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Synthesis of Hollow Biomineralized CaCO3-Polydopamine Nanoparticles for Multimodal Imaging-Guided Cancer Photodynamic Therapy with Reduced Skin Photosensitivity Ziliang Dong, Liangzhu Feng, Yu Hao, Muchao Chen, Min Gao, Yu Chao, He Zhao, Wenwen Zhu, Jingjing Liu, Chao Liang, Qiao Zhang, and Zhuang Liu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b11036 • Publication Date (Web): 28 Jan 2018 Downloaded from http://pubs.acs.org on January 28, 2018

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Journal of the American Chemical Society

Synthesis of Hollow Biomineralized CaCO3-Polydopamine Nanoparticles for Multimodal Imaging-Guided Cancer Photodynamic Therapy with Reduced Skin Photosensitivity Ziliang Dong†, Liangzhu Feng†*, Yu Hao†, Muchao Chen†, Min Gao†, Yu Chao†, He Zhao‡, Wenwen Zhu†, Jingjing Liu†, Chao Liang†, Qiao Zhang†, Zhuang Liu†* †

Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, 199 Ren'ai Road, Suzhou, 215123, Jiangsu, PR China. ‡

Department of Radiology, Children's Hospital of Soochow University, Suzhou 215003, Jiangsu, PR China. Supporting Information ABSTRACT: The development of activatable nanoplatforms to simultaneously improve diagnostic and therapeutic performances while reducing side effects is highly attractive for precision cancer medicine. Herein, we develop a one-pot, dopamine-mediated biomineralization method using a gas diffusion procedure to prepare calcium carbonate-polydopamine (CaCO3-PDA) composite hollow nanoparticles as a multifunctional theranostic nanoplatform. Because of the high sensitivity of such nanoparticles to pH, with rapid degradation under a slightly acidic environment, the photoactivity of the loaded photosensitizer, i.e., chlorin e6 (Ce6), which is quenched by PDA, is therefore increased within the tumor under reduced pH, showing recovered fluorescence and enhanced singlet oxygen generation. In addition, due to the strong affinity between metal ions and PDA, our nanoparticles can bind with various types of metal ions, conferring them with multimodal imaging capability. By utilizing pH-responsive multifunctional nanocarriers, effective in vivo anti-tumor photodynamic therapy (PDT) can be realized under the precise guidance of multimodal imaging. Interestingly, at normal physiological pH, our nanoparticles are quenched and show much lower phototoxicity to normal tissues, thus effectively reducing skin damage during PDT. Therefore, our work presents a unique type of biomineralized theranostic nanoparticles with inherent biocompatibility, multimodal imaging functionality, high anti-tumor PDT efficacy, and reduced skin phototoxicity.

INTRODUCTION To date, multifunctional nanoplatforms with integrated diagnostic and therapeutic functions have been extensively explored, aiming at precision cancer nanomedicine with great specificity and efficacy 1. Toward this end, a variety of activatable nanoplatforms, which can be switched from the “silent state” before reaching the tumor to the “activated state” within the tumor in response to either exogenous stimuli (e.g., light, temperature, ultrasound) and/or endogenous stimuli (variations in pH, redox gradients and enzyme concentrations), have received tremendous attention in recent years 2-7. Compared with conventional nonresponsive nanoplatforms, activatable nanoplatforms not only can perform treatment precisely at the desired time and location (e.g., only within lesions) but can also minimize the side effects to normal tissues. Thus, developing activatable nanoplatforms with excellent biocompatibility is of great interest, particularly for cancer nanomedicine. Photodynamic therapy (PDT) has emerged as a potent noninvasive therapeutic method in which photosensitizers (PS), upon light irradiation, are able to transform surrounding molecular oxygen into cytotoxic singlet oxygen (SO) and reactive oxygen species (ROS) for the effective treatment of cancers or other diseases 8-10. However, the poor tumor selectivity of conventional PDT agents, especially in terms of skin accumulation, can lead to severe damage to the skin and other healthy tissues during PDT upon exposure to light 11,12. Thus, to reduce the phototoxicity of PDT while maintaining its therapeutic efficacy, activata-

ble PDT agents that are inactive in normal tissues but become active within the tumor via either exogenous stimuli or tumorassociated endogenous stimuli would be rather attractive 3. Recently, several different groups, including ours, have reported the construction of near-infrared (NIR)-light-activatable nanoagents for selective cancer PDT with efficient skin protection. In those systems, the PDT effect of loaded PS was blocked by the coloaded NIR dye molecules via fluorescence resonance energy transfer (FRET) and recovered after NIR light irradiation to induce photobleaching of the NIR dyes, realizing NIR-controllable selective cancer PDT 13,14. However, those strategies still have some limitations; such activatable PDT nanosystems, which are externally controlled by a laser rather than the internal characteristics of tumors, cannot precisely distinguish tumors from normal tissues. Thus, designing activatable PDT nanosystems that can be activated by tumor-specific endogenous stimuli would be of great interest for the next generation of cancer PDT. Biomimetic and mineralized materials, such as eumelanin-like polydopamine (PDA) 15-19, calcium carbonate (CaCO3) 20,21 and calcium phosphate 22,23, are appealing candidate materials for bioapplications owing to their excellent biocompatibility 24,25. Herein, we report a simple approach for preparing biodegradable and biocompatible CaCO3-PDA hollow nanoparticles and utilizing them as a multifunctional molecular-loading nanoplatform for imaging-guided cancer PDT with efficient skin photoprotection (Figure 1). In our design, a dopamine-mediated biomineralization method using a one-pot gas diffusion process was developed to synthesize hybrid CaCO3-PDA hollow nanoparticles. After being modified with polyethylene glycol (PEG), the Ca-

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CO3-PDA-PEG hollow nanoparticles acquired great physiological stability and exhibited pH-dependent degradation under a slightly acidic environment. Thus, the photoactivity of the loaded chlorin e6 (Ce6), an effective photosensitizer that is quenched by the strong absorption of PDA under neutral pH, could be activated with recovered fluorescence and enhanced singlet oxygen generation ability under reduced pH within the tumor microenvironment. In addition, these hollow nanoparticles could intrinsically bind with different types of metal ions (e.g., Fe3+, Zn2+, Mn2+ and Co2+), producing nanocomposites with multimodal imaging functionality. As simultaneously discovered by various imaging modalities, our Ce6@CaCO3-PDA-PEG could efficiently accumulate in the tumor after intravenous (i.v.) administration, leading to a great anti-cancer effect by PDT. Additionally, the skin phototoxicity during PDT could be significantly suppressed by these nanoparticles. Therefore, these Ce6@CaCO3-PDA-PEG nanoparticles with biodegradability, a pH-responsive PDT effect, multimodal imaging functionality, and minimal skin phototoxicity are promising for next-generation of PDT.

