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Carrier-Free, Chemo-Photodynamic Dual Nanodrugs via Self-Assembly for Synergistic Antitumor Therapy Ruiyun Zhang, Ruirui Xing, Ti-Feng Jiao, Kai Ma, Chengjun Chen, Guanghui Ma, and Xuehai Yan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02416 • Publication Date (Web): 13 May 2016 Downloaded from http://pubs.acs.org on May 15, 2016

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ACS Applied Materials & Interfaces

Carrier-Free, Chemo-Photodynamic Dual Nanodrugs via Self-Assembly for Synergistic Antitumor Therapy Ruiyun Zhang,†,‡,# Ruirui Xing,†,‡,§,# Tifeng Jiao,*,†,‡ Kai Ma,†,‡ Chengjun Chen,*,§,ǁ Guanghui Ma,§ and Xuehai Yan*,§,ǁ

†

State Key Laboratory of Metastable Materials Science and Technology, Yanshan

University, Qinhuangdao 066004, China. ‡

Hebei Key Laboratory of Applied Chemistry, School of Environmental and Chemical

Engineering, Yanshan University, Qinhuangdao 066004, China §

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering,

Chinese Academy of Sciences, Beijing 100190, China ǁ

Center for Mesoscience, Institute of Process Engineering, Chinese Academy of Sciences,

Beijing 100190, China

1 ACS Paragon Plus Environment


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ABSTRACT There are tremendous challenges from both tumor and its therapeutic formulations affecting the effective treatment of tumor, including tumor recurrence and complex multi-step preparations of formulation. To address these issues, herein a simple and green approach based on the self-assembly of therapeutic agents including a photosensitizer (chlorine e6, Ce6) and a chemotherapeutic agent (doxorubicin, DOX) was developed to prepare carrier-free nanoparticles (NPs) with the ability to inhibit tumor recurrence. The designed NPs were formed by self-assembly of Ce6 and DOX associated with electrostatic, π-π stacking and hydrophobic interactions. They have a relatively uniform size of average 70 nm, surface charge of −20 mV and high drug encapsulation efficiency, which benefits the favorable accumulation of drugs at the tumor region through a potential enhanced permeability and retention (EPR) effect as compared to their counterpart of free Ce6 solution. In addition, they could eradiate tumors without recurrence in a synergistic way following one treatment cycle. Furthermore, the NPs are safe without any activation of inflammation or immune response in separated organs. Taken together, the rationale of these pure nanodrugs via the self-assembly approach might open an alternative avenue and give inspiration to fabricate new carrier-free nanodrugs for tumor theranostics, especially for two small molecular anti-tumor drugs with the aim of combinational anti-tumor therapy in a synergistic way.


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KEYWORDS: Nanodrugs, self-assembly, co-delivery, photodynamic therapy, antitumor therapy



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1. INTRODUCTION Tumors as a devastating disease have currently become one of the most common causes of death all over the world.1 It mains a critical challenge to eradicate tumors without any recurrence following one treatment cycle.2 In general, most tumors cannot be effectively inhibited or still grow slowly by mono-therapeutic modality such as chemotherapy only. Another obstacle contributing to the failure of treatment is the multidrug resistance (MDR) during every treatment cycle, which greatly compromises the chemotherapeutic efficiency likely due to the drug efflux from tumor cells.3 After decades of experimental and clinical trials, combinational therapy procedure has been demonstrated as a more preferable and effective treatment modality for tumor therapy.4,5 However, some patients are still threatened by death resulted from the tumor recurrence despite the fact that they have taken therapeutic modalities which only temporarily suppressed the growth of the tumor.6 Thus, it is highly desirable to develop a combinational modality that can both overcome the drug resistance and eradicate the tumor without recurrence in one treatment cycle. Photodynamic therapy (PDT) has recently gained great attention due to its minimal systematic toxicity of photosensitizer to non-specific tissues and harmlessness of activating light.7-11 PDT is a US Food and Drug Administration approved non-invasive theranostic modality which induces singlet oxygen generation to damage tumor cells through necrotic and apoptotic pathways.12,13 Another important advantage of PDT is that


