Light-Responsive Nanoparticles for Highly Efficient Cytoplasmic

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Light-Responsive Nanoparticles for Highly Efficient Cytoplasmic Delivery of Anticancer Agents Yangyun Wang, Yibin Deng, Huanhuan Luo, Aijun Zhu, Hengte Ke, Hong Yang, and Huabing Chen ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b05214 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 15, 2017

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Light-Responsive Nanoparticles for Highly Efficient Cytoplasmic Delivery of Anticancer Agents Yangyun Wang,†,# Yibin Deng‡,# Huanhuan Luo,‡,# Aijun Zhu,‡ Hengte Ke,‡ Hong Yang,† and Huabing Chen†,‡,* †

School of Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation

Center of Radiation Medicine of Jiangsu Higher Education Institutions, and School of Radiation Medicine and Protection, Soochow University, Suzhou 215123, China ‡

Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases,

College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China

#

These authors contributed equally.

*To whom correspondence should be addressed: E-mail: [email protected]

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ABSTRACT: Stimuli-responsive nanostructures have shown great promise for intracellular delivery of anticancer compounds. A critical challenge remains in the exploration of stimuliresponsive nanoparticles for fast cytoplasmic delivery. Herein, near-infrared (NIR) lightresponsive nanoparticles were rationally designed to generate highly efficient cytoplasmic delivery of anticancer agents for synergistic thermo-chemotherapy. The drug-loaded polymeric nanoparticles of selenium-inserted copolymer (I/D-Se-NPs) were rapidly dissociated in several minutes through reactive oxygen species (ROS)-mediated selenium oxidation upon NIR light exposure, and this irreversible dissociation of I/D-Se-NPs upon such a short irradiation promoted continuous drug release. Moreover, I/D-Se-NPs facilitated cytoplasmic drug translocation through ROS-triggered lysosomal disruption, and thus resulted in highly preferable distribution to the nucleus even in 5 min post-irradiation, which were further integrated with light-triggered hyperthermia for achieving synergistic tumor ablation without tumor regrowth.

KEYWORDS: light-responsive nanoparticles, micelles, cytoplasmic delivery, photothermal therapy, synergistic therapy


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Nanoparticles have emerged as effective drug vehicles for intracellular delivery in the field of cancer nanomedicine.1-8 To maximize the anticancer efficiency, a variety of nanoparticles have been extensively explored to achieve enhanced intracellular delivery of anticancer agents such as chemotherapeutic compounds, photosensitizers, and siRNA through enhanced tumor accumulation, preferable endocytosis, as well as effective cytoplasmic drug translocation.1-5 In particular, stimuli-responsive nanoparticles have also been exploited to smartly release these payloads in response to endogenous stimuli such as pH, enzyme, and reactive oxygen species (ROS) at tumor.5,9-23 To precisely promote drug release, some external stimuli such as light, ultrasound, magnetic field, and temperature might continuously be applied to cause spatiotemporal disassembly of nanoparticles, resulting in accelerated or ON/OFF drug release kinetics.9,15,24-30 Moreover, multiple stimuli have been rationally employed to cause the disassembly of nanoparticles including the combination of intrinsic and external stimuli, thereby facilitating cytoplasmic drug translocation ability for flexible control of intracellular delivery.3141

These smart nanoparticles are often able to generate significant dissociation of nanostructures

that cause effective drug release under continuous stimuli in a few hours. Essentially, endocytic compartments undergo intracellular trafficking in a limited lifespan of several hours,42-44 and abundant lysosomal enzymes might also digest endocytosed agents.45-47 Recently, several nanoparticles have been explored to immediately release encapsulated agents under continuous external stimuli, thus inducing effective anticancer efficiency with minimal release at normal tissues.12 Unfortunately, these types of nanoparticles usually demand continuous stimuli owing to their reversible response characteristics, and also require complex or specific designs in compositions. Consequently, stimuli-responsive nanoparticles that possess a fast and irreversible