RESULTS AND DISCUSSION Here, for the first time, we developed a simple one-pot method by the gas diffusion procedure to prepare organic-inorganic hybrid CaCO3-PDA nanoparticles. As illustrated in Figure 2a, in an enclosed chamber, the CO2 and NH3 gas produced by the natural decomposition of NH4HCO3 would continuously diffuse into the ethanol solution containing Ca2+ ions and dopamine, simultaneously providing the CO32- source and an alkaline environment to trigger the formation of CaCO3 26. Interestingly, the introduced dopamine could accelerate the growth of CaCO3, which together with PDA oxidized from dopamine would form CaCO3-PDA composite nanoparticles 27. Notably, the nanoparticle formation time was shortened from 72 h for bare CaCO3 nanoparticles formed in the absence of dopamine to 24 h for CaCO3-PDA nanoparticles formed in the presence of dopamine. The morphology of the as-prepared CaCO3-PDA nanoparticles could be precisely tuned by simply adjusting the feeding ratios of dopamine and calcium chloride (CaCl2) (Figure 2b). Unlike bare CaCO3, which showed a solid spherical morphology with a smooth surface under observation by transmission electron microscopy (TEM), the obtained CaCO3-PDA showed a gradual morphology evolution from solid nanospheres with a rough surface, prepared at a mass feeding ratio of dopamine:CaCl2 at 1:150, to hollow nanoparticles upon increasing the dopamine content. With uniform and well-defined hollow structures, CaCO3-PDA nanoparticles prepared at a mass feeding ratio of dopamine:CaCl2=2:150 were selected for further experiments. The CaCO3-PDA nanoparticles showed an average diameter of 168 nm and a shell thickness of 44 nm by TEM (Figure 2c), as well as a mesoporous structure (Inserted in Figure 2c). The pore size and specific surface area were calculated to be 6.2 nm and 252 m2 g-1, respectively, according to Brunauer-Emmett-Teller (BET) measurements (Figure S1). Furthermore, as analyzed by elemental mapping based on high-angle annular dark-field scanning TEM (HAADF-STEM), N, O and Ca were found to be homogenously distributed in the CaCO3-PDA nanoparticles (Figure 2d&e). This fabrication method could easily be scaled up to the gram-scale production of CaCO3-PDA nanoparticles with high quality via a one-pot reaction (Figure 2f-h). Then, the detailed formation mechanism of the CaCO3-PDA hollow nanoparticles was carefully studied. During the prepara-

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tion of CaCO3-PDA, it was found that the growth solution rapidly changed from colorless to blue, which could be attributed to the coordination between Ca2+ and polymerized dopamine (Figure S2). Therefore, the function of oxygen, a pivotal factor that triggers the polymerization of dopamine, in the growth of CaCO3-PDA was investigated. CaCO3-PDA nanoparticles prepared in the presence of oxygen showed a well-defined hollow structure, while no well-defined nanoparticles were observed upon preparation in the absence of oxygen (Figure 3a). In contrast, for bare CaCO3 nanoparticles prepared in the absence of dopamine, oxygen had no obvious influence on the morphology of as-made CaCO3 nanostructures under the corresponding conditions. These results indicate that the polymerization of dopamine is a key factor for mediating the growth of CaCO3-PDA hollow nanoparticles. While polymerized dopamine would facilitate the growth of CaCO3, the coordination of Ca2+ with dopamine in the absence of oxygen-mediated polymerization would interrupt the formation of the CaCO3 nanostructures. Furthermore, the time-dependent formation of the hollow CaCO3-PDA nanoparticles was studied by TEM analysis of nanoparticles collected from the growth solution at different time intervals. At 2 h after the beginning of the reaction, irregular solid nanoparticles with an average size of ~40 nm were observed (Figure 3b), and their PDA content was determined to be ~ 47% by thermogravimetric analysis (TGA) (Figure 3c). After 6 h, hollow nanoparticles with small solid cores were observed (Figure 3b). After 24 h, the nanoparticles had evolved into welldefined hollow nanoparticles. Moreover, the size of the CaCO3PDA nanoparticles gradually increased from ~40 nm at 2 h to ~80 nm at 6 h and ~170 nm at 24 h post-initiation of this reaction (Figure S3), while their corresponding PDA contents gradually dropped from 47% to 21% and 7.2% at the three respective time points (Figure 3c). Therefore, based on these observations, the formation mechanism of the CaCO3-PDA hollow nanoparticles is proposed to follow a three-step process (Figure 3d). First, dopamine undergoes rapid oxidative polymerization in the presence of oxygen, generating PDA that coordinates with Ca2+ to form Ca-PDA coordination nanocomplexes. Then, CO2 originating from the decomposition of NH4HCO3 gradually dissolves into the CaCl2/dopamine solution to interact with Ca2+ in the form of carbonate ions, leading to the deposition of CaCO3 on the surface of the Ca-PDA core. Owing to the stronger binding affinity between Ca2+ and CO32- than between the phenolic hydroxyl groups of dopamine and Ca2+, the Ca2+ in the solid core gradually diffuses out to coordinate with CO32-, leading to the dissolution of core and the formation of CaCO3-PDA hollow nanoparticles via an inside-out ripening process 28. This one-pot method is simple and reliable for preparing well-defined CaCO3 hollow nanoparticles compared with previously developed micelle template methods for fabricating hollow nanostructures 29,30. Next, the nanoparticles were subjected to surface modification. As-prepared CaCO3-PDA nanoparticles were modified with lipid bilayers composed of 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA), 1,2-dihexadecanoyl-sn-glycero-3phosphocholine (DPPC), and cholesterol and were further coated with PEG-conjugated 1,2-distearoyl-sn-glycero-3phosphoethanolamine (DSPE-PEG) via a two-step method according to our previous work (Figure 4a) 31. In this approach, the phosphate head of DOPA coordinates with the calcium ions of CaCO3-PDA nanoparticles, forming a hydrophobic lipid layer to enable further modification by DPPC and DSPE-PEG with a

