pH-Responsive Apoferritin Nanocage for

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A Self-targeting, Dual ROS/pH-Responsive Apoferritin Nanocage for Spatiotemporally Controlled Drug Delivery to Breast Cancer Bin Du, Shaona Jia, Qinghui Wang, Xiaoyu Ding, Ying Liu, Hanchun Yao, and Jie Zhou Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00012 • Publication Date (Web): 17 Feb 2018 Downloaded from http://pubs.acs.org on February 19, 2018

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A Self-Targeting, Dual ROS/pH-Responsive Apoferritin Nanocage for Spatiotemporally Controlled Drug Delivery to Breast Cancer Bin Du,†,‡,§ Shaona Jia,† Qinghui Wang,† Xiaoyu Ding,† Ying Liu,† Hanchun Yao,* ,†,‡,§ and Jie Zhou*,†,‡,§ †

School of Pharmaceutical Sciences, Zhengzhou University, 100 Science Road, Zhengzhou

450001, China ‡

Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan

Province, 100 Science Road, Zhengzhou 450001, China §

Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province,

100 Science Road, Zhengzhou 450001, China ABSTRACT: In this study, an intelligent pH and ROS dual-responsive drug delivery system based on apoferritin (AFt) nanocage was prepared. This therapeutic system can specifically selftargeting to 4T1 breast cancer cells by exploiting L-apoferritin receptor SCARA 5, avoiding the nonspecific binding or aggregation of nanoparticles due to the chemical functionalization for targeting. The characteristics of AFt were utilized for the simultaneous delivery of anticancer drug doxorubicin (DOX) and photosensitizer rose bengal (RB). RB exhibited efficient reactive oxygen species (ROS) generation, which can be applied to photodynamic therapy. Meanwhile, AFt nanocage was prone to undergoing peptide backbone cleavage when oxidized by ROS. Therefore, by combining the intrinsic pH-responsive property of AFt, the dual ROS/pHresponsive system was developed. The time and location of drug release can be controlled by the combination of internal and external stimulus, which avoids the incomplete drug release under 1

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single stimulus response. The drug release rate increased significantly (from 26.1% to 92.0%) under low-pH condition (pH 5.0) and laser irradiation. More DOX from AFt entered the nucleus and killed the tumor cells, and the cell inhibition rate was up to ~83% (DOX concentration: 5 µg/mL) after 48 h incubation. In addition, the biodistribution and the in vivo antitumor efficacy (within 14 d treatment) of the nanosystem were investigated in 4T1 breast cancer BALB/c mice. The results indicated that the system is a promising therapeutic agent involving ROS/pH dual response, self-targeting and chemo-photodynamic therapy. Key words: Apoferritin; Protein nanocage; ROS/pH responsive; Self-targeting

■ INTRODUCTION Although many researchers have spared no effort to seek effective ways for tumor therapy, tumor is still a serious threat to human health. As we all know, those conventional drugs are widely distributed throughout the body and can adversely affect other cells and tissue other than the targeted treatment area.1 New therapeutic technique with high specificity is eagerly required to release anticancer agents intelligently to the cancerous cells.2 To address the problems, the drug delivery systems (DDSs) based on nanomaterials have been designed and shown promising therapeutic efficacy.3,4 However, most of them are not biocompatible and biodegradable, which form potential toxicity.5 In addition, these materials usually have no intrinsic tumor targeting properties. We have to functionalize them with specific ligands (such as antibodies,6 peptides7 or small molecules8) to improve their targeting capability.9 But the chemical functionalization for targeting tends to make the preparation process troublesome, resulting in nonspecific binding and aggregation of nanoparticles.10 Biological nanoparticles are perfect candidates with precisely defined dimensions, biocompatibility and biodegradability.11-13 2