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disassembly hold a great promise to preferably maximize cytoplasmic translocation for efficient intracellular delivery of anticancer compounds. Light has been considered as a precise, flexible, and spatiotemporal stimulus in the field of smart nanoparticles for cancer therapy.29,48-50 To date, UV- or visible light-labile moieties such as nitrobenzyl group, perylene, cardiogreen, and riboflavin chromophores have been employed to enhance drug release from nanoparticles.25,29,51-53 However, this wavelength range of less than 650 nm often suffers from limited anticancer efficacy owing to its poor tissue transparency. Recently, near-infrared (NIR) light of 650-900 nm is applied to produce on-demand or ON/OFF drug release from the nanoparticles through the disruption of nanostructures mediated by lightinduced photothermal effect or reactive oxygen species (ROS).26,27,54-61 Furthermore, the NIR light-responsive nanoparticles can exert synergistic cancer therapy through rational integration of activatable drug release for chemotherapy and phototherapy under light irradiation.62-66 Despite the progress of NIR-responsive nanoparticles with sensitive ON/OFF drug release,26,61,67 only few type of nanoparticles can efficiently encapsulate drugs while quickly release their cargo in response to short-term NIR light exposure.56,68 In our previous studies, NIR light-responsive nanoparticles were found to effectively enhance drug release through hyperthermia-mediated phase separation or destabilization of π-π interaction for in vivo cancer therapy.39,40,48,49,69-75 In the nanoparticles, the encapsulated cyanine dyes such as indocyanine green (ICG) were used to generate ROS to disrupt lysosomal membranes through photochemical internalization effect for intracellular translocation of chemotherapeutic compounds such as doxorubicin from lysosomes to cytoplasma upon light exposure.39,72 Presumably, the light-triggered ROS might also act as an oxidizing agent to disassemble ROS-responsive nanoparticles for further promoting drug release.76,77 Herein, NIR light-responsive nanoparticles are rationally fabricated to produce


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highly efficient disassembly for highly effective cytoplasmic delivery of anticancer compounds (Scheme 1). The polymeric nanoparticles of selenium-inserted copolymer with 64 nm in diameter (I/D-Se-NPs) are found to rapidly dissociate in several minutes through light-induced selenium oxidation upon NIR light exposure, and thus promote effective drug release, followed by cytoplasmic drug translocation through ROS-mediated lysosomal disruption. Thus, I/D-SeNPs resulted in highly preferable distribution of doxorubicin (DOX) to the nucleus even in 5 min post-irradiation, which were further integrated with light-triggered hyperthermia for synergistic thermo-chemotherapeutic anticancer efficacy with total tumor ablation.

RESULTS AND DISCUSSION Synthesis and Characterization. Briefly, we firstly synthesized ROS-responsive amphiphilic selenium-inserted copolymer (Se-polymer) via sequential one-pot coupling reactions of poly(ethylene glycol) (PEG), hexamethylene diisocyanate (HDI), and bis(hydroxypropyl) selenide (Figure 1A). In contrast, an inert amphiphilic copolymer with the replacement of selenium by carbon (C-polymer) was also synthesized for fabricating selenium-free nanoparticles (I/D-C-NPs) as the control using a similar procedure (Figure S1). The chemical structure, molecular weight, and polydispersity index (PDI) were characterized using 1H NMR (Figure 1B,S2) and GPC (Figure S3), indicating that Se-polymer and C-polymer had the molecular weights of 22602 and 17138 at the PDI levels of 1.06 and 1.02, respectively. Subsequently, I/D-Se-NPs were prepared through the assembly of photothermal ICG and chemotherapeutic DOX with ROS-responsive Se-polymer, while I/D-C-NPs consisting of ICG, DOX and inert C-polymer were obtained as a control in a similar manner. The transmission electron microcopy (TEM) imaging shows that I/D-Se-NPs and I/D-C-NPs possessed spherical 5 ACS Paragon Plus Environment

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micellar nanostructures with ~64.0 nm (Figure 2A) and ~131.0 nm (Figure S4) in diameter, respectively, and also exhibited corresponding hydrodynamic diameters of 131.0 nm and 178.6 nm according to dynamic light scattering measurements (Figure 2C), respectively. The absorption spectra of I/D-Se-NPs and I/D-C-NPs with 5.0 µg mL-1 ICG indicate that both ICG and DOX were successfully encapsulated in the micelles (Figure S5). I/D-Se-NPs and I/D-C-NPs were also found to have the ICG and DOX loading levels of 10% and 10%, respectively, indicating that Se-polymer and C-polymer are effectively assembled with them. Interestingly, no spherical nanostructure was observed after I/D-Se-NPs suffered from 3 min light irradiation (785 nm, 1.0 W cm-2) (Figure 2B), thus indicating that light exposure effectively causes the disassembly of the nanoparticles. In contrast, I/D-C-NPs maintained their spherical shape after treatment of 3 min light irradiation (785 nm, 1.0 W cm-2) (Figure S6). To confirm the responsive mechanism of this nanostructure, Se-polymer was assembled into the blank micelles that were further suffered from H2O2 treatment. Distinctly, I/D-Se-NPs showed the obvious increase of turbidity, while this treatment caused no obvious change in turbidity for I/D-C-NPs (Figure S7). To further verify the oxidation of Se groups, FTIR and NMR were used to characterize the Sepolymer treated with 1%wt H2O2 solution for 2 h or not. The characteristic absorption bands of the oxidized product appeared at 924 and 881 cm-1 corresponding to asymmetric and symmetric stretching vibrations of O=Se=O groups (Figure S8). Moreover, the oxidation of Se-polymer resulted in a change of chemical shift (three methylene protons between Se atom and O atom) to higher resonance peaks (Figure S9), indicating that the three methylene protons between Se atom and the O atom were located in a more polar chemical environment after oxidation. Reasonably, the rapid micellar dissociation is attributed to the Se oxidation and subsequent hydrophobicityto-hydrophilicity transition in the backbone of Se-polymer in response to ROS.78,79