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lipid bilayer structure. From the thermogravimetric analysis (TGA), we estimated that the lipid content in the PEGylated CaCO3-PDA nanoparticles (CaCO3-PDA-PEG) was approximately 9.1% (Figure S4). Moreover, the obtained CaCO3-PDA-PEG exhibited excellent stability in water, saline solution, fetal bovine serum, and cell culture medium (Figure S5). Decomposition profiles of the obtained CaCO3-PDA-PEG were then investigated by immersing the nanoparticles in phosphate buffered saline (PBS) at pH 5.5, 6.5 and 7.4. As shown by TEM, CaCO3-PDA-PEG was stable at pH 7.4 but dissociated under mildly acidic conditions (e.g., pH 5.5, 6.5) (Figure 4b). Interestingly, With decreasing pH, CaCO3-PDA-PEG showed not only increased dissociation but also decreased absorption (Figure 4c) and enhanced transmittance (%T) (Figure 4d). These optical changes are primarily due to the decomposition of the PDA skeleton that occurs together with the dissolution of CaCO3 under mildly acidic conditions. Collectively, our results demonstrate that the structures and optical properties of CaCO3-PDA-PEG nanoparticles are highly sensitive to pH, which explains the efficient degradation under mildly acidic conditions. Due to the inspiring pH-responsive decomposition behavior, the capability of CaCO3-PDA-PEG nanoparticles as an efficient pH-responsive drug carrier was then studied. By simply mixing CaCO3-PDA-PEG with Ce6 solutions at a series of concentrations overnight under neutral pH and subsequently washing the material to remove unbound Ce6 by repeated centrifugation, Ce6 could be successfully loaded onto those nanoparticles, as demonstrated by the obvious Ce6 characteristic absorbance peaks of the obtained product (Figure 4e). The Ce6-loading capacity of CaCO3-PDA-PEG increased as the feeding amounts of Ce6 increased and reached a rather high loading capacity of ~140% (Ce6:CaCO3, w/w) (Figure 4f). Because of the existence of a PDA skeleton, CaCO3-PDA-PEG could effectively protect the loaded Ce6 molecules from being photobleached upon irradiation for 1 h with a high-intensity ultraviolet lamp (365 nm, 220240 volts, 50 Hz) (Figure 4g). Taken together, these results indicate that CaCO3-PDA-PEG nanoparticles may be a promising nanoplatform for the delivery of PS to enable PDT. The release profiles of Ce6 from the obtained Ce6-loaded CaCO3-PDA-PEG nanoparticles (Ce6@CaCO3-PDA-PEG) were quantitatively studied by incubating Ce6@CaCO3-PDA-PEG in PBS or serum under various pH values for different time intervals. Approximately 69.1% of the loaded Ce6 was released from Ce6@CaCO3-PDA-PEG incubated at pH 5.5 for 1 h, much higher than the ~34.8%, 6.2% and 1.5% release from Ce6@CaCO3PDA-PEG incubated in PBS at pH 6.5 and 7.4 and serum, respectively (Figure 4h), as the result of the pH-dependent decomposition of CaCO3-PDA-PEG. The fluorescence of Ce6 in Ce6@CaCO3-PDA-PEG was also enhanced upon gradually lowering the incubation pH level due to the pH-responsive nanoparticle decomposition weakening the quenching of Ce6 fluorescence by PDA (Figure 4i). The photosensitizing ability of Ce6@CaCO3-PDA-PEG under a variety of pH values at the same Ce6 concentration was evaluated by comparing their SO generation efficiencies upon exposure to a 660-nm light emitting diode (LED) (5 mW cm-2, 40 min). The generation of SO from the Ce6@CaCO3-PDA-PEG also showed a significant increase under reduced pH at 6.5 and 5.5 (Figure 4j). Therefore, these pH-activatable Ce6@CaCO3-PDA-PEG nanoparticles would be particularly suitable for treating tumors with PDT via the acidic tumor microenvironment.

In addition, CaCO3-PDA hollow nanoparticles could also act as a versatile nanoplatform for the loading and delivery of chemotherapeutics. As shown in Figure S6, with increasing feeding drug:CaCO3-PDA weight ratios, the amounts of loaded drug also increased. Later, the pH-responsive cellular uptake profile of the Ce6@CaCO3-PDA-PEG nanoparticles was carefully studied. For comparison, its non-pH-responsive counterpart, Ce6@liposomes, in which hydrophobic Ce6 was encapsulated within PEGylated liposomes using the same lipid content as that used to functionalize CaCO3-PDA nanoparticles, was prepared according to our previously developed method 32. As shown in Figure S7a&b, the as-obtained Ce6@liposomes exhibited a similar hydrodynamic size and size distribution to Ce6@CaCO3PDA-PEG, suggesting that Ce6@liposomes are ideal candidates for comparison. First, confocal laser scanning microscopy (CLSM) revealed strong intracellular Ce6 fluorescence in 4T1 murine breast cancer cells after incubation with Ce6@CaCO3PDA-PEG, which could diffuse out from the endosomes/lysosomes, as indicated by staining with LysoTracker (Figure 5a) 33. In contrast, only weak Ce6 fluorescence signals co-localized with the LysoTracker fluorescence signals were observed in cells incubated with Ce6@liposomes. Quantitative analysis by flow cytometry further confirmed the much higher cellular uptake of Ce6@CaCO3-PDA-PEG than of Ce6@liposomes (Figure 5b). Our results demonstrate that pHactivatable Ce6@CaCO3-PDA-PEG nanoparticles are efficiently internalized by cells and are capable of effective endosome/lysosome escape. It has been speculated that the consumption of protons occurring when CaCO3 decomposes within acidic lysosomes results in a “proton sponge” effect 34,35, which may lead to disruption of the lysosomal membrane and the subsequent escape of Ce6 from endosomes/lysosomes into the cytoplasm, which would be beneficial for PDT-induced cancer cell death. Based on the above observations, we then evaluated the differences between Ce6@CaCO3-PDA-PEG and Ce6@liposomes in intracellular ROS generation via 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) staining. As expected, the cells treated with Ce6@CaCO3-PDA-PEG showed a much higher level of ROS generation than did those treated with Ce6@liposomes after being irradiated with 660-nm LED light (Figure 5c). The enhanced ROS generation by Ce6@CaCO3-PDA-PEG could efficiently accelerate cell death, as indicated by the confocal fluorescence imaging of calcein AM and propidium (PI) co-stained cells (Figure S8). The standard cell viability assay was performed to evaluate the PDT efficacy of the Ce6@CaCO3-PDA-PEG nanoparticles. In accordance with the CLSM observations, Ce6@CaCO3-PDAPEG showed greater PDT efficacy than free Ce6 and Ce6@liposomes under the same Ce6 concentration and light irradiation parameters (Figure 5d). All three Ce6 formulations, including free Ce6, Ce6@CaCO3-PDA-PEG and Ce6@liposomes, induced no obvious toxicity in the cells even at a high incubation concentration of Ce6 (Figure 5e). The higher phototoxicity of Ce6@CaCO3-PDA-PEG than that of free Ce6 and Ce6@liposomes may be explained by the more efficient cellular uptake as well as the pH-responsive decomposition of those hollow nanoparticles in the acidic lysosomes enabling the lysosomal escape of the PS. Therefore, Ce6@CaCO3-PDA-PEG nanoparticles could serve as a biocompatible and pH-responsive nanoplatform for highly efficient PDT.