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it could also avoid tumor drug resistance and the associated tumor recurrence likely due to its direct damage to proteins responsible for drug resistance.14-17 However, it should be noted that the efficiency of PDT relies on the efficiency of carriers.18-21 It remains a challenge to design functional anti-tumor drug delivery system (DDS) with desired merits for effective treatment of tumors.22-24 Despite great advances in the novel design of DDS, there are still many problems for facilitating their real world applications.25-27 In some cases, smart carriers are prepared by complex multi-step preparation processes and/or with toxic organic solvents, and thus cause problems such as adverse effects originating from organic solvents, low cost-effectiveness and difficulties in the scale-up process of formulation.28 In addition, the safety of carriers, such as the possible toxicity from carriers or their biodegradation problems, is still of major concerns, and for this reason some functional formulations did not get approved by US Food and Drug Administration (FDA). Accordingly, formulations should not only be preferably prepared from substances that are generally recognized as safe (GRAS), but also in simple and green procedures.29,30 In light of the above considerations, herein we have developed a simple and green approach to carrier-free nanoparticles by self-assembly of doxorubicin (DOX) molecules and chlorine e6 (Ce6) (Figure S1) molecules associated with the electrostatic, π-π stacking and hydrophobic interactions without the involvement of any organic solvent (Scheme 1). DOX is one of the most common chemicals for the treatment of tumor, while



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Ce6 is widely used in PDT as a very safe and efficient therapeutic drug. Therefore, potential toxicity from carriers is of less concern in this study as both two species have the therapeutic effects. Meanwhile, the designed NPs are expected to achieve a synergistic therapeutic effect, achieving effective eradication of tumors without recurrence by the combination of chemotherapy (DOX) and PDT (Ce6) following one treatment cycle.

2. EXPERIMENTAL SECTION 2.1 Materials. Doxorubicin hydrochloride (DOX) and cholrin e6 (Ce6) were purchased from sigma-Aldrich. MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] was provided by Merck. MCF-7 human breast cancer cell was purchased from the Institute of Basic Medical Science, Chinese Academy of Medical Sciences (Beijing, China). All cell-culture-related reagents were obtained from Macgene Biotech Co., Ltd. (Beijing, China) unless stated otherwise. 2.2 Preparation of Nanoparticles. The DOX/Ce6 NPs were prepared in a green and simple self-assembly approach between these two molecules. In brief, 100 µL of Ce6 aqueous solution (1 mg mL-1, pH = 12) was firstly diluted with 880 µL of water, followed by the addition of 20 µL of DOX solution (10 mg mL-1) with stirring. After aging the solution for at least 24 h, the DOX/Ce6 NPs were finally formed and further used for characterization. Other formulations with different molar ratios of Ce6 to DOX were prepared in the same way. The size and surface charge of NPs can be finely modulated by 6 ACS Paragon Plus Environment

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changing some parameters such as ratio of Ce6 to DOX and the concentration of drug solutions. 2.3 Characterization. SEM images were obtained using a Hitachi S-4800 SEM working at 15 kV or a Philips XL30 electron microscope at an accelerating voltage of 3 kV. TEM measurements were carrier by a Zeiss EM 912 Omega transmission electron microscope operated at 120 kV, for which samples were carefully placed onto the carbon-coated copper grids.31 Confocal images of both nanoparticles and cells were obtained by an Olympus FV1000 +IX481 microscope or a confocal laser scanning microscopy (CLSM, Leica TCS SP). A Zetasizer (Malvern Instrument) was used to determine the size distribution and zeta potential of the prepared nanoparticles. UV data were recorded at room temperature using an UV/Vis spectrophotometer (U3010, Hitachi). The turbidity of the NPs was determined in the form of absorbance at 650 nm by an UV/Vis spectrophotometer. A fluorescence spectrometer (Hitachi F-4500) was used to measure the photoluminescence of free Ce6, free DOX and their co-assembled nanoparticles in water in a 1.0 cm quartz cuvette. The encapsulation efficiency (EE) was determined by an ultrafiltration method. The free drug in the filtrate was determined and EE was calculated according to the following formula: EE (%) =

