 256-TF, iTheramedical, Germany) at an excitation wavelength of 680 nm. Polyethylene tubes (I.D. ~ 3.0 mm) were held submerged in water and the ultrasound transducer placed across them. The micelle solution was added into the tubes and the PA measurements were conducted at three locations across the tubes. The averaged PA signal intensity for each group was then plotted against the micelle concentration to give the relative PA signal intensities. For MSOT imaging in vivo, the nude mice implanted with MCF-7/ADR tumor were i.v. injected with 100 µL of PDPC or PTBC micelle suspension at an identical Ce6 dose of 5.0 mg/kg. The mouse was then placed in a horizontal position in an animal holder under isoflurane anesthesia that allows for translation of the mouse through the imaging plane using a linear stage. The PA images were acquired at different time point p.i. and the averaged PA signals of the tumor regions were extracted with the ViewMSOT software. Biodistribution and Pharmacokinetics In Vivo. The organ biodistribution of the PDOX-loaded micelles was examined using MCF-7/ADR tumor bearing nude mice. The mice were randomly grouped (n = 3) when the tumor volume reached 200 mm3, and intravenously injected with 100 µL of PBS solution of DOX, PDOX or PDOX/PDPA micelles at an identical DOX dose of 5.0 mg/kg. The mice were sacrificed at 2 or 24 h p.i.. All major organs (i.e. heart, liver, spleen, lung, kidney and tumor) were collected, weighted and homogenized in 0.5 mL of DMSO per 100 mg tissue. The supernatant was collected by centrifugation at 10,000 rpm for 10 min. The DOX content in each tissue was measured using a fluorescence spectrophotometric


ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

method, and normalized with that of tissue weight. To investigate the pharmacokinetics of DOX prodrug, SD rats were randomly divided into three groups (n = 3). Each group was i.v. injected with 1.0 mL aliquot of DOX, PDOX or PDOX/PDPA solution with an identical DOX concentration of 5.0 mg/kg. Blood samples were collected from the orbital venous plexus with heparinized tube at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h 12 h and 24 h post administration, The supernatant of the blood sample was collected by centrifugation at 10,000 rpm for 10 min, and the DOX content in the supernatant was determined using fluorescence spectrophotometric measurement. Anti-Tumor Effect and Biosafety Assay In Vivo. For anti-tumor studies in vivo, nude mice were orthotopicly implanted with MCF-7/ADR tumor. The mice were randomly grouped (n = 6) when the tumor volume reached 150 mm3, and treated with PBS, free DOX, PDOX, PDPC, or PDOX/PDPC hybrid micelles respectively, at a DOX or Ce6 dose of 5.0 mg/kg. Two hours p.i., the tumors in the PDPC and PDOX/PDPC-injected groups were locally treated with 655 nm laser at photo density of 2.0 W/cm2 for 2 min. The treatment was repeated for three times at a time interval of three days. Body weight and tumor volume were monitored every 3 days over a whole period of 23 days. The tumor volume was calculated by following formula: V = (L×W×W)/2 (L, the longest dimension; W, the shortest dimension), and expressed as the relative tumor growth rate by normalizing with the initial tumor volume. Animal death was recorded when the tumor volume reached 2000 mm3 according to the protocol of animal study. All mice were sacrificed at the 23th day post first treatment. To evaluate the biosafety of the hybrid micelles, the major organs (heart, liver, spleen, lung, kidney and tumor) were collected and examined by Hematoxylin-Eosin (H&E) staining. Statistical Analysis. Results are given as Mean ± S.D. One way analysis of variance (ANOVA) was used to determine the significance of the difference. Statistical significance was set at p < 0.05 (* p < 0.05, ** p < 0.01).