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As one of the most attractive protein architectures, apoferritin (AFt) has a hollow nanocage with an outer diameter of 12 nm and inner cavity diameter of 8 nm.14-16 The cavity provides a space for loading molecular cargoes.17 In addition, this cage-like structure has self-targeting properties. Especially, horse spleen ferritin (contains over 80% L-ferritin) can selectively transport drug to cancer cells over-expressing L-ferritin receptor SCARA 5.18,19 The receptor is often over-expressed in breast cancer cells.20,21 The cell uptake of AFt provides a biological pathway to facilitate cellular delivery of any drugs through receptor-mediated endocytosis.22-24 Interestingly, at low pH, AFt cages disassemble into subunits, allowing the dispersed drugs to assemble into the AFt cage. When the pH value is turned back to neutral, the subunits are reconstituted into nanocage structures, almost in an intact manner.25,26 So AFt can be used for targeting delivery and controlling drug release based pH response.27-29 Although the swelling channels of AFt at the acidic microenvironment of tumor cells bring the drugs into cancer cells. However, the drug release rate is limited. If the drug is not completely released in time, it will result in poor treatment effect. Recently, it was found that amino acids such as histidine, arginine, lysine, glutamic acid and proline residues in peptides are especially prone to undergoing peptide backbone cleavage when oxidized, causing protein fragmentation.30,31 Therefore, we connected a photosensitizer rose bengal (RB) to the surface of AFt. RB is notable for its high triplet quantum yield, fairly long lived triplet state (t1/2 =0.1–0.3 ms), and high singlet oxygen quantum yield.32,33 We exploit the powerful oxidation capacity of ROS generated by RB to destruct the structure of AFt. So ROS can act as the second stimulus to trigger drug (loaded into AFt internal cavity) release. In addition to oxidizing protein cage, the photosensitizer can also achieve photodynamic therapy (PDT). PDT is widely used in clinics because it is non-invasive medical treatment with few side effects.34,35 Through the 3



administrations of photosensitizer and light irradiation at the tumor site, PDT selectively eradicates tumor and has low toxicity.36 In this study, a self-targeting, dual-responsive (ROS and pH) nanosystem based on AFt for drug delivery was synthesized. First of all, by using its reversible dissociation-reassemble characters upon pH changes, DOX was encapsulated in the cavity of AFt (DOX@AFt) and released specifically response to the acidic microenvironment of tumor cells. Then, in order to produce more ROS in the tumor environment to further destroy protein cages and photodynamic therapy, we introduced photo-sensitizer RB onto AFt surface (DOX@AFt-RB). By exploiting the intrinsic tumor-targeting properties of AFt, the nanocomposites can specifically target tumor tissues without additional targeting ligands or molecules. In general, this work developed a unique mechanism of drug release and provided a biological approach for targeted drug delivery, aimed at realizing an effective treatment of cancer by combining chemotherapy and photodynamic therapy.

■ MATERIALS AND METHODS Materials. DOX (purity>98%) was obtained from Beijing Yi-He Biotech Co. Ltd. (Beijing, China). AFt from horse spleen (A3641, 50 mg/mL), Rose Bengal, N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl-N’-ethylcarbodiimide) hydrochloride (EDC-HCl) and annexin-Vfluos staining kit were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). 2,7dichlorofluorescin diacetate (DCFH-DA) was from Beyotime Co. (Shanghai, China). Other reagents were acquired from China National Medicines Corporation Ltd. (Beijing, China). All the solvents were of analytical grade. All other chemicals were commercially obtained and used without further purification. 4