total drug - free drug × 100 total drug

2.4 Drug Release Test. The drug release profiles were determined by a dialysis method. All samples were sealed in dialysis bags (Sigma, 14 000 MW cutoff) and



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immersed in phosphate-buffered saline (PBS) solutions (pH 7.4 or 5.0). The tubes were maintained at 37°C in a water bath shaker at 100 rpm. Samples were withdrawn at predetermined time intervals (replaced with an equivalent volume of release medium) and the release amount of DOX and Ce6 were analyzed using high performance liquid chromatography (HPLC). All the experiments were carried out in triplicate. 2.5 Flow Cytometry Analysis. MCF-7 cells with a density of 4×105 cells per well were seeded onto 6-well plate and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS). After 24 h incubation, the cells were washed with PBS twice. Complete RPMI 1640 medium containing different formulations (free DOX and NPs) were then separately added to each well with incubation for 12 h. After the detachment of cells with trypsin, the samples were washed thrice and the mean fluorescence intensity of DOX per 1×104 cells was measured using flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA). The untreated cells were used as the control. 2.6 Cell Viability Test. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The cytotoxicities of various formulations on MCF-7 cells were measured by the MTT assay. In brief, a stock solution of MTT (5 mg mL-1) in PBS was prepared and sterile-filtered. Cells were cultured with 100 µL of fresh medium containing 10 µL of MTT stock solution in the incubator for 4 h. After that, the supernatant was removed, and then DMSO (100 µL) was added. The solutions on the cells were incubated for 10 min at room temperature without movement. The absorbance at 490 nm was


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measured in each well using a microplate reader. The cell viability (%) was determined by comparing the absorbance at 490 nm with control wells merely containing cell culture medium. Data are shown as the mean and standard error on the basis of six independent experiments. The combination index (CI) analysis was calculated based on the following formula:32,33 CI = [CA,x/ICx,A]+ [CB,x/ICx,B] Where CA,x and CB,x are the concentrations of agent A and B, respectively, when achieving x% drug effect in combination treatment. ICx,A and ICx,B are single agent concentrations when it was alone used to achieve the same drug effect. There is a synergism if CI is smaller than 1. 2.7 In Vivo Fluorescence Imaging. Old male BALB/c female mice (6–8 weeks) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products. All care and handling of animals was performed with the approval of Institutional Authority for Laboratory Animal Care. The tumor-bearing mice were intravenously injected with the different NPs. At pre-determined time intervals, the mice were anesthetized and scanned using an In Vivo Imaging System (FX Pro, Kodak, Japan). For resected organ imaging, the animals were euthanized, and then the tumors or organs were excised and imaged (λex =635 nm). 2.8 In Vivo Anti-tumor Experiment. MCF-7 cells (2 × 106) were inoculated subcutaneously in the left ﬂank of each Balb/c nude mouse. The tumor volume was



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calculated using the following formula: volume (mm3) = length×width2/2. The treatment started when the tumor volume reached approximately 150 mm3. The female balb/c nude mice xenografted MCF-7 tumors were randomly divided into five groups (1) 5 % glucose solution as a control, (2) DOX aqueous solution, (3) DOX/Ce6 NPs without irradiation, (4) Ce6 aqueous solution, and (5) DOX/Ce6 NPs with irradiation (laser irradiation: λ = 635 nm, 90 J/cm2, 150 mW/cm2 for 10 min), and injected intravenously every four days for three times. Body weight and tumor size of each mouse were also measured every day. Hematoxylin and eosin (H&E) staining assay were used to stain the tumor and organs slices. Tumor tissues and organs from each group which were harvested 1 d after different treatments and fixed in 10% neutral buffered formalin embedded into paraffin and sliced into 8 µm thick sections. The obtained slices were then stained with H&E and examined under a digital microscope.