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Photothermal Effect, Light-Responsive Drug Release and ROS Generation. To demonstrate the capacity of I/D-Se-NPs for photothermal conversion, their photothermal effect was evaluated under 785 nm irradiation at 1.0 W cm-2. I/D-Se-NPs caused the temperature elevations of 7.8 °C at the dose of 2.0 µg mL-1 ICG, and also exhibited a concentrationdependent photothermal effect (Figure 2D). I/D-C-NPs exhibited a similar photothermal behavior under light irradiation as compared to I/D-Se-NPs (Figure S10). Reasonably, the effective photothermal conversion results from the enhanced non-radiative transition of excited ICG afforded by the micellar structure, and enhanced photostability of ICG (Figure S11).39 The obvious hyperthermia under irradiation implies that I/D-Se-NPs can act as a favorable photothermal agent for subsequent photothermal cell damage. To demonstrate light-responsive drug release, I/D-Se-NPs suffered from 3, 5, 7, 10, and 13 min, respectively, followed by subsequent drug release measurement. I/D-Se-NPs exhibited a sustained release of DOX in the absence of light irradiation, while the drug release was significantly increased for I/D-Se-NPs under 785 nm irradiation along with light exposure time, indicating a light-exposure-dependent release behavior during 13 min (Figure 3A). We fitted the drug release curves, and calculated the derivatives at time zero point as the drug release rates. Distinctly, a longer light exposure is able to accelerate release rate in a linear manner during 7 min (Figure 3B). In contrast, I/D-C-NPs exhibited no significant change in their drug release regardless of light exposure as compared to I/D-Se-NPs (Figure 3E). Thus, I/D-Se-NPs have an ability to generate irreversibly enhanced continuous drug release in response to NIR light even in several minutes. To confirm the generation of ROS that triggers the oxidation of Se under irradiation, the generation of singlet oxygen was evaluated using 1,3-diphenyliso-benzofuran (DPBF). I/D-Se-


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NPs exhibited concentration-dependent singlet oxygen generation as indicated by the decrease of DPBF absorbance (Figure 3C), and simultaneously possessed a light-exposure-dependent ROS generation rate (Figure 3D).39 Similarly, I/D-C-NPs can effectively generate ROS under irradiation as well (Figure S12). As a result, the Se oxidation might further improve the hydrophilicity of Se-polymer that is responsible for the disassembly of I/D-Se-NPs. Obviously, the ROS generation is highly correlated with the release rate of DOX responding to NIR light irradiation. The facilitated drug release from I/D-Se-NPs under irradiation is possibly attributed to their disassembly of micellar structure triggered by light-induced ROS. To further validate the influence of ROS on drug release of I/D-Se-NPs, H2O2 as a ROS donor was further employed to trigger the dissociation of nanoparticles (Figure 3F).80 Obviously, I/D-Se-NPs showed a much more significant release of DOX in the presence of H2O2, while I/D-C-NPs as a control had a similar drug release regardless of H2O2 treatment. Thus, ROS is highly responsible for Se oxidation-mediated disassembly of nanoparticles and subsequent enhanced continuous drug release in an irreversible manner. Cellular Uptake, Synergistic Cytotoxicity, and Intracellular Distribution. To demonstrate the endocytosis of I/D-Se-NPs, their cellular uptake was evaluated on 4T1 murine cells (Figure 4A). Distinctly, I/D-Se-NPs exhibited an obvious increase in their cellular uptake after 24 h incubation as compared to free ICG/DOX (Free I/D), indicating a preferable cellular internalization. To evaluate the cell damage of I/D-Se-NPs, we incubated 4T1 cells with I/D-SeNPs for 24 h, followed by 5 min light exposure (785 nm, 1.0 W cm-2) or not. In the absence of light irradiation, both I/D-Se-NPs and I/D-C-NPs exhibited the IC50 values of about 2.7 µg mL-1 DOX (Figure 4B), primarily resulting from chemotherapeutic cytotoxicity of DOX. Interestingly, I/D-Se-NPs and I/D-C-NPs resulted in the cytotoxicity at the IC50 levels of 0.6 µg mL-1 and 2.0