Acknowledgement. We thank Dr. Jianfeng Zeng at School for Radiological and Interdisciplinary Sciences (RAD-X) from Soochow University for MSOT examination. Financial supports from the National Basic Research Program of China 20 ACS Paragon Plus Environment

Page 20 of 33

Page 21 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(2013CB932704), the National Natural Science Foundation of China (81521005, 81373359 and 11275050), and the Youth Innovation Promotion Association CAS are gratefully acknowledged. Supporting Information Available: Materials, methods for synthesis of PDOX prodrug, Ce6-conjugated PDPA diblock copolymer, and additional figures are available free of charge via the Internet at http://pubs.acs.org. REFERENCES AND NOTES 1. Chauhan, V. P.; Jain, R. K. Strategies for Advancing Cancer Nanomedicine. Nat. Mater. 2013, 12, 958—962. 2. Lim, E. K.; Kim, T.; Paik, S.; Haam, S.; Huh, Y. M.; Lee, K. Nanomaterials for Theranostics: Recent Advances and Future Challenges. Chem. Rev. 2015, 115, 327—394. 3. He, C.; Lu, J.; Lin, W. Hybrid Nanoparticles for Combination Therapy of Cancer. J. Control. Release. 2015, 219, 224—236. 4. Whitehead, K. A.; Langer, R.; Anderson, D. G. Knocking Down Barriers: Advances in siRNA Delivery. Nat. Rev. Drug Discov. 2009, 8, 129—138. 5. Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada, Y.; Kano, M. R.; Miyazono, K.; Uesaka, M.; Nishiyama, N.; Kataoka, K. Accumulation of Sub-100 nm Polymeric Micelles in Poorly Permeable Tumours Depends on Size. Nat. Nanotech. 2011, 6, 815—823. 6. Jain, R. K.; Stylianopoulos, T. Delivering Nanomedicine to Solid Tumors. Nat. Rev. Clin. Onco. 2010, 7, 653—664. 7. Gatenby, R. A.; Gillies, R. J. A Microenvironmental Model of Carcinogenesis. Nat. Rev. Cancer 2008, 8, 56—61. 8. Gottesman, M. M.; Fojo, T.; Bates, S. E. Multidrug Resistance in Cancer: Role of ATP-Dependent Transporters. Nat. Rev. Cancer 2002, 2, 48—58. 9. Bock, C.; Lengauer, T. Managing Drug Resistance in Cancer: Lessons from HIV Therapy. Nat. Rev. Cancer 2012, 12, 494—501. 10. Frangioni, J. V. In Vivo Near-Infrared Fluorescence Imaging. Curr. Opin. Chem. Bio. 2003, 7, 626—634. 11. Huh, Y. M.; Jun, Y. W.; Song, H. T.; Kim, S.; Choi, J. S.; Lee, J. H.; Yoon, S.; Kim, K. S.; Shin, J. S.; Suh, J. S.; Cheon, J. In Vivo Magnetic Resonance Detection of Cancer by Using Multifunctional Magnetic Nanocrystals. J. Am. Chem. Soc. 2005, 127, 12387—12391. 12. Wang, Y.; Yang, T.; Ke, H.; Zhu, A.; Wang, J.; Shen, J.; Liu, G.; Chen, C.; Zhao, Y.; Chen, H. Smart Albumin-Biomineralized Nanocomposites for Multimodal Imaging and Photothermal Tumor Ablation. Adv. Mater. 2015, 27, 3874—3882. 21 ACS Paragon Plus Environment