As a mussel-inspired polymer, PDA has been widely explored for various biomedical applications, including chelating metals (e.g., Fe3+, Mn2+, 64Cu2+) to enable different modalities of molecular imaging 27,36. Here, the capability of our hybrid CaCO3PDA hollow nanoparticles for metal ion chelation was explored by simply incubating as-prepared CaCO3-PDA-PEG nanoparticles with different metal ions pre-dissolved in ethanol (Figure 6a). After removing unbound metal ions by centrifugation, the morphologies and elemental compositions of the obtained CaCO3-PDA(M) (M=Fe, Zn, Mn, Co) nanocomposites were carefully analyzed using TEM and HAADF-STEM, respectively. The morphology of the CaCO3-PDA(M) composite nanoparticles was not obviously different from that of the original CaCO3PDA nanoparticles (Figure 6b). Moreover, strong and homogenous signals of Fe3+, Zn2+, Mn2+ or Co2+ were observed on the respective composite nanoparticles by HAADF-STEM mapping and EDX spectroscopy (Figure 6b&S9), demonstrating the efficient binding of metal ions with the PDA-containing nanoparticles. In addition, by semi-quantitative energy dispersive spectrometer (EDS) analysis, we found a much higher capacity for metal ion loading for CaCO3-PDA nanoparticles than for bare CaCO3 nanoparticles (Table S1). With further PEGylation, the CaCO3-PDA-PEG nanoparticles with chelated metal ions were used for bioimaging. Motivated by the excellent T1-contrasting ability of Mn2+ in magnetic resonance (MR) imaging, the T1-weighted MR imaging performance of Mn2+-loaded CaCO3-PDA-PEG (CaCO3PDA(Mn)-PEG) nanoparticles was evaluated using a 3-T MR imaging system. CaCO3-PDA(Mn)-PEG showed obvious Mn2+concentration-dependent brightening effects, and its corresponding longitudinal relaxation (r1) was calculated to be 9.5 mM-1 s-1 (Figure S10a&b), which was much higher than that of Magnevist (4.25 mM-1 s-1), a commercially used Gd3+-based contrast agent 37. Then, upon systemic administration, CaCO3-PDA(Mn)PEG showed strong T1-weighted MR signals on tumors grown in BALB/c mice as imaged by an MRI scanner at 24 h post injection (p.i.), which were significantly enhanced compared with those acquired before injection under the same imaging parameter setting (Figure 6c&d). Photoacoustic (PA) imaging, which is enabled by the conversion of absorbed light to ultrasonic signals, has recently attracted much research interest for structural and functional imaging in many preclinical studies due to its higher spatial resolution, deeper tissue penetration and higher optical contrast than conventional optical imaging, as well as its relatively low tissue scattering 38,39. Owing to the presence of PDA in our CaCO3PDA-PEG, we found that CaCO3-PDA-PEG showed strong absorbance in the NIR region (Figure S11). A Visualsonics Vevo 2100 LAZR PA imaging system was used to examine the CaCO3-PDA-PEG nanoparticles, which showed a concentrationdependent PA contrasting ability (Figure S12a&b). Furthermore, after the i.v. injection of CaCO3-PDA-PEG, the tumors of 4T1 tumor-bearing mice showed significantly increased PA signals (Figure 6e&f), further demonstrating the high tumor specificity of our CaCO3-PDA-PEG nanoparticles. By tracking the fluorescence of Ce6 using the IVIS® Spectrum in vivo optical imaging system, we found that after the i.v. administration of Ce6@CaCO3-PDA-PEG, the 4T1 tumor-bearing mice showed efficient tumor accumulation (Figure 7a). Furthermore, to quantitatively understand the in vivo behavior of these nanoparticles, we measured Ce6 fluorescence in homogenized tissue lysate samples. Analysis of the detailed pharmaco-