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µg mL-1 DOX under irradiation, respectively. In addition, the empty micelles consisting of Sepolymer as a control without DOX and ICG showed no significant cytotoxicity against 4T1 cells (Figure S13). Obviously, I/D-Se-NPs led to more significant cytotoxicity as compared to I/D-CNPs under irradiation, reasonably owing to their enhanced continuous drug release in response to NIR light. To evaluate the contribution of ROS-induced toxicity under this irradiation, we evaluated the cell damage of I/D-Se-NPs and I/D-C-NPs in solutions in the presence of vitamin C (Vc) as a ROS scavenger. The cytotoxicity of I/D-Se-NPs was distinctly suppressed in the presence of Vc (Figure S14), while I/D-C-NPs showed a slight change in their cytotoxicity regardless of Vc (Figure S15). This further demonstrates that the generated ROS under irradiation mainly trigger drug release for promoting anticancer efficiency, while ROS might play a slight role in their cytotoxicity. Moreover, I/D-Se-NPs also had the enhanced cell damage under irradiation as compared to that without irradiation, suggesting that the photothermal effect also contributes to their enhanced cytotoxicity through hyperthermia-mediated necrosis and apoptosis.81 To demonstrate the ability of I/D-Se-NPs to achieve intracellular delivery of DOX under light exposure, their intracellular distributions in 4T1 cells were observed at various post-irradiation time using confocal laser scanning microscopy (CLSM). In the absence of light irradiation, I/DSe-NPs exhibited the co-localization of 76.9% with the lysosomes after 30 min incubation, and no red fluorescence from DOX was observed in the nuclei (Figure 4C). However, upon 5 min irradiation at 1.0 W cm-2, the co-localization between DOX from I/D-Se-NPs and lysosomes was decreased to 38.4% at 5 min post-irradiation, and DOX was firstly found to be translocated into the nucleus (22.7% co-localization of red and blue colors), probably owing to the ROS-mediated lysosomal disruption through the photochemical internalization effect.39,72,74 The cytoplasmic


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delivery of DOX facilitated its accessibility into the nucleus. As expected, 65.9% co-localization of red DOX and blue stained nucleus was observed at 15 min post-irradiation, and their colocalization were increased to 80.3% at 30 min post-irradiation. More importantly, DOX was also distributed into the whole nucleus at 30 min post-irradiation (Figure 4C). Thus, DOX from I/D-Se-NPs could mainly accumulate in the nucleus in the range of 5~30 min post-irradiation, indicating an accurate timeline for intracellular delivery (Scheme 1). In contrast, I/D-C-NPs only induced a much lower distribution of DOX in the nucleus (Figure S16). Significantly, I/D-SeNPs are able to cause the enhanced continuous DOX release and subsequent translocation of DOX into the cytoplasm, thus facilitating its rapid accessibility to nucleus for subsequent chemotherapeutic damage. To demonstrate the generation of intracellular ROS that accounts for the Se oxidation and lysosomal disruption, the dihydroehtidium (DHE) staining was applied to monitor the intracellular ROS from I/D-Se-NPs and I/D-C-NPs (Figure S17). Apparently, I/DSe-NPs were found to effectively produce ROS in a dose-dependent manner, thereby accounting for both light-triggered drug release and subsequent translocation of DOX into the cytoplasm for further accessing the nucleus.82 I/D-C-NPs could generate ROS in 4T1 cells under irradiation as well, but much less DOX penetrated into the nucleus (Figure S16), possibly due to the limited drug release. As a result, I/D-Se-NPs possess a preferable cytotoxicity through their enhanced continuous drug release mediated by the rapid disassembly of nanoparticles and subsequent cytoplasmic translocation triggered by ROS-induced lysosomal disruption under light exposure even in 5 min post-irradiation. Biodistribution, In Vivo Tissue Distribution and Anticancer Efficacy. To demonstrate the ability of I/D-Se-NPs to accumulate at tumor, their biodistribution was evaluated in the mice bearing 4T1 tumors. I/D-Se-NPs exhibited a distinctly enhanced tumor accumulation at 24 h