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

13. Pu, K.; Shuhendler, A. J.; Jokerst, J. V.; Mei, J.; Gambhir, S. S.; Bao, Z.; Rao, J. Semiconducting Polymer Nanoparticles as Photoacoustic Molecular Imaging Probes in Living Mice. Nat. Nanotech. 2014, 9, 233—239. 14. Cui, L.; Lin, Q.; Jin, C. S.; Jiang, W.; Huang, H.; Ding, L.; Muhanna, N.; Irish, J. C.; Wang, F.; Chen, J.; Zheng, G. A PEGylation-Free Biomimetic Porphyrin Nanoplatform for Personalized Cancer Theranostics. ACS Nano 2015, 9, 4484—4495. 15. Paulus, M. J.; Gleason, S. S.; Easterly, M. E.; Foltz, C. J. A Review of High-Resolution X-ray Computed Tomography and Other Imaging Modalities for Small Animal Research. Lab Animal 2001, 30, 36—45. 16. Shokeen, M.; Anderson, C. J. Molecular Imaging of Cancer with Copper-64 Radiopharmaceuticals and Positron Emission Tomography (PET). Acc. Chem. Res. 2009, 42, 832—841. 17. Sun, X.; Huang, X.; Yan, X.; Wang, Y.; Guo, J.; Jacobson, O.; Liu, D.; Szajek, L. P.; Zhu, W.; Niu, G.; Kiesewetter, D. O.; Sun, S.; Chen, X. Chelator-Free (64)Cu-Integrated Gold Nanomaterials for Positron Emission Tomography Imaging Guided Photothermal Cancer Therapy. ACS Nano 2014, 8, 8438—8446. 18. Shin, T. H.; Choi, Y.; Kim, S.; Cheon, J. Recent Advances in Magnetic Nanoparticle-Based Multi-Modal Imaging. Chem. Soc. Rev. 2015, 44, 4501—4516. 19. Li, Y.; Lin, T. Y.; Luo, Y.; Liu, Q.; Xiao, W.; Guo, W.; Lac, D.; Zhang, H.; Feng, C.; Wachsmann-Hoqiu, S.; Walton, J. H.; Cherry, S. R.; Rowland, D. J.; Kukis, D.; Pan, C.; Lam, K. S. A Smart and Versatile Theranostic Nanomedicine Platform Based on Nanoporphyrin. Nat. Commu. 2014, 5, 4712. 20. Wang, J.; Sun, X.; Mao, W.; Sun, W.; Tang, J.; Sui, M.; Shen, Y.; Gu, Z. Tumor Redox Heterogeneity-Responsive Prodrug Nanocapsules for Cancer Chemotherapy. Adv. Mater. 2013, 25, 3670—3676. 21. Zheng, X.; Wang, X.; Mao, H.; Wu, W.; Liu, B.; Jiang, X. Hypoxia-Specific Ultrasensitive Detection of Tumours and Cancer Cells in Vivo. Nat. Commu. 2015, 6, 5834. 22. Chen, H.; Tian, J.; He, W.; Guo, Z. H2O2-Activatable and O2-Evolving Nanoparticles for Highly Efficient and Selective Photodynamic Therapy Against Hypoxic Tumor Cells. J. Am. Chem. Soc. 2015, 137, 1539—1547. 23. Ma, X,; Wang, Y.; Zhao, T.; Li, Y.; Su, L. C.; Wang, Z.; Huang, G.; Sumer, B. D.; Gao, J. Ultra-pH-Sensitive Nanoprobe Library with Broad pH Tunability and Fluorescence Emissions. J. Am. Chem. Soc. 2014, 136, 11085—11092. 24. Ling, D.; Park, W.; Park, S. J.; Lu, Y.; Kim, K. S.; Hackett, M. J.; Kim, B. H.; Yim, H.; Jeon, Y. S.; Na, K.; Hyeon, T. Multifunctional Tumor pH-Sensitive Self-Assembled Nanoparticles for Bimodal Imaging and Treatment of Resistant Heterogeneous Tumors. J. Am. Chem. Soc. 2014, 136, 5647—5655. 25. Wagner, E. Polymers for siRNA Delivery: Inspired by Viruses to be Targeted, Dynamic, and Precise. Acc. Chem. Res. 2012, 45, 1005—1013.