3. RESULTS AND DISCUSSION Among the patterns of fabricating drug delivery carriers, self-assembly plays a pivotal role in the fields of both biomedicine and nanotechnology.34-37 In this study, synergistic anti-tumor NPs were prepared and optimized based on a simple and green self-assembly approach. It is interesting to note that the final size of self-assembled NPs could be modulated ranging from nanometer to micrometer by adjusting the molar ratio of Ce 6 to DOX (Figure S2A). In particular, the NPs measured approximately 70 nm in size (Figure 1A) with the optimal ratio of Ce6 to DOX (1:2, molar ratio), which could 10 ACS Paragon Plus Environment

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render a good enhanced permeability and retention (EPR) effect if these particles were administrated into the blood. More importantly, the encapsulation efficiency (EE) of drug was very high for both compounds with this ratio less than 3 (Table S1). More than 95% of Ce6 molecules and ca. 99% DOX molecules were incorporated into their self-assembled NPs. With this optimal ratio, the surface charge of NPs was approximately −20 mV (Figure 1B). Some reports suggested that negatively charged nanoparticles were easy to display a slow or reduced opsonization by reticular endothelial system (RES), which facilitates the elongation of blood circulation time.38-40 Hence, NPs with this optimal ratio of Ce6 to DOX (1:2, molar ratio) was chosen for further investigations. Also, these NPs displayed spherical shapes with mono-dispersed morphology even in a dry state, as shown in the both transmission electron microscopy (TEM) (Figure 1C) and scanning electron microscopy (SEM) (Figure 1D) images, indicating good colloidal stability of their co-assembled NPs. The colloidal stability greatly depends on the ratio between Ce6 and DOX. If this ratio reaches above 4, the size of the self-assembled particles significantly increases and have a distribution of 2~5 µm, as observed in the DLS measurement (Figure S2A). Also, the surface charge of NPs changes from –25 mV to +10 mV by altering the ratio of Ce6 to DOX from 1:2 to 1:5 (Figure S2B). This observation suggests the strong electrostatic coupling between Ce6 and DOX. The significant increase in size with the ratio of Ce 6/DOX from 1:3 to 1:4 is due to the charge neutralization at the ratio of 1:4, leading to



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the aggregation of NPs. This is also confirmed by the SEM and TEM observation (Figure S3). To further prove the existence of electrostatic interactions, the turbidity of the NPs was determined with addition of various amounts of NaCl.41 The disassembly of some NPs (reflected by the decreased turbidity of the NPs) could take place as a function of increased ionic strength (Figure S4). This suggests that electrostatic interactions contribute to the co-assembly of NPs. All these above results infer that the final NPs are co-assembled partly due to the electrostatic interaction between these two species. UV spectra (Figure 1E) revealed that the co-assembled NPs had the typical absorbance peaks from both DOX (ca. 233 nm and 480 nm) and Ce6 (ca. 402 nm and 655 nm). This implies that NPs were indeed self-assembled by these two components, where they interacted with each other and were constructed into NPs at nanoscale.42,43 A considerably broader and red-shifted Soret band of Ce6, together with a red-shifted absorption band of DOX (Figure 1E), indicates that the pyrrole groups of Ce6 are able to interact with aromatic ring of DOX by hydrophobic and π-π interactions.41 When sodium dodecyl sulfate (SDS, 0.2% w/v) was added to the NPs aqueous solution, changes in UV-vis absorption of NPs (Figure S5), as an indicator for changes in the co-assembled architecture of NPs, were observed likely due to the additionally hydrophobic interactions by SDS.44 The intensity of fluorescence emission peaks of Ce6 and DOX inside the NPs were dramatically decreased compared to monomeric Ce6 and DOX (Figure 1F), indicating excitonic migration among stacked Ce6 or DOX molecules.41 The results