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post-injection as compared to Free I/D and I/D-C-NPs (Figure 5A), indicating that I/D-Se-NPs possess a preferable distribution at tumor via enhanced permeability and retention (EPR) effect due to their relatively smaller size. To further confirm the intracellular distribution of DOX in the tumor, the tumor slices, excised from tumor-bearing mice treated with 3 min light irradiation or not, were stained with Hoechst 33342 at 24 h post-injection. Apparently, I/D-Se-NPs caused a co-localization of 35.1% between DOX and nuclei with blue fluorescence in the absence of irradiation (Figure 5B), while the co-localization was increased to 66.6% between DOX and nucleus at 15 min post-irradiation, indicating that I/D-Se-NPs can effectively facilitate the in vivo distribution of DOX from the lysosomes into the nucleus in 15 min through immediate drug release and cytoplasmic translocation after 3 min light exposure. Furthermore, the hematoxylin & eosin (H&E) staining was applied to monitor the damage of I/D-Se-NPs against the tumor at 6 h post-irradiation. I/D-Se-NPs resulted in severe hemorrhagic and destructive necrosis of tumor cells, and I/D-C-NPs only displayed a mild damage on the tumor (Figure S18), indicating that I/D-Se-NPs are able to cause potent damage on the tumor cells through their preferable distribution of DOX into the nucleus. In addition, I/D-Se-NPs showed no obvious damage on the normal tissues including heart, liver, spleen, lung, and kidney (Figure S19). To demonstrate the in vivo anticancer efficacy, I/D-Se-NPs were intravenously injected into the tumor-bearing mice at the dose of 7.5 mg kg-1 DOX, followed by 785 nm irradiation (5 min, 1.0 W cm-2) at 24 h post-injection. The tumor volumes were monitored during subsequent 16 days (Figure 5C). PBS as a control resulted in ~13-fold increase in tumor volume regardless of light exposure, suggesting a negligible influence on the tumor growth. The 11.5-fold volume increase was observed for Free I/D, while I/D-C-NPs and I/D-Se-NPs caused 9-fold and 10-fold increases of tumor volume without light irradiation, respectively, suggesting their slight


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influence on their anticancer efficacies probably owing to their sustained release of DOX (Figure 5C). Under irradiation, I/D-C-NPs resulted in significant tumor inhibition at 6 day postirradiation, but still exhibited a significant tumor regrowth afterwards, while I/D-Se-NPs resulted in the total tumor ablation without any regrowth under irradiation (Figure 5C,D). Distinctly, I/DSe-NPs achieved the maximized anticancer efficacy through the cytoplasmic translocation of DOX and effective photothermal effect of ICG, although the hyperthermia contributes to enhanced anticancer efficacies of both I/D-Se-NPs and I/D-C-NPs. Reasonably, the higher distribution of DOX from I/D-Se-NPs in the nucleus is responsible for their preferable anticancer efficacy under light irradiation. As a result, NIR light-responsive nanoparticles with ultrafast disassembly act as a key role in their synergistic thermo-chemotherapy.

CONCLUSION In conclusion, we report light-responsive polymeric nanoparticles with ultrafast disassembly for highly effective synergistic thermo-chemotherapy. The established I/D-Se-NPs could produce fast irreversible disassembly through ROS-mediated selenium oxidation, thus promoting the continuous drug release in response to such a short light exposure. Moreover, I/D-Se-NPs are able to generate the cytoplasmic translocation of DOX through ROS-mediated lysosomal disruption under NIR light exposure, thereby causing the preferable in vivo distribution of DOX into the nucleus even in 5 min post-irradiation. In addition, I/D-Se-NPs possess potent photothermal effect, preferable cellular uptake, and enhanced tumor accumulation, finally facilitating the synergistic anticancer efficacy with total tumor ablation. The rational design of NIR light-responsive nanoparticles represents a promising paradigm for highly efficient cytoplasmic delivery of anticancer agents, and potentially acts as a complementary approach to 12 ACS Paragon Plus Environment