Page 22 of 33

Page 23 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

26. Weissleder, R.; Tung, C. H.; Mahmood, U.; Bogdanov, A. Jr. In Vivo Imaging of Tumors with Protease-Activated Near-Infrared Fluorescent Probes. Nat. Biotech. 1999, 17, 375—378. 27. Dai, J.; Lin, S.; Cheng, D.; Zou, S.; Shuai, X. Interlayer-Crosslinked Micelle with Partially Hydrated Core Showing Reduction and pH Dual Sensitivity for Pinpointed Intracellular Drug Release. Angew. Chem. Int. Ed. 2011, 50, 9404—9408. 28. Wang, Y.; Zhou, K.; Huang, G.; Hensley, C.; Huang, X.; Ma, X.; Zhao, T.; Sumer, B. D.; Deberardinis, R. J.; Gao, J. A Nanoparticle-Based Strategy for the Imaging of a Broad Range of Tumours by Nonlinear Amplification of Microenvironment Signals. Nat. Mater. 2014, 13, 204—212. 29. Du, J.; Du, X.; Mao, C.; Wang, J. Tailor-Made Dual pH-Sensitive Polymer–Doxorubicin Nanoparticles for Efficient Anticancer Drug Delivery J. Am. Chem. Soc. 2011, 133, 17560—17563. 30. Jinhwan, K.; Mi, L. Y.; Youngnam, K.; Jong, K. W. Tumor-homing, size-tunable clustered nanoparticles for anticancer therapeutics. ACS Nano 2014, 8, 9358—9367. 31. Liu, Y.; Zhang, D.; Qiao, Z. Y.; Qi, G. B.; Liang, X. J.; Chen, X. G.; Wang, H. A Peptide Network Weaved Nanoplatform with Tumor Microenvironment Responsiveness and Deep Tissue Penetration Capability for Cancer Therapy. Adv. Mater. 2015, 27, 5034—5042. 32. Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12, 991—1003. 33. Yu, H.; Zou, Y.; Wang, Y.; Huang, X.; Huang, G.; Sumer, B. D.; Boothman, D. A.; Gao, J. Overcoming Endosomal Barrier by Amphotericin B-Loaded Dual pH-Responsive PDMA-b-PDPA Micelleplexes for siRNA Delivery. ACS Nano 2011, 5, 9246—9255. 34. Yu, H.; Cui, Z.; Yu, P.; Guo, C.; Feng, B.; Jiang, T.; Wang, S.; Yin, Q.; Zhong, D.; Yang, X.; Zhang, Z.; Li, Y. pH- and NIR Light-Responsive Micelles with Hyperthermia-Triggered Tumor Penetration and Cytoplasm Drug Release to Reverse Doxorubicin Resistance in Breast Cancer. Adv. Funct. Mater. 2015, 25, 2489—2500. 35. Allison, R. R.; Downie, G. H.; Cuenca, R.; Hu, X. H.; Childs, C. J.; Sibata, C. H. Photosensitizers in Clinical PDT. Photodiagn. Photodyn. 2004, 1, 27—42. 36. Alakhova, D. Y.; Kabanov, A. V. Pluronics and MDR Reversal: an Update. Mol. Pharma. 2014, 11, 2566—2578. 37. Yu, H.; Xu, Z.; Wang, D.; Chen, X.; Zhang, Z.; Yin, Q.; Li, Y. Intracellular pH-Activated PEG-b-PDPA Wormlike Micelles for Hydrophobic Drug Delivery. Polym. Chem. 2013, 4, 5052—5055. 38. MacDonald, T. D.; Liu, T. W.; Zheng, G. An MRI-Sensitive, Non-Photobleachable Porphysome Photothermal Agent. Angew. Chem. Int. Ed. 2014, 53, 6956—6959. 39. Zhang, X. A.; Lovejoy, K. S.; Jasanoff, A.; Lippard, S. J. Water-Soluble Porphyrins as a Dual-Function Molecular Imaging Platform for MRI and Fluorescence Zinc Sensing. Proc. Nat. Acad. Sci. U.S.A. 2007, 104, 10780—10785. 23 ACS Paragon Plus Environment