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suggest that π-π stacking and hydrophobic interactions may be one of main driving forces for formation of the NPs. These associative forces, including electrostatic, π-π stacking and hydrophobic interactions, also ensured a good storage stability of self-assembled NPs in the aqueous state. It was found that there were no significant changes in both size (Figure 2A) and zeta potential (Figure 2B) of NPs during storage for up to 8 days. In addition, the co-assembled NPs (Ce6/DOX=1:2) still retain good colloidal stability (Figure 2C) in the cell culture RPMI-1640 medium containing 10% fetal bovine serum (FBS). Their particle size increases to approximately 100 nm with a little broader size distribution as compared to the original one (70 nm). It has been reported the site of tumor appears to be slightly acidic.45 Thus, we investigated the assembly and disassembly of NPs in vitro under different pH environments. The PBS solution (pH = 7.4) mimics the condition in the bloodstream, whereas the PBS solution (pH = 5.0) simulates the acidic environment inside the tumor region. There are slight changes in terms of size and zeta potential if the NPs were located in different pH environments (Figure S6). Both DLS analysis (Figure S6A) and SEM observation (Figure S7) show a broader size distribution of NPs at pH of 5.0, indicating the disassembly of some NPs. Furthermore, a minor reduction in zeta potential of NPs (Figure S6B) might be closely correlated to the increased protonation of amino groups from DOX under the acidic condition.46



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The in vitro release profile of NPs was monitored at different pH environments. There is no any difference in the release character for free DOX and free Ce6 solutions under the following conditions such as pH = 7.4 and pH = 5.0. All the drugs are completely released during the release period of three hours (Figure S8). In contrast, the release rate turns slower in the form of NPs under the above same conditions. Approximately 30% of Ce6 and 30% of DOX are released from NPs in PBS solution (pH = 7.4) during 3 hours (Figure 2D). Under the acidic solution of pH = 5.0, the release rate of DOX gets a little faster while that of Ce6 becomes slower. There are above 90% of DOX and almost 70% of Ce6 released from NPs at pH of 5.0 during 48 hours, both of which might benefit the effectiveness of chemotherapy and PDT. The relatively faster release rate of DOX might result from the fact that the amino group of DOX is more readily protonated under the acidic condition, which increases its solubility and accelerates its diffusion in the aqueous solution.46 Similarly, the slightly slower release of Ce6 is attributed to its reduced solubility associated with the carboxyl groups under the acidic condition. It is known that good cell internalization of drugs is crucial to the efficiency of both PDT and chemotherapy.47 We found that the cellular uptake of NPs increased gradually as a function of the incubation time (Figure. 3A). A little fraction of NPs was initially absorbed onto the surface of cells within 1 h of incubation, and some NPs arrived in the intracellular environments following 4 h of incubation. However, much more NPs could


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be taken up into the cells with longer time. For example, the uptake of NPs reached saturation in 12 h. We further quantified the intracellular DOX content by flow cytometer. The results show that NPs have a good cellular uptake of DOX after 12 h incubation in vitro, comparable to that of the free DOX solution (Figure S9). The good cell internalization of NPs here is probably related to a previously reported mechanism that negatively charged NPs are still able to bind at a small amount of cationic sites located in the cell membrane, which would further initiate cell internalization in a relatively slow process (also described as “adsorptive endocytosis” pathway).48,49 More importantly, the NPs could reach the same tumor cells together (Figure 3A), which ensured their synergistic function at the same site. Still, DOX and Ce6 can be observed by confocal laser scattering microscope (CLSM) even with an incubation period of 24 h, implying that an effective release of drug from the NPs were fulfilled in the intracellular environment. To further test the anti-tumor efficiency of NPs in vitro, the cytotoxicity of NPs in the dark or under laser irradiation was evaluated. It was obvious that the DOX/Ce6 NPs under laser irradiation achieved the best efficiency in killing tumor cells (Figure 3B). The calculated combination index32,33 (CI < 1) indicates a synergism in combination of PDT (Ce6) and chemotherapy (DOX) (Table S2). In particular, no significant dark cytotoxicity was observed for Ce6 solution in MCF-7 cells. On the contrary, the cell viability reduced gradually with increased amount of Ce6 concentration when cells were exposed to light irradiation. We also found that the anti-tumor efficiency