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amplify

cytoplasmic

delivery

efficiency

upon

integration

with

intrinsically

tumor

microenvironment-responsive strategy.83,84

MATERIALS AND METHODS Synthesis of Se-polymer. Initially, (1.35 mmol) bis(hydroxypropyl) selenide was dissolved in 5 mL of anhydrous THF in a 20 mL flask and sealed with a rubber plug. The flask was then degassed by argon for 20 min. A solution of 1.49 mmol hexamethylene diisocyanate (HDI, 98%) in 2 mL anhydrous THF was injected into the flask under argon flow. The system was transferred into an oil bath at 50 °C to reaction for 12 h under stirring. 0.086 mmol PEG (Mw = 5000 Da) was then dissolved in 2 mL of anhydrous THF and injected into the flask under argon flow and the reaction was carried out for 12 h. The resulting products were collected by precipitating in diethyl ether and filtration for three times, followed by drying under the vacuum to a constant weight, affording a yield of over 79%. Preparation of Polymeric Nanoparticles. One milligram of ICG, 1.0 mg of DOX, and 8.0 mg of Se-polymer were dissolved in 0.4 mL DMSO under ultrasonication, followed by the addition of 2.0 µL Triethylamine (TEA). Then, the mixed solution was added to 4.0 mL deionized water under 5 min ultrasonication. Subsequently, the ICG/DOX-loaded Se-polymer nanoparticles (I/D-Se-NPs) were purified through 24 h dialysis (Cut-off 3.5 kDa Mw). The empty Se-polymer nanoparticles without ICG/DOX and the ICG/DOX-loaded C-polymer nanoparticles (I/D-C-NPs) as the control without stimuli-responsiveness were prepared using the same procedures. Free I/D as the mixture of free ICG and DOX dissolved in 5% DMSO was prepared as control.


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Characterization. The morphology of samples was observed using a transmission electron microscope (TEM, Tecnai-G20). The hydrodynamic diameters and size distributions were measured at 25 °C using dynamic light scattering (DLS, Zetasizer ZS90, Malvern). The drug loading and entrapment efficiency of ICG and DOX within I/D-Se-NPs were determined using centrifugal ultrafiltration (Cut-off 10 kDa Mw). The absorbance spectra of ICG and fluorescence spectra of DOX were measured using UV-vis Spectrometer (UV2600, Shimadzu) and Fluorescence Spectrometer (LS 55, PerkinElmer), respectively. Drug Release. The drug release behaviors of DOX from I/D-Se-NPs under various conditions were evaluated using dialysis method. Free I/D and I/D-C-NPs were used as the control. The various formulations (1.0 mL for each) were separately added to dialysis bags incubated in 10 mL release buffer. Then, the release was performed in air Contrast Temperature Oscillator shaker at 37 °C. Each 1.0 mL release medium was taken at 0.17, 0.5, 1, 2, 4, 8, and 12 h, followed by the addition of fresh medium. The DOX concentrations in all samples were measured using Fluorescence Spectrometer. The results are expressed as mean ± SD (n = 3). Photothermal Effect. The solutions of I/D-Se-NPs at the concentrations of 2.0, 5.0, 10, 25, and 50 µg mL-1 ICG in 0.5 mL glass vials were irradiated for 5 min by 785 nm laser, respectively. Simultaneously, the temperature of solutions was recorded using a thermometer at an interval of 30 s. Singlet Oxygen Generation. Singlet oxygen generation was measured using 1,3diphenylisobenzofuran (DPBF) as the singlet oxygen probe. The various samples at the concentration of 0 to 1.0 µg mL-1 ICG were mixed with 30.0 µM DPBF, respectively, followed by 13 min irradiation (785 nm laser) with a fixed intensity (1.0 W cm-2). Meanwhile, the absorbance of DPBF at 415 nm was monitored during the irradiation.