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Synthesis of DOX@AFt-RB. DOX@AFt was prepared using the disassembly/reassembly method. 9.6 mg DOX was added to 960 µL of AFt (50 mg/mL) and 4.8 mL PBS, and 1 M hydrochloric acid was slowly added to decrease the pH of the solution and disintegrate the AFt. The solution was mixed for 15 min, then the solution pH was adjusted to 7.4 with 0.1 M sodium hydroxide added dropwise. The mixture was continuously stirred for 2 h to encapsulate DOX inside AFt (creating DOX@AFt). To covalently conjugate RB to DOX@AFt, RB (400 µg) was activated by EDC and NHS (molar ratio of RB/EDC/NHS: 1/1/1.5) in 2 mL PBS for 30 min at room temperature, then DOX@AFt (2 mg) was added and reacted overnight at room temperature and protected from light. The resultant mixture was purified through a 100 kDa Amicon filter (Millipore) and washed several times with PBS buffer. Characterization. A dynamic light scattering (DLS) (Zetasizer Nano ZS-90, Malvern, UK) and Transmission electron microscopy (TEM) (Tecnai G2 20, FEI, USA) were used for characterizing particle size, zeta potential, and morphology, respectively. The optical properties of DOX@AFt-RB was characterized using an UV-Vis spectrometer (Lambda 35, Perkin Elmer, USA) and FTIR spectrometer (NicoletiS10, Thermo, USA). Detection of extracellular ROS. The generation of extracellular ROS was detected by the chemical probe 1, 3-diphenyl isobenzofuran (DPBF).37 In a typical experiment, 80 µg DPBF and 1 mg AFt-RB were mixed in DMSO (4 mL) solution, and then transferred into a cuvette. The mixture was irradiated with a 532 nm laser at a power density of 1 W/cm2 for 10 min, and the absorption intensity of DPBF at 417 nm was recorded at every minute. For comparison, the ROS generation ability of free RB was also assessed using the same method. 5



Dual-stimuli triggered DOX release. To investigate the dual stimuli responsive release, the AFt@DOX-RB samples containing 1mg DOX in dialysis bags (MWCO of 8, 000) were dialyzed in 50 mL PBS (pH 7.4 or 5.0) at 37 ℃ with a stirring rate of 100 rpm. Then the samples were treated with or without 532 nm laser irradiation (1 W/cm2) for 10 min every 4 h. At specified time intervals, 1 mL samples of the release solution were taken out and replaced with an equal volume of fresh fluid. The release of DOX from the buffer solution was quantified by high performance liquid chromatography. Native-PAGE. The analysis of the damage degree of AFt at different conditions was carried out by native polyacrylamide gel-electrophoresis (Native-PAGE). Samples were prepared by laser irradiation at different times or incubation at different pH values for 10 min and then mixed well with an equal volume of sample loading buffer. The prepared samples were analyzed on 8% Native-PAGE. Coomassie brilliant blue staining was used for 2h with subsequent destaining for 2 h incubation (30% ethanol, 10% acetic acid, 60% Millipore water) or overnight incubation. The software program Image J was used to quantify the relative gray value. Cellular experiments. 4T1 (mice breast cancer) cell line was gained from Chinese Academy of Sciences Cell Bank (Catalog No. HYC3204, Beijing, China). Cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin. The cells were trypsinized using trypsin-EDTA and maintained in a humidified atmosphere supplemented with 5% CO2 at 37 ℃. The detection of SCARA 5 and binding of AFt to SCARA 5. The cells were seeded on glass coverslips and allowed to adhere on this substrate overnight at 37 ℃ and 5% CO2. The next