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kinetic profiles obtained by recording the Ce6 fluorescence in lysed blood samples showed that the blood circulation of Ce6@CaCO3-PDA-PEG followed a classical two-compartment model with a long blood half-life (t1/2(α)=1.39 ± 0.05 h, t1/2(β)=16.41 ± 2.26 h) (Figure 7b). Moreover, based on the biodistribution data, the tumor accumulation of Ce6 in mice after the i.v. administration of Ce6@CaCO3-PDA-PEG reached as high as ~10.0% ID/g (percentage of injected dose per gram tissue) at 24 h p.i. (Figure 7c), which was only slightly lower than 11.6% ID/g in the liver, which clears foreign nanoparticles. Notably, the tumor-to-liver uptake ratio achieved by the Ce6@CaCO3-PDA-PEG nanoparticles is competitive with that of many other drug delivery nanocarriers achieved by passive tumor homing and the enhanced permeability and retention (EPR) effect 40,41. Furthermore, the kidneys showed rather high signals, and the remaining Ce6@CaCO3-PDA-PEG nanoparticles in the liver and kidneys rapidly decayed over time, indicating that Ce6@CaCO3-PDA-PEG nanoparticles might be quickly removed from the body via the renal metabolism. Then, the in vivo PDT effect of Ce6@CaCO3-PDA-PEG was examined in a 4T1 tumor-bearing mouse model. First, mice with an average tumor size of ~100 mm3 were randomly divided into several groups (n=6), as follows: PBS (1), free Ce6 + light exposure (2), Ce6@liposomes + light exposure (3), Ce6@CaCO3PDA-PEG (4), and Ce6@CaCO3-PDA-PEG + light exposure (5). In each group, 200 µL of agents at equivalent concentrations of Ce6 (6 mg kg-1) were intravenously injected. Then, mice in groups 2, 3 and 5 were exposed to 660-nm LED light (5 mW cm2 ) at 24 h post i.v. injection for 1 h. The tumor sizes and body weights of the treated mice were recorded after the various treatments were initiated (Figure 8a). Compared with the untreated mice, mice treated with PDT with free Ce6 (group 2) showed minimal tumor suppressive effects owing to the limited tumor uptake of free Ce6 molecules after i.v. injection. The other two Ce6 nanoformulations showed effective tumor growth inhibition after PDT. Interestingly, the PDT efficacy of Ce6@CaCO3-PDA-PEG (group 5) was greater than that of Ce6@liposomes (group 4) (Figure 8a). Such a superior PDT therapeutic effect of Ce6@CaCO3-PDA-PEG over Ce6@liposomes is consistent with our in vitro results and may be attributed to the pH-dependent decomposition of those nanoparticles enabling efficient endosomal/lysosomal escape. In addition, it should be noted that the mice injected with Ce6@liposomes after light exposure showed an obvious drop in body weight (~11%), which could probably result from the severe phototoxicity of Ce6 in the Ce6@liposome formulation. In contrast, no significant body weight changes were observed in the other treatment groups (Figure 8b). To further verify the therapeutic effect, slices of tumors collected at 24 h after various treatments were subjected to classical hematoxylin and eosin (H&E) staining (Figure 8c). Severe morphological damage appeared on the tumor slices from mice postPDT with Ce6@CaCO3-PDA-PEG or Ce6@liposomes. In contrast, the tumor slices of the other two groups showed cell morphologies similar to that of samples from the untreated control group. Furthermore, the sections of major organs (including the heart, liver, spleen, kidneys and lungs) collected from the control group (group 1) and the Ce6@CaCO3-PDA-PEG-treated group (group 5) showed negligible morphological changes (Figure S13), demonstrating the excellent biosafety of Ce6@CaCO3PDA-PEG hollow nanoparticles.


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Traditional photodynamic agents usually give rise to severe phototoxicity to the skin and other superficial tissues exposed to light in treated patients 11,12. Due to the excellent tumor-specific pH-activatable photodynamic effect of the tested materials, the skin photosensitivity of our Ce6@CaCO3-PDA-PEG nanoparticles was carefully evaluated, with Ce6@CaCO3-PEG (without PDA, prepared according to previously reported methods 31) and Ce6@liposomes as examples of traditional photodynamic nanoagents used for comparison 32. Healthy female BALB/c mice with shaved hair were divided into four groups (n=9): PBS injection (control), Ce6@CaCO3-PDA-PEG injection, Ce6@CaCO3-PEG injection and Ce6@liposome injection. In this experiment, the Ce6 dose was 6 mg kg-1. First, to determine the effective amounts of Ce6 within the skin, three mice from each group were sacrificed to collect skin samples from their backs at 1 h p.i. As shown by ex vivo fluorescence imaging, the skin collected from mice treated with Ce6@CaCO3-PDA-PEG exhibited much weaker fluorescence signals than skin collected from mice treated with Ce6@CaCO3PEG and Ce6@liposomes (Figure 9a&b). In contrast, the absolute amounts of Ce6 within the skin treated with those nanoparticles were nearly equivalent, indicating the significant Ce6 fluorescence quenching effect of the PDA skeleton in Ce6@CaCO3PDA-PEG nanoparticles (Figure 9c). The remaining mice in each group were exposed to 660 nm light (5 mW cm-2, 30 min) at 1 h p.i. The traditional PDT nanoagents (both Ce6@CaCO3-PEG and Ce6@liposomes) caused obvious edema after light exposure, whereas only slight edema was observed in mice treated with Ce6@CaCO3-PDAPEG. For quantitative analysis, a piece of skin with a fixed size (1.5 cm × 1.5 cm) was collected from the back of each mouse from different groups at 4 h post-light exposure. Notably, the average skin weight of mice treated with Ce6@CaCO3-PEG and Ce6@liposomes were ~150% and ~120% greater than that of mice in the control group, respectively, while there was ~50% increase in skin weight after treatment with Ce6@CaCO3-PDAPEG (Figure 9d), suggesting the minimal photosensitivity of Ce6@CaCO3-PDA-PEG under the protection of the PDA skeleton. Moreover, such severe edema would disappear and gradually progress to erythema. Severe skin erythema was observed on the backs of mice treated with Ce6@CaCO3-PEG and Ce6@liposomes 4 days post-light irradiation, while only minimal erythema was observed in mice treated with Ce6@CaCO3PDA-PEG (Figure 9e). Ex vivo H&E staining was conducted to evaluate the histological changes of the skin after various treatments at 4 days postlight irradiation. The skin of mice with Ce6@CaCO3-PDA-PEG injection remained largely intact after light exposure. In marked contrast, severe skin damage was found in mice treated with Ce6@CaCO3-PEG or Ce6@liposomes 4 days post-light irradiation (Figure 9f). Moreover, with greatly reduced phototoxicity, the Ce6@CaCO3-PDA-PEG nanoparticles showed an in vivo PDT therapeutic effect comparable to that of Ce6@CaCO3-PEG without PDA (Figure S14a&b). Collectively, these results demonstrate that our Ce6@CaCO3-PDA-PEG nanoparticles are promising as a tumor acidity-activatable nanophotosensitizer capable of highly efficient and precise cancer PDT with remarkably reduced skin photosensitivity.