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

40. Yu, P.; Yu, H.; Guo, C.; Cui, Z.; Chen, X.; Yin, Q.; Zhang, P.; Yang, X.; Cui, H.; Li, Y. Reversal of Doxorubicin Resistance in Breast Cancer by Mitochondria-Targeted pH-Responsive Micelles. Acta Biomater. 2015, 14, 115—124. 41. Mi, P.; Dewi, N.; Yanagie, H.; Kokuryo, D.; Suzuki, M.; Sakurai,Y.; Li, Y.; Aoki, I.; Ono, K.; Takahashi, H.; Cabral, H.; Nishiyama, N.; Kataoka, K. Hybrid Calcium Phosphate-Polymeric Micelles Incorporating Gadolinium Chelates for Imaging-Guided Gadolinium Neutron Capture Tumor Therapy. ACS Nano 2015, 9, 5913—5921 42. Ng, K.; Lovell, J.; Vedadi, A.; Hajian, T.; Zheng, G. Self-Assembled Porphyrin Nanodiscs with Structure-Dependent Activation for Phototherapy and Photodiagnostic Applications. ACS Nano 2013, 7, 3484—3490. 43. Peng, Z.; Kopecek, J. Enhancing Accumulation and Penetration of HPMA Copolymer-Doxorubicin Conjugates in 2D and 3D Prostate Cancer Cells via iRGD Conjugation with an MMP-2 Cleavable Spacer. J. Am. Chem. Soc. 2015, 137, 6726—6729. 44. Li, J.; Yu, X.; Wang, Y.; Yuan, Y.; Xiao, H.; Cheng, D.; Shuai, X. A Reduction and pH Dual-Sensitive Polymeric Vector for Long-Circulating and Tumor-Targeted siRNA Delivery. Adv. Mater. 2014, 26, 8217—8224. 45. Guo, C.; Yin, S.; Yu, H.; Liu, S.; Dong, Q.; Goto, T.; Zhang, Z.; Li, Y.; Sato, T. Photothermal Ablation Cancer Therapy Using Homogeneous CsxWO3 Nanorods with Broad Near-Infrared Absorption. Nanoscale 2013, 5, 6469—6478. 46. Wang, X.; Li, J.; Wang, Y.; Cho, K.; Kim, G.; Gjyrezi, A.; Koenig, L.; Giannakakou, P.; Shin, H.; Tighiouart, M.; Nie, S.; Chen, Z.; Shin, D. HFT-T, a Targeting Nanoparticle, Enhances Specific Delivery of Paclitaxel to Folate Receptor-Positive Tumors. ACS Nano 2009, 3, 3165—3174.


Page 24 of 33

Page 25 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Scheme 1. (A) Self-assemble and structural composition of the acid-switchable micelles. The micelles were composed of pH-responsive diblock copolymer PEG-b-PDPA, gadolinium-coordinated photosensitizer Ce6 and a pluronic prodrug of DOX; (B) Schematic illustration of the versatile micelle as a robust nanoplatform for multimodal imaging and combinational therapy of drug resistant tumor.

ACS Paragon Plus Environment

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Characterization of the intracellularly acid-switchable multifunctional micelles. (A) TEM images of the PTBC and PDPC micelles examined at pH 7.4 and 6.0 (scale bar 100 nm); (B) pH-responsive fluorescence property of the PDPC and PTBC micelles, the insert shows the fluorescence images of the PDPC (up panel) and PTBC (bottom panel) micelles examined at different pH values; (C) pH-dependent T1weighted MR properties of the PDPC micelles; (D) 655 nm laser-triggered ROS generation of PDPC and PTBC micelles; (E) Photothermal profile of PDPC micelles vs. Ce6 concentration (black curve, pH 7.4) or pH value (red curve, Ce6 concentration 50 µg/mL); (F) PA signal intensity vs. Ce6 concentration at pH 6.0 or 7.4, the insert shows the PA images of the PDPC micelles examined at pH 6.0. 42x22mm (300 x 300 DPI)