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day, cells were incubated with AFt-FITC on ice for 1h, then internalization was stopped by adding cold PBS/0.02% sodium azide. Cells were fixed in 4% formalin solution for 10 min and subsequently rinsed twice with PBS, and non-specific binding was blocked with 10% bovine serum albumin (BSA, from Sigma-Aldrich) in PBS for 20 min at room temperature. AntiSCARA 5 antibody (5 µg/mL) was diluted in PBS containing 1% BSA and added to the coverslips overnight at 4 ℃. Cells were rinsed twice with PBS and then incubated with secondary antibody Cy3-conjugated goat anti-rabbit (5 µg/mL) in PBS containing 1% BSA for 1 h at room temperature. Cell nuclei were counter stained with DAPI. The coverslips were mounted with fluoromount mounting medium and visualized with a confocal laser scanning microscope (CLSM) (Leica TCS SP5, Germany). To further examine the role of SCARA 5 in binding of AFt to 4T1 cells, cells were incubated on ice for 30 min with or without anti-SCARA 5 (50 µg/mL) to specifically block binding of AFt to SCARA 5, which was followed by the addition of AFt-FITC (50 µg/mL) further incubation was on ice for 90 min. Uptake and intracellular distribution of the nanoparticles. 4T1 cells were seeded in 6well plates at a density of 2×105 cells per well and cultured for 24 h, followed by treatment with DOX and DOX@AFt-RB in normal culture medium. Then the laser-triggered release experiments were performed with 532 nm laser (1 W/cm2) irradiation for 5 min. The concentration of DOX in each group was 5 µg/mL. After 4h incubation, the DAPI staining solution (5 µg/mL) was added to each well of the plate to stain the nucleus of cells for 15 min, followed by soaking for 15 min in 4% paraformaldehyde, and then washed with PBS. The cells were imaged using a fluorescence microscope (Zeiss LSM 510, Thornwood, NY, USA).

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To quantitatively evaluate the intracellular uptake capacity of the above samples to cells, all operations were same as above. In addition, the cell samples were collected and quantitatively analyzed by using flow cytometry (Epics XL, Coulter, USA). In vitro cytotoxicity. For in vitro cytotoxicity studies, 4T1 cells were seeded at 5×103 cells per well in 96-well plates and then incubated for 24 h. After that, cells were incubated with AFt, DOX, RB/laser, DOX@AFt-RB (with laser or not) for 48 h with a series of concentration (DOX concentration: 0.625, 1.25, 2.5, 5, 10, 20 µg/mL), and then the cells were irradiated with or without 532 nm laser (1 W/cm2) for 10 min. After total 48 h incubation, the cell viabilities were measured by a standard MTT assay. Detection of intracellular ROS.

The intracellular ROS level was measured using an

oxidation sensitive fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA). DCFH-DA can be deacetylated by nonspecific esterase to form DCFH, which can be oxidized by hydrogen peroxide or low-molecular-weight peroxides to produce a stable fluorescent ROS-sensitive compound 2,7-dichlorofluorescein (DCF).38, 39 In this study, 4T1 cells were seeded at a density of 2×105 cells per well in 6-well plates. Following incubation with DOX@AFt-RB (DOX concentration: 5 µg/mL) for 4 h, DCFH-DA was loaded in the cells. After 30 min incubation, cells were washed twice with PBS followed by exposure to 532 nm irradiation for 0, 5, 10 and 15 min, respectively. After irradiation, the fluorescence images of the treated cells were acquired using a Fluorescence Microscope (Zeiss LSM 510, Thornwood, NY, USA). Cell apoptosis assay. Apoptosis was monitored using an Annexin-V-Fluos Staining kit.40 4T1 cells were treated with DOX, DOX@AFt-RB, DOX@AFt-RB/laser 10 min for 48 h at 37 ℃. Cells were harvested and washed three times with PBS, then resuspended in 500 µL binding buffer. After adjusting the cell density to 1×106 cells/mL, 5 µL Annexin-V-FITC and 5 µL PI 8