CONCLUSIONS In summary, we report for the first time a one-pot dopaminemediated biomineralization method for preparing pH-responsive

hybrid CaCO3-PDA hollow nanoparticles via a gas diffusion procedure. After PEGylation, the CaCO3-PDA nanoparticles become an efficient molecular nanocarrier for both imaging ions (e.g., Mn2+) and therapeutic molecules (e.g., Ce6) upon simple mixing. Due to the existence of CaCO3, Ce6@CaCO3-PDA-PEG nanoparticles show acidic pH-responsive decomposition and could be released within the acidic tumor microenvironment, enabling the effective treatment of tumors by PDT with greatly reduced skin phototoxicity. As a multifunctional nanoplatform, our CaCO3-PDA hybrid hollow nanoparticles show several unique advantages: (1) Because both CaCO3 and PDA are endogenous components, our nanoparticles would be highly biocompatible without long-term side effects. (2) The biomineralization synthesis method presented here is rather simple, with a high production scale and yield, and is superior to previously reported methods in the preparation of well-defined hollow CaCO3 nanoparticles. (3) These hybrid CaCO3-PDA-PEG hollow nanoparticles allow the simultaneous loading of both imaging and therapeutic molecules for imaging-guided cancer therapy. (4) The intriguing pH-responsive decomposition and optical property alterations of those nanoparticles enable not only enhanced PDT efficacy via the effective lysosomal release of PS but also obviously reduced phototoxicity to normal tissues, such as skin. Therefore, our hybrid CaCO3-PDA hollow nanoparticles may indeed be a promising nanocarrier with great potential for future clinical translation.

EXPERIMENTAL SECTION Materials. NH4HCO3 and CaCl2.2H2O were obtained from Sinopharm Chemical Reagent Co., Ltd., China. Ce6, cholesterol, zinc chloride (ZnCl2), manganese chloride (MnCl2), cobalt chloride (CoCl2) and iron chloride hexahydrate (FeCl3.6H2O) were obtained from J&K Chemical Co. Dopamine was purchased from Sigma-Aldrich. DOPA, DPPC and DSPE-PEG5k were purchased from Avanti Lipids Polar, Inc. Synthesis of CaCO3-PDA Hollow Nanoparticles. Hollow CaCO3-PDA nanoparticles were synthesized by adopting the one-pot gas diffusion process with modifications. Briefly, 150 mg of CaCl2.2H2O was mixed with 1, 2, or 4 mg of dopamine and then dissolved in a beaker containing 100 mL of anhydrous ethanol; the mixture was then placed in a sealed container with 5 g of NH4 HCO3 and kept at 40 °C. After 24 h, the bluish CaCO3PDA nanoparticles were collected and purified by repeated centrifugation at 8000 rpm. Finally, the obtained CaCO3-PDA nanoparticles were dispersed in ethanol and kept at room temperature for further use. To study the influence of oxygen on the growth of CaCO3-PDA, the aforementioned ethanol mixture of CaCl2 and dopamine were placed in another container under vacuum while keeping the other experimental conditions the same as previously mentioned. Bare CaCO3 nanoparticles were prepared in the presence or absence of air as were the CaCO3-PDA nanoparticles, just without the addition of dopamine. PEGylation of CaCO3-PDA Hollow Nanoparticles. CaCO3PDA nanoparticles were modified following our procedure previously used for the surface modification of bare CaCO3 nanoparticles 31. Briefly, 20 mg of CaCO3-PDA in 5 mL of ethanol and 4 mg of DOPA in 1 mL of chloroform were mixed and sonicated in a water bath sonicator for 20 min. Then, the DOPAmodified CaCO3-PDA nanoparticles were purified by centrifuga-



tion and re-dispersed in 2 mL of chloroform, followed by mixing with DPPC, cholesterol and DSPE-PEG5k at concentrations of 8, 4 and 16 mg mL-1, respectively. After vigorous stirring overnight, the chloroform was fully removed using a rotary evaporator. The residual solid was hydrated by adding 2 mL of PBS and sonicated for 5 min. Finally, we collected those CaCO3-PDA-PEG nanoparticles by centrifugation. Characterization. The morphologies and elemental maps of the as-prepared CaCO3-PDA nanoparticles were observed using a Tecnai F20 transmission electron microscope (FEI). The UVVis-NIR spectrum was detected using a Lambda750 spectrophotometer (PerkinElmer). The dynamic light scattering (DLS) size distribution of CaCO3-PDA-PEG nanoparticles was measured by a Malvern Zetasizer (ZEN3690, Malvern). The content of Ca2+ in the CaCO3-PDA nanoparticles was determined by inductively coupled plasma mass spectrometry (ICP-MS, Jena, PlasmaQuant ® MS), while the content of PDA was estimated using thermogravimetric analysis (TGA, Mettler Toledo). Ce6 Loading. For Ce6 loading, different amounts of Ce6 (10 mg/mL) dissolved in dimethyl sulfoxide (DMSO) were added to CaCO3-PDA-PEG solutions (5 mg/mL in PBS) and then stirred at room temperature overnight. Then, the excess Ce6 molecules were removed by ultra-filtration (Millipore) with the molecular weight cut off (MWCO) at 100 kDa. The amounts of Ce6 loaded were quantitatively evaluated by UV-Vis-NIR spectrometry with a molar extinction coefficient of 96037 L mol-1 cm-1 at 404 nm. The loading capacity of Ce6 was calculated using the following equation: Loading capacity of Ce6 (%)=weight of loaded Ce6/weight of CaCO3-PDA-PEG×100%.