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of DOX was not affected by co-assembly with Ce6, as DOX solution and NPs showed similar dark cytotoxicities. Encouraged by the excellent therapeutic efficiency of carrier-free nanodrugs (NPs) in vitro, the distributions of NPs were also monitored in vivo by fluorescence imaging. Both free Ce6 and NPs were well distributed all over the body in mice at early time points (such as 1 h) since these formulations have been intravenously administrated. However, only groups administrated with NPs still had accumulated drugs in the tumor site at 12 h or 24 h time points in comparison with those of free Ce6 solution (Figure 4A), indicating better circulation time in blood stream as well as better EPR effect for the designed NPs. The excised tumor via ex vivo imaging further confirmed that NPs groups had higher drug accumulation in tumors than that of free Ce6 solution group (Figure 4B). Another difference between these two groups is that free Ce6 was readily distributable to kidneys, while a certain amount of NPs were prone to distribution into lungs (Figure 4B). This might be ascribed to the different size or disparate state of existence for the Ce6. To further verify the therapeutic efficiency of carrier-free nanodrugs in depth, therapeutic efficiency of various formulations was measured in vivo using female balb/c nude mice xenografted MCF-7 tumors. The inhibition of tumor growth (Figure 5A) show that DOX solution and NPs without irradiation have similar therapeutic effects, both significantly suppressing the tumor growth when compared to the negative control group (5% glucose solution). Ce6 groups under irradiation (Ce6+laser) suppress tumor growth


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more efficiently than those with DOX solution and NPs without irradiation. Under irradiation,

carrier-free

nanoparticles

containing

both

photosensitizer

and

chemotherapeutic agent can act as one effective combinational therapeutic modality to achieve the best and synergistic anti-tumor effect (Figure 5A and see more details in Figure S10 and S11). The excised tumor tissues after 14 days further confirmed the best tumor growth inhibition efficacy of combinational therapy by NPs under irradiation with a tumor elimination rate of up to 80% (Figure 5B). This best therapeutic efficiency might result from the combinational effect of both two drugs. For preliminary safety evaluation, there were no significant changes of body weight in all mice during the entire experimental period (Figure 5C). The body skin of mice can get recovered with a faint scar left following the combinational therapy of carrier-free nanodrugs (Figure S12). Moreover, hematoxylin and eosin (H&E) staining studies showed that the designed combined therapeutic modality was safe without any activation of inflammation or immune response in separated organs (Figure S13). Hence, these carrier-free nanodrugs are demonstrated to be safe and effective therapeutic modalities for the tumor treatment in a synergistic way with significantly lowered recurrence rates.

4. CONCLUSION In conclusion, carrier-free nanoparticles with an average size distribution of 70 nm and zeta potential of −20 mV have been prepared via a simple and green self-assembly approach between DOX and Ce6. These co-assembled pure nanodrugs (NPs) revealed 17 ACS Paragon Plus Environment


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good cellular uptake, reached the site of action together with a higher tumor accumulation likely due to the EPR effect, enabled a synergistic anti-tumor effect in vivo and eventually eradicated the tumor during one treatment cycle. Therefore, these NPs might be a promising candidate for the chemo-photodynamic dual therapy against tumor in their preclinical studies. Importantly, the preparation process did not involve the use of any harmful organic solvent for dissolution of hydrophobic precursors, thus addressing all safety concerns (such as possible toxicity) from either carriers or organic solvents, which might facilitate its translation into clinics. The rationale of these pure nanodrugs might open an alternative avenue and give inspiration to fabricate new carrier-free nanodrugs for tumor theranostics, especially from two small molecular anti-tumor drugs with the aim of combinational anti-tumor therapy in a synergistic way.