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were added and incubated with the cells for 15 min in the dark. Finally, the stained cells were analyzed by Flow Cytometry. The cells without any treatment were used as the negative control group. Animal experiments. All animal experiments were performed under a protocol approved by Henan Laboratory Animal Center. The Life Science Ethics Committee (Zhengzhou University) reviewed and approved the entire animal protocol prior to initiation of the experiments. The tumor models were generated by subcutaneous injection of 1×106 4T1 cells in 0.1 mL saline into the right shoulder of BALB/c female mice (18–20 g, Henan Laboratory Animal Center). The mice were used when the tumor volume reached 60 – 100 mm3 (≈5 d after tumor inoculation). Biodistribution and tumor-targeting ability of DOX@AFt-RB in tumor-bearing mice. Fluorescence images were obtained using an in vivo imaging system (FX PRO, Bruker, USA). In the experiment, near-infrared dye IR783 was loaded in NPs according to the method in the drug loading section. The loading content was measured using UV-Vis spectrometry. IR783-labeled NPs or free IR783 were injected intravenously via the tail vein into mice at an equivalent IR783 dose of 0.5 mg per kg mouse. The imaging data were collected at 1, 2, 4, 8, 12 and 24 h after tail vein injection of the nanoparticles, respectively. At 4 h after treatments, mice were dissected, their organs and tumors were harvested and visualized by the imaging system. The signal of IR783 was represented by the fluorescent intensity. The acquisition parameters were λem = 790 nm, λex = 720 nm, binning = 1, and exposure time = 0.1 s. In vivo antitumor efficacy. The mice were randomly divided into five groups (five mice per group). (i) Saline group (Blank), (ii) DOX, (iii) RB/laser, (iv) DOX@AFt-RB, (v) DOX@AFt9



RB /laser (DOX dose: 5 mg/kg). All five groups were intravenously injected into mice via the tail vein every 2 d. The drug release and PDT were controlled by irradiating tumors with 532 nm laser (1 W/cm2, laser-treated groups) for 10 min at 4 h post-injection. The mice were observed daily for clinical symptoms and the tumor sizes were measured using a caliper every other day and calculated as volume = (tumor length)×(tumor width)2/2. After treatment for 14 d, the mice were killed to collect heart, liver, spleen, lung, kidney and tumor. Then the collected tissues were soaked in a 10% formalin solution, embedded with paraffin for H&E staining. Morphological changes were observed under a microscope (Zeiss LSM 510, Thornwood, NY, USA).

■ RESULTS AND DISCUSSION Preparation and characterization. The synthetic process of the DOX@AFt-RB nanoparticles was summarized in Figure 1. DOX was encapsulated in the cavity of AFt by using its ability to disassemble and reassemble under pH control. Then the self-targeting AFt was coupled with RB by amide bond connection. The nanocomposites were able to specifically target to 4T1 breast cancer cells by exploiting L-apoferrit in receptor SCARA 5. The protein cage slightly disassembled in response to acidic environment, causing DOX released slowly. With 532 nm laser irradiation, the protein cage can be degraded into small fragments due to ROS, promoting the complete release of DOX. The released DOX entered the nucleus and intercalated in the DNA double-helix, which gradually induced the cell death.

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Figure 1. Synthetic routes of the of DOX@AFt-RB nanocomposites.

The UV-Vis spectra of DOX, AFt and DOX@AFt were shown in Figure 2A. Pure DOX had an obvious peak at 480 nm in the UV absorption spectrum. Similarly, DOX loaded with AFt showed two peaks at 280 nm and 480 nm, which indicated that DOX was successfully encapsulated in AFt. The FTIR spectra of RB, AFt and AFt-RB were shown in Figure 2B. After RB was attached to AFt, the peak of RB at 1739 cm-1 disappeared, which indicated that the free carboxyl group of RB bound to the amino group of AFt to form an amide bond. The stronger absorption at 1,654 cm-1 could be attributed to the stretching vibration of C=O (the so-called Amide I vibrational stretch). And the peak at 1,546 cm-1 could be ascribed to the bending vibration N-H and stretching vibration of C-N (the so-called Amide II vibration).41 The results confirmed that RB was successfully attached to the surface of AFt through the amidation process. DLS measurement indicated that the sizes of AFt and DOX@AFt-RB were 11.40 ±2.1 nm and 23.75±3.42 nm, respectively (Figure 2C). The zeta potentials of AFt and DOX@AFt-RB were -2.09±1.1 mV and -21.10±2.3 mV, respectively (Figure 2D). TEM images indicated that AFt had a nanocage structure of about 11 nm and DOX@AFt-RB nanoparticles had spherical shape about 23 nm (Figure 2E and F).