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2 h. Later, the cells were washed with fresh PBS twice and then re-incubated with fresh medium containing LysoTracker (100 nM, Invitrogen) (1 h, 37 °C) before being imaged by a Leica SP5 confocal microscope. After being treated with trypsin, the cells were further quantitatively analyzed with a FACS Calibur flow cytometer (BD Biosciences, USA). Intracellular ROS generation was examined using the DCFH-DA (Sigma Aldrich) probe following the recommended protocol. The standard methyl thiazolyl tetrazolium (MTT) assay was carried out to determine relative cell viabilities after various treatments. For in vitro PDT, 4T1 cells were incubated with Ce6@CaCO3PDA-PEG, Ce6@CaCO3-PEG or Ce6@liposomes at varying concentrations for 2 h. Then, those cells were washed with PBS several times and then subjected to 660 nm LED light exposure at a power density of 5 mW cm-2 for 15 min. After an additional 24 h incubation, the MTT assay was carried out. In addition, 4T1 cells after irradiation with 660 nm LED light were co-stained by calcein AM and PI for confocal fluorescence imaging. Tumor Model. Our animal experiments were conducted following protocols approved by the Soochow University Laboratory Animal Center. The 4T1 tumor modal was generated by the standard subcutaneous inoculation on female BALB/c mice.

Preparation of Ion-Doped CaCO3-PDA Hollow Nanoparticles. To prepare metal ion-doped CaCO3-PDA nanoparticles, the as-synthesized CaCO3-PDA was dissolved in ethanol solutions containing FeCl3, ZnCl2, CoCl2 or MnCl2. After 1 h of vigorous stirring, the unbound ions were removed by washing with fresh ethanol more than three times.

Multimodal Imaging. For MR imaging, different concentrations of CaCO3-PDA(Mn)-PEG nanoparticles were dissolved in water and then scanned by a 3-T clinical MR scanner (Bruker Biospin). In vivo MR imaging of mice was performed under the same MR scanner using a small-animal imaging coil. For PA imaging, a CaCO3-PDA-PEG sample filled in a special polyurethane tube was scanned by the Visualsonics Vevo® 2100 LAZR system with an excitation wavelength of 700 nm. Then, the tumor regions of 4T1 tumor-bearing mice were imaged using the Visualsonics Vevo® 2100 LAZR system before and 24 h after the i.v. injection of CaCO3-PDA-PEG nanoparticles with an excitation wavelength of 700 nm. For in vivo fluorescence imaging, mice (n=3) with 4T1 tumors were intravenously injected with Ce6@CaCO3-PDA-PEG nanoparticles. Later, the mice were imaged at different time intervals (1 h, 2 h, 4 h, 8 h, 12 h and 24 h) by a Lumina III in vivo imaging system (PerkinElmer). In Vivo Pharmacokinetic Study. To evaluate the blood circulation profile of the Ce6@CaCO3-PDA-PEG nanoparticles, 3 healthy mice were intravenously injected with Ce6@CaCO3PDA-PEG at a Ce6 dose of 6 mg kg-1. Thereafter, ~20 µL of blood was withdrawn from each mouse at different time points, dissolved in lysis buffer and subjected to microreader measurements of Ce6 fluorescence. To evaluate the biodistribution profile of Ce6@CaCO3-PDAPEG nanoparticles, 9 mice intravenously injected with Ce6@CaCO3-PDA-PEG (at a Ce6 dose of 6 mg kg-1) were sacrificed at 12 h, 24 h and 48 h p.i. (n=3 per time point). Then, the tumors and main organs were collected, weighed, homogenized and measured with a microreader.

Cellular Experiments. 4T1 cells were cultured under standard conditions (37 °C and 5% CO2). To evaluate the cellular uptake and intracellular trafficking profiles of Ce6@CaCO3-PDA-PEG, 4T1 cells were incubated with Ce6@CaCO3-PDA-PEG or Ce6@liposomes at the same Ce6 concentration of 10 g mL-1 for

In Vivo Cancer PDT. A total of 30 mice bearing 4T1 tumors (~100 mm3 in size) were randomly divided into five groups (n=6 per group). Each mouse was intravenously injected with 200 µL of the various agents, as described in Figure 8a. At 24 h p.i., all mice were exposed to 660-nm LED light (5 mW cm-2) for 1 h.

pH-Responsive Release of Ce6. The pH-responsive release profile of Ce6 from Ce6@CaCO3-PDA-PEG nanocomposites was obtained using a widely adopted procedure 42. Briefly, Ce6@CaCO3-PDA-PEG nanoparticles (2 mg, in terms of Ce6) were diluted with 10 mL of PBS (pH 5.5, 6.5 or 7.4) or serum and then incubated at 37 °C. At different time points, a 0.5-mL aliquot was withdrawn from each sample and centrifuged to collect the supernatant; the absorbance at 404 nm of the supernatant was recorded, and the amount of Ce6 was calculated as previously mentioned. pH-Responsive Singlet Oxygen Generation. The SO generation was measured by singlet oxygen sensor green (SOSG) according to our previously reported procedure 43. An LED light (660 nm, 5 mW cm-2) was used to irradiate various samples.


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The length (L) and width (W) of each tumor were monitored to calculate the tumor volume (V): V=LW2/2. At 24 h post-LED light exposure, one mouse from each group was sacrificed to collect its tumor for H&E staining. At 12 days post-treatment, mice from groups 1 and 5 were sacrificed to collect their main organs for H&E staining to evaluate the biocompatibility of Ce6@CaCO3-PDA-PEG at the injected dose. In Vivo Skin Photosensitivity Evaluation. To evaluate the in vivo photosensitivity of Ce6@CaCO3-PDA-PEG compared with Ce6@CaCO3-PEG and Ce6@liposomes, healthy BALB/c mice were depilated and randomly divided into four groups (n=9, each group). Mice in group 1 were intravenously injected with 200 µL of PBS. Mice in the other three groups were intravenously injected with 200 µL of Ce6@CaCO3-PDA-PEG, Ce6@CaCO3PEG or Ce6@liposomes at a Ce6 dose of 6 mg -kg-1. At 1 h p.i., three mice from each group were sacrificed to collect the skin from their back for ex vivo skin imaging. The skin samples were weighed and homogenized in lysis buffer containing 0.3% collagenase. The skin lysate samples were then centrifuged at 8000 rpm to obtain the supernatant. Free Ce6 was extracted from the nanoparticles by adding 1% hydrochloric acid to Ce6@CaCO3PDA-PEG and Ce6@CaCO3-PEG solutions and methyl alcohol to Ce6@liposome solution. Their fluorescence was then measured to quantify the amounts of Ce6. Meanwhile, at 1 h p.i., the remaining mice in each group were exposed to 660-nm LED light (5 mW cm-2) for 0.5 h. At 4 h post-light irradiation, three mice from each group were sacrificed to collect a skin sample from their back 1.5 cm × 1.5 cm in size for weighing. The remaining three mice from each group were imaged and then sacrificed to collect their skin for H&E staining for the evaluation of light-induced skin damage at four days post-treatment.