Page 26 of 33

Page 27 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 2. (A) Fluorescence images of MCF-7/ADR cells incubated with the PDPC micelle. The intracellular fluorescence signal was quenched with Baf-A1 pre-incubation; (B) CLSM images of intracellular ROS generation in vitro; (C) Schematic illustration of protonation dependent fluorescence and PDT properties of PDPC micelles; (D&E) Flow cytometric examination of intracellular ROS generation of PDPC micelles; (F&G) Phototoxicity of PDPC micelles in MCF-7/ADR cells determined by (F) MTT assay and (G) apoptosis analysis, respectively. 76x98mm (300 x 300 DPI)


ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. (A) Fluorescence images of MCF-7/ADR tumor-bearing mice post i.v. injection of the PDPC or PTBC micelles, the bottom panel showed the ex-vivo images examined at 4h p.i.; (B) Fluorescence signal to background (S/B) ratio vs. the injection time; (C) T1-weighted MR images of MCF-7/ADR tumor-bearing mice p.i. of the PDPC or PTBC micelles; (D) Time course quantifications of T1-weighted MR signal intensity; (E) PA imaging of the tumor-bearing mice injected with the PDPC micelles; (F) Time course quantifications of the PA signal intensity. The PA intensity was normalized with the tumor area and the intratumoral distribution area, respectively (the dashed cycles showed the tumor margin). 70x81mm (300 x 300 DPI)


Page 28 of 33

Page 29 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 4. (A) Quantitative analysis of DOX accumulation in MCF-7/ADR cells; (B) Intracellular DOX distribution in MCF-7 and MCF-7/ADR cells (scale bar 50 µm for all images); (C) Cytotoxicity assay of PDOX/PDPC micelles in MCF-7/ADR cells, PDOX and PDOX/PDPC micelles both significantly combated DOXresistance of MCF-7/ADR cells. 40x32mm (300 x 300 DPI)


ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. (A) Pharmacokinetics of PDOX/PDPC hybrid micelles; (B) Organ distribution of PDOX in MCF-7/ADR tumor bearing nude mice; (C) Ce6 concentration dependent photothermal property of the PDOX/PDPC micelles in vivo at a Ce6 dose of 2.5 mg/kg (LD) or 5.0 mg/kg (HD); (D) Infrared thermal images of MCF7/ADR tumor treated with 655 nm laser for 2 min; (E) Laser-triggered tumor penetration of PDOX/PDPC micelles (scale bar 100 µm). 54x49mm (300 x 300 DPI)


Page 30 of 33

Page 31 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 6. PDT effect of the PDOX/PDPC micelles in vivo. The CLSM images demonstrated that laser illumination induced significant ROS generation and cytosol release of DOX. 67x56mm (300 x 300 DPI)


ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7. (A) Photographs of MCF-7/ADR tumor bearing nude mice and the tumors taken at the end of antitumor studies, the white arrows indicated the tumor location; (B) Tumor growth inhibition profiles of PDOX/PDPC micelles in combination with NIR laser irradiation, the red arrows indicated the time points for micelle administration, # the mice were sacrificed when the tumor volume reached 2000 mm3); (C) Body weight change of the tumor-bearing mice measured during the anti-tumor studies (1#: PBS, 2#: PDPC; 3#: DOX; 4#: PDOX; 5#: PDOX/PDPC; 6#: PDPC+ Laser; 7#: PDOX/PDPC + Laser); (D) H&E and (E) Tunnel staining of the tumor sections (scale bar = 200 µm for all images). 38x20mm (300 x 300 DPI)


Page 32 of 33

Page 33 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Intracellularly acid-switchable multifunctional micelles with laser-enhanced tumor penetration capability were presented. This micelle-based nanoplatform could be used for acid-activatable photodynamic/photothermal/chemotherapy of the drug resistant cancer, as well as acid-triggered fluorescence, T1-weighted magnetic resonance and photoacoustic multimodal tumor imaging.


Intracellularly Acid-Switchable Multifunctional ... - ACS Publications

Recommend Documents