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A

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B

DOX@AFt-RB

DOX@AFt-RB

C

D AFt AFt

E

F

Figure 2. Characterization of the different formulations. (A) UV-Vis spectra of DOX, AFt, DOX@AFt, respectively. (B) FT-IR spectra of AFt, RB and AFt-RB, respectively. (C) The sizes of AFt (inset) and DOX@AFt-RB. (D) The zeta potentials of AFt (inset) and DOX@AFt-RB. (E) and (F) TEM images of AFt and DOX@AFt-RB, respectively.

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Detection of extracellular ROS. In this study, ROS generation was the critical step in the photo-triggered DOX release and photodynamic therapy, so the ROS generation abilities of RB and AFt-RB were very important indexes to illustrate the potential application. The generated ROS was captured by DPBF, then caused DPBF bleaching. The absorption decreased at 417 nm (417 nm: the maximum absorption wavelength of DPBF) was measured to reflect the ROS generation.42,43 As can be seen in Figure 3A, RB began to generate the detectable amount of ROS at a concentration under 532 nm laser irradiation (1 W/cm2), indicating that RB was highly capable of inducing singlet oxygen. In addition, AFt-RB was also found to generate singlet oxygen under laser irradiation at 532 nm. Although its production efficiency of ROS was lower than that from RB due to the consumption of part of ROS by the reaction of AFt with RB, it still retained about 72.7% of free RB, which allowed us to use it for PDT treatment of cancer cells. Dual-stimuli triggered DOX release.

In order to evaluate DOX release in vitro, we

incubated the nanocomposites in PBS (pH 7.4 or 5.0) with or without 532 nm laser irradiation. As shown in Figure 3B, the release of DOX from DOX@AFt-RB was low at normal physiological pH 7.4, where 48 h incubation led to the drug release less than 30%. Acidification of the buffer to pH 5.0 caused a much higher release rate, resulting from the swelling channels of the AFt at pH 5.0. In addition, we found that the release of DOX from the nanocomposites after laser irradiation was higher and quicker than that without laser irradiation under the same conditions. The reason can be attributed to the fact that the ROS generated by RB could degrade AFt, facilitating DOX release, which indicated that laser radiation could act as the second stimulus to trigger DOX release. The accumulative DOX release rate was 92% after the combination of laser irradiation and low pH 5.0. These values, which were significantly higher

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than that achieved by alone using pH 5.0 or laser irradiation, confirmed that DOX release inside cells can be improved by combining the effect of low pH and laser radiation. Native-PAGE.

ROS and acidic pH resulted in highly efficient degradation of AFt as

evidenced by Native-PAGE. First, we examined the effect of different irradiation time on the protein structure, using commercial horse spleen AFt as reference standard. The results were shown in Figure 3C and D. Commercial horse spleen AFt without any treatment appeared as single band on Native-PAGE. However, a new band of lower molecular weight protein fragment appeared on Native-PAGE when the samples were irradiated with 532 nm laser due to degradation. Moreover, as the irradiation time increased, the apparent intensity of the new band increased. This phenomenon can be explained as follows: with the extension of laser irradiation time, the yield of ROS increased, and consequently the damage degree of AFt increased, even AFt was cracked into protein fragment. In addition, we also investigated the effect of different pH values on protein structure. As can be seen from Figure 3E and F, about 20% of AFt were cleaved at pH 5.0 (cancer cells environment), which endowed the protein with pH-triggered drug release property. Most of AFt were dissociated at pH 2 and then the AFt gave major band (85%) that correspond to the native apoferritin’s 24-meric structure when the pH was turned back to neutral. Its ability to disassemble and reassemble under pH control has allowed for the loading of a large number of therapeutic agents.