ASSOCIATED CONTENT Supporting Information Figures S1-S14 & Table S1 can be found in the supporting information.

AUTHOR INFORMATION Corresponding Author [email protected], [email protected]

Author Contributions Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was partially supported by the National Natural Science Foundation of China (51525203), the National Research Programs from the Ministry of Science and Technology (MOST) of China (2016YFA0201200), the China Postdoctoral Science Foundation (2017M610348), the Collaborative Innovation Center of Suzhou Nano Science and Technology, the 111 Program from the Ministry of Education of China, and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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Figure 1. Schematic illustration of Ce6@CaCO3-PDA-PEG nanoparticles as a tumor acidic pH-activatable nanoplatform for multimodal imaging-guided cancer PDT with reduced skin phototoxicity.



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Figure 2. The preparation and characterization of CaCO3-PDA hollow nanoparticles. (a) Scheme showing the synthesis of CaCO3-PDAPEG hollow nanoparticles. (b) Typical TEM images of CaCO3 and CaCO3-PDA nanoparticles prepared at different feeding ratios of dopamine and CaCl2. (c) Representative TEM image of CaCO3-PDA hollow nanoparticles prepared with a feeding ratio of dopamine:CaCl2 of 2:150. (d) STEM mapping analysis of CaCO3-PDA hollow nanoparticles in the dark field showing the distribution of N, O and Ca in the as-synthesized CaCO3-PDA hollow nanoparticles. (e) EDX spectrum of CaCO3-PDA hollow nanoparticles. The inset shows the table of C, N, O and Ca contents in the as-prepared CaCO3-PDA hollow nanoparticles. (f) Digital images of the 1000-mL beaker used for the largescale synthesis of CaCO3-PDA hollow nanoparticles. (g) Image showing the gram-scale production of CaCO3-PDA hollow nanoparticles. (h) Typical TEM image of CaCO3-PDA hollow nanoparticles prepared on the gram scale.


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Figure 3. Formation mechanism study of CaCO3-PDA hollow nanoparticles. (a) TEM images of CaCO3-PDA hollow nanoparticles and bare CaCO3 prepared in the presence and absence of oxygen. (b) TEM images of the time-dependent morphology evolution of CaCO3PDA hollow nanoparticles taken at reaction times of 2 h, 6 h and 24 h. (c) TGA curves of CaCO3 and CaCO3-PDA hollow nanoparticles collected at different reaction times. (d) Proposed time-dependent growth mechanism of the CaCO3-PDA hollow nanoparticles.



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Figure 4. Characterization of CaCO3-PDA-PEG hollow nanoparticles. (a) Scheme showing the PEGylation process of CaCO3-PDA nanoparticles. (b) Scheme illustrating the acid-responsive decomposition of CaCO3-PDA-PEG hollow nanoparticles and their corresponding TEM images after incubation in PBS with different pH values (5.5, 6.5 and 7.4) for 2 h. (c) Time-dependent absorbance change of CaCO3PDA-PEG nanoparticles after incubation within PBS at different pH values for 2 h. (d) Transmittance spectra of CaCO3-PDA-PEG nanoparticles measured after incubation at different pH values for 2 h. (e) UV-Vis-NIR spectra of Ce6-loaded CaCO3-PDA-PEG at different feeding ratios of Ce6 to CaCO3-PDA-PEG. (f) Quantification of Ce6-loading capacities of CaCO3-PDA-PEG at different Ce6:CaCO3PDA-PEG feeding ratios. (g) The absorbance curves of free Ce6 and Ce6@CaCO3-PDA-PEG at 404 nm after being exposed to 365-nm UV light. Insets are representative fluorescence images of Ce6@CaCO3-PDA-PEG before (1) and after (3) UV irradiation, as well as free Ce6 before (2) and after UV irradiation (4) taken by a digital camera under 365-nm light irradiation. (h) Time-dependent release profiles of Ce6 from Ce6@CaCO3-PDA-PEG incubated in PBS or serum at different pH values. (i) pH-dependent fluorescence intensity of Ce6@CaCO3-PDA-PEG after being incubated in PBS at various pH values for 2 h. Inset is the corresponding fluorescence image of Ce6@CaCO3-PDA-PEG after being incubated at various pH values. (j) Evaluation of singlet oxygen generation capacity of Ce6@CaCO3PDA-PEG after being incubated at different pH values for 2 h. Error bars are based on at least triplicate measurements.


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Figure 5. In vitro pH-responsive cellular uptake, intracellular trafficking and photodynamic effect of Ce6@CaCO3-PDA-PEG. (a) Confocal images of 4T1 cells treated with Ce6@CaCO3-PDA-PEG or Ce6@liposomes for 2 h. The endosomes/lysosomes were stained with LysoTracker. (b) Flow cytometry data for cells incubated with Ce6@CaCO3-PDA-PEG or Ce6@liposomes for 2 h. (c) Confocal images of 4T1 cells treated with Ce6@CaCO3-PDA-PEG or Ce6@liposomes and then stained using DCFH-DA (green) to evaluate intracellular ROS generation. The cell nuclei were stained using DAPI (blue). (d) In vitro PDT treatment of 4T1 cells by Ce6@CaCO3-PDA-PEG and Ce6@liposomes under 660-nm light irradiation (5 mW/cm2, 15 min). (e) Relative cell viability data determined by MTT assay after culturing 4T1 cells with Ce6@CaCO3-PDA-PEG and Ce6@liposomes at various concentrations for 24 h in the dark. P values in (d) were calculated by Tukey's post-test (***p

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