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Figures

Figure 1. Characterization of carrier-free DOX/Ce6 nanoparticles (NPs, DOX:Ce6 = 1:2, molar ratio). Particle size (A), zeta potential (B), TEM (C) and SEM image (D) of NPs. (E) UV-vis absorption spectra of free DOX, free Ce6 and NPs. (F) Fluorescence spectra of free DOX, free Ce6 and NPs.



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Figure 2. The measurement of particle size (A) and zeta potential (B) of carrier-free DOX/Ce6 nanoparticles (NPs, Ce6/DOX=1:2, molar ratio) during storage. (C) The particle size of NPs in RPMI-1640 medium containing 10% fetal bovine serum (FBS). (D) Cumulative drug release profiles of NPs under different pH environments.


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Figure 3. (A) CLSM images showing the intracellular trafficking process of DOX (red) and Ce6 (green) in MCF-7 cells after incubation with NPs for different time periods (BF, bright field). (B) Viability of MCF-7 cells after incubation with various formulations with (+L) or without laser irradiation (λ = 635 nm, 150 mW/cm2, 1 min).



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Figure 4. (A) In vivo fluorescence images of free Ce6 solution and Dox/Ce6 nanoparticles (NPs). The black circle area represents tumor tissue. (B) Representative ex vivo fluorescence imaging of tumor and organs excised from Balb/c nude mice xenografted MCF-7 tumor at 24 h post-injection.


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Figure 5. (A) Suppression of tumor growth after intravenous injection of different formulations (dose of DOX: 4 mg/kg and/or dose of Ce6: 2 mg/kg) with or without laser irradiation (laser power: 90 J/cm2, 150 mW/cm2 for 10 min, n = 5). Arrows indicate the treatment of laser irradiation. (B) Images of the excised MCF-7 tumor tissues at 14 days post-implantation. These red circles in this graph represent that the tumors were totally disappeared after the treatment, therefore, no excised tumor samples were obtained eventually. (C) Body weight changes of tumor-bearing mice during one treatment cycle (n = 5). 23 ACS Paragon Plus Environment


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SCHEMES

Scheme 1. Schematic representation of carrier-free nanoparticles (NPs) via co-assembly between DOX and Ce6.


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ASSOCIATED CONTENT Supporting Information. Chemical structures, particle size, zeta potential, encapsulation efficiency, turbidity of NPs, UV-vis absorption spectra, TEM and SEM images, drug release profiles, intracellular uptake, tumor images, and H&E staining images.

AUTHOR INFORMATION Corresponding Author ∗Authors to whom correspondence should be addressed. E-mail: [email protected]; [email protected]; [email protected]. Homepage: www.yan-assembly.org Author Contributions #

These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors acknowledge financial support from the National Natural Science Foundation of China (Project Nos. 21522307, 21473208, 91434103, and 8140287), Beijing Natural Science Foundation (No. 7154220), Youth Innovation Promotion Association CAS, the CAS visiting professorships for senior international scientists (Project No. 2016VTA042) and the Chinese Academy of Sciences (CAS). This work was



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also financially supported by the Natural Science Foundation of China (No. 21473153), the Science Foundation for the Excellent Youth Scholars from Universities and Colleges of Hebei Province (No. YQ2013026), the Support Program for the Top Young Talents of Hebei Province, the Post-graduate’s Innovation Fund Project of Hebei Province, the China Postdoctoral Science Foundation (No. 2015M580214), and the Scientific and Technological Research and Development Program of Qinhuangdao City (No. 201502A006). X.Y. is great indebted to Prof. Möhwald for his support.

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