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Cellular Uptake: 4T1 tumor cells were seeded on the six-well plates (3 × 105 cells/well) and incubated for 24 h in RPMI 1640 medium containing 10% FBS. I/D-Se-NPs, I/D-C-NPs, and Free I/D at the dose of 10 µg mL-1 ICG were added into the wells, respectively. After 24 h incubation, the cells were incubated with 0.5 mL trypsin for 3 min after PBS washing, followed by the centrifuging for collecting the cells prior to resuspended in 2.5 mL PBS. Next, the cells were disrupted under the probe ultrasonication, and then ICG and DOX were further extracted from the cell lysis (1.0 mL) using 2.5 mL methanol. The ICG and DOX concentrations were measured using UV-vis Spectrometer and Fluorescence Spectrometer, respectively. The results are expressed as mean ± SD (n = 3). MTT Assay. 4T1 tumor cells were seeded on the 96-well plates (5 × 104 cells/well) and incubated for 12 h in RPMI 1640 medium containing 10% FBS 4T1 cells. I/D-Se-NPs, I/D-CNPs, and Free I/D at the dose of 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 4.0 µg mL-1 ICG were added into the wells, respectively. After 24 h incubation, the cells were washed using PBS, followed by 5 min irradiation (785 nm, 1.0 W cm-2). The laser head was fixed with a distance of 15 cm above the 96-well plate, and the light spot (1.0 cm2) was controlled to cover the whole well (0.32 cm2). Each well of the plate was treated with a similar procedure. After further incubation for 24 h, the cell viability was evaluated using MTT assay. The IC50 was obtained by fitting the in vitro log (concentration)-response data following the symmetrical sigmoidal model using OriginPro® 8. Intracellular Distribution. 4T1 cells (5 × 104 cells/well) were seeded in a glass-bottom dish with 1.0 mL culture medium. After 24 h incubation, the cells were incubated with the I/D-SeNPs at the dose of 10 µg mL-1 ICG for 0.5 h at 37 °C, and then the cells were washed using PBS. For the sample without irradiation, the cells were incubated for 10 min incubation at 37 °C with 1.0 mL Hoechst 33342 (1.0 µg mL-1), followed by another 5 min incubation with Lysotraker


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Green DND-26 (1.0 mL, 50 nM) and fixation using 4% paraformaldehyde. For the sample observed at 5 min post-irradiation, the cells were incubated for 10 min at 37 °C with 1.0 mL Hoechst 33342 (1.0 µg mL-1), and then suffered from 5 min irradiation at 785 nm or not (1.0 W cm-2), followed by 5 min incubation with Lysotraker Green DND-26 (1.0 mL, 50 nM) before subjected to fixation using 4% paraformaldehyde. For the sample observed at 15 min or 30 min post-irradiation, the cells were suffered from 5 min irradiation at 785 nm (1.0 W cm-2), followed by 0 min or 15 min incubation. Subsequently, the cells were incubated for 10 min at 37 °C with 1.0 mL Hoechst 33342 (1.0 µg mL-1) and another 5 min incubation with Lysotraker Green DND26 (1.0 mL, 50 nM) prior to fixation using 4% paraformaldehyde. Finally, the cells were washed using PBS before CLSM observation (Zeiss LSM710). Free DOX or DOX encapsulated in I/DSe-NPs or I/D-C-NPs was imaged with the excitation of 514 nm and the emission between 535 and 673 nm. The co-localization in CLSM images was calculated using Image-Pro® Plus. Biodistribution. 4T1 cells (2.0 × 106 cells/mice) were subcutaneously transplanted into the female Balb/c mice (16-18 g) for constructing tumor-bearing mice (60-70 mm3). The mice bearing 4T1 tumors were intravenously injected with I/D-Se-NPs, I/D-C-NPs, and Free I/D at the dose of 7.5 mg kg-1, respectively. At 24 h post-injection, the various tissues including heart, liver, spleen, lung, kidney, and tumor were extracted from the mice, and homogenized in 1.0 mL PBS. Next, chloroform (4.0 mL) and methanol (1.0 mL) were added to extract DOX from the solutions. Finally, the amounts of DOX in different tissues were determined by HPLC. In Vivo Tissue Distribution. The Balb/c mice bearing 4T1 tumors were injected with I/D-SeNPs, I/D-C-NPs, and Free I/D at the dose of 10.0 mg kg-1 ICG, followed by 785 nm irradiation (5 min, 1.0 W cm-2) or not at 24 h post-injection. Subsequently, the tumors were dissected at 15 min post-irradiation, and the obtained tumors were frozen and then sliced at the thickness of 40


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µm using a cryostat microtome (Leica CM1950). Subsequently, 1 µg mL-1 Hoechst was added into the slices with 10 min incubation at 37 ºC. Finally, the slices were washed by PBS and prepared on glass slides for CLSM (Zeiss LSM710) observation. In Vivo Antitumor Efficacy. I/D-Se-NPs, I/D-C-NPs, and Free I/D at the dose of 7.5 mg kg-1 ICG were intravenously injected into the mice bearing 4T1 tumors (60-70 mm3) on day 0, 2, 4, respectively. The tumors suffered from 785 nm irradiation for 5 min (1.0 W cm-2) at 24 h after each injection, respectively. Then, the tumor volume (V) was calculated according to the equation of V = (L × W2)/2, where W and L are the tumor size at the widest and longest dimensions, and monitored during subsequent 16 days. The relative tumor volumes are obtained through the normalization against their original volumes at day 0. Finally, the tumors were extracted at 16 days post-injection for optical observation.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.” Additional experiments and figures (Figure S1-19). AUTHOR INFORMATION Corresponding Author *[email protected] Author Contributions #

These authors contributed equally. The authors declare no competing financial interest.