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B

A

D

C

1

2

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F

E

1

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Figure 3. Investigation on the dual responsive properties of DOX@AFt-RB. (A) Time-course generation of singlet oxygen detecting by the bleaching of DPBF absorption at 417 nm under 532 nm laser irradiation. (B) In vitro release profiles of DOX. (C, D) The electrophoresis and relative gray value of AFt after different irradiation times. Lane 1: AFt Lane 2-5: AFt-RB under 532 nm laser irradiation for 0, 5, 10, 15 min. (E, F) The electrophoresis and relative gray value of 15



AFt in different pH values. Lane 1: AFt/pH 7; Lane 2: AFt/pH 5; Lane 3: AFt/pH 2; Lane 4: pH 2→pH 7.

The detection of SCARA 5 and binding of AFt to SCARA 5. To determine whether native SCARA 5 is expressed on the 4T1 cells membrane and binds to AFt, we added AFt-FITC at 4 ℃ to 4T1 cells and examined the expression of L-AFt receptor SCARA 5 by immunofluorescence. As shown in Figure 4A, a co-localization of AFt-FITC and SCARA 5 was observed (orange, overlap of the red fluorescence of SCARA 5 with green fluorescence of FITC), indicating that SCARA 5 and AFt interacted at the cell surface. We further examined the role of SCARA 5 in binding of AFt to 4T1 cells, using the capacity of the anti-SACRA 5 to specifically block binding of AFt to SCARA 5. As shown in Figure 4B, when cells were incubated with AFt-FITC on ice for 1h, there was a large amount of AFt-FITC bound to the surface of 4T1 cells membrane. Cells were incubated with excess anti-SCARA 5 in advance, however, the binding of AFt to 4T1 cells was substantially blocked by anti-SCARA 5. It again showed the specific binding of AFt to SCARA 5 and it excluded the possibility that this binding was mediated or facilitated by SCARA 5.

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AFt-FITC

DAPI

SCARA 5

Merged

A

DAPI

Merge

AFt-FITC

B AntiSCARA 5(-)

AntiSCARA 5(+)

Figure 4. The detection of SCARA 5 and the binding of AFt to SCARA 5. (A) Confocal microscopy images of 4T1 cells incubated for 1 h at 4 ℃ with AFt-FITC (green). Then L-ferritin receptor SCARA 5 were recognized with anti-SCARA 5 antibody and labeled with an anti-rabbit secondary antibody conjugated to Cy3 (red). (B) 4T1 cells incubated or not with excess antiSCARA 5 antibody for 0.5 h at 37 ℃ and then treated with AFt-FITC 1.5 h.

Uptake and intracellular distribution of the nanoparticles.

The cellular uptake and

intracellular distribution of the nanoparticles were performed to compare the penetrating capacity by the fluorescent microscope and flow cytometry. The results were illustrated in Figure 5. 17



Nucleus was stained as blue fluorescence by DAPI. DOX was observed as red fluorescence representing uptake of nanoparticles (Figure 5A). After 4 h incubation, the cellular uptake of DOX@AFt-RB was significantly higher than that of free DOX. Since free DOX just entered the cells by passive diffusion, the nanocomposites were mediated by SCARA 5 receptor endocytosis. Meanwhile, the DOX fluorescence of DOX@AFt-RB/laser was mainly distributed in the nucleus, producing almost perfectly overlapped purple-fluorescent spots in the merged images. The phenomenon was explained that tumor cells could quickly endocytose DOX@AFt-RB nanoparticles and these nanoparticles locally released DOX in response to low pH and ROS generated by RB upon laser irradiation. Then the released DOX entered the cell nuclei and specifically affiliated with DNA. However, the DOX fluorescence of DOX@AFt-RB without laser irradiation was evenly distributed in the whole cell including the nucleus and cytoplasm, probably resulting from the incompletely release of DOX from the nanocomposites under low pH stimulus alone. After culturing for 4 h, the quantitative analysis of red fluorescence intensity based on flow cytometry revealed the following tendency: DOX

pH-Responsive Apoferritin Nanocage for

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