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ACKNOWLEDGMENT This work was supported by National Basic Research Program (2014CB931900), National Natural Science Foundation of China (31671016, 51473109, 51503139, 31422021, and 31500811), Natural Science Foundation of Jiangsu Province of China (BK20150324), Natural Science Foundation for Colleges and Universities in Jiangsu Province of China (15KJB150025), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Scheme 1. Schematic illustration of selenium-inserted polymeric nanoparticles (I/D-Se-NPs) with immediate drug release and highly efficient cytoplasmic translocation properties under NIR light exposure for synergistic thermo-chemotherapy.


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Figure 1. (A) Synthetic route of Se-polymer. (B) 1H NMR spectrum of Se-polymer


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Figure 2. (A) TEM image of I/D-Se-NPs. (B) TEM image of disassembled I/D-Se-NPs after 3 min irradiation (785 nm, 1.0 W cm-2). (C) Size distribution of intact I/D-Se-NPs and I/D-C-NPs. (D) Temperature elevation of I/D-Se-NPs at the concentrations of 2, 5, 10, 25, and 50 µg mL-1 ICG under irradiation.


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Figure 3. (A) Release profiles of DOX from I/D-Se-NPs and Free I/D at pH 7.4 under irradiation for various time (n = 3). (B) Release rate of DOX at zero point after various irradiation time according to release profiles. (C) Normalized absorbance of DPBF at 410 nm and (D) corresponding ROS generation rate in the presence of I/D-Se-NPs at the doses of 0.1, 0.2, 0.5, and 1.0 µg mL-1 ICG under 785 nm irradiation or not. (E) Release profiles of DOX from I/D-Se32 ACS Paragon Plus Environment

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NPs, I/D-C-NPs, and Free I/D at pH 7.4 under 10 min irradiation (785 nm, 1.0 W cm-2) or (F) H2O2 treatment (n = 3).


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Figure 4. (A) Internalized amounts of ICG and DOX from I/D-Se-NPs, I/D-C-NPs, and Free ICG/DOX at the dose of 10 µg mL-1 ICG after 24 h incubation (n = 3). (B) Normalized viability 34 ACS Paragon Plus Environment

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of 4T1 cells treated with I/D-Se-NPs, I/D-C-NPs, and Free I/D at various doses of DOX after 24 h incubation under 5 min irradiation or not (785 nm, 1.0 W cm-2), n = 3. (C) CLSM images of 4T1 cells stained with Lysotracker Green DND-26 and Hoechst 33342 after 0.5 h incubation with I/D-Se-NPs at the dose of 10.0 µg mL-1 ICG before the irradiation, as well as those at 5, 15, and 30 min post-irradiation (5 min, 1.0 W cm-2).


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Figure 5. (A) Ex vivo biodistribution of DOX at various tissues of the mice bearing 4T1 tumors treated with I/D-Se-NPs, I/D-C-NPs, and Free I/D at the dose of 7.5 mg kg-1 DOX. (B) CLSM images of in vivo tumor slices from the mice treated with I/D-Se-NPs at the dose of 10.0 mg kg-1 ICG under 3 min irradiation (785 nm, 1.0 W cm-2) or not at 24 h post-injection. (Scale bar: 100 µm) (C) Tumor growth profiles of the mice treated with I/D-Se-NPs, I/D-C-NPs, and Free I/D at the dose of 7.5 mg kg-1 DOX under 5 min irradiation or not (785 nm, 1.0 W cm-2), and (D) Photograph of the tumors extracted from the mice at 16 day post-irradiation. *p < 0.05 and **p < 0.01 (Student’s t-test), n = 5.


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The table of contents entry: We rationally designed polymeric nanoparticles of seleniuminserted copolymer to generate highly efficient cytoplasmic delivery of anticancer agents for synergistic thermo-chemotherapy

Authors: Yangyun Wang, Yibin Deng, Huanhuan Luo, Aijun Zhu, Hengte Ke, Hong Yang, and Huabing Chen

Title: Light-Responsive Nanoparticles for Highly Efficient Cytoplasmic Delivery of Anticancer Agents

ToC figure


Light-Responsive Nanoparticles for Highly Efficient Cytoplasmic

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