Domino-Like Intercellular Delivery of Undecylenic Acid-Conjugated

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Domino-Like Intercellular Delivery of Undecylenic Acid-Conjugated Porous Silicon Nanoparticles for Deep Tumor Penetration Tuying Yong,§ Jun Hu,§ Xiaoqiong Zhang, Fuying Li, Hao Yang, Lu Gan,* and Xiangliang Yang National Engineering Research Center for Nanomedicine, Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China S Supporting Information *

ABSTRACT: Improving the intratumoral distribution of anticancer agents remains the critical challenge for developing efficient cancer chemotherapy. Luminescent porous silicon nanoparticles (PSiNPs) have attracted considerable attention in the biomedical field especially in drug delivery. Here, we described the lysosomal exocytosis-mediated domino-like intercellular delivery of undecylenic acid-conjugated PSiNPs (UA-PSiNPs) for deep tumor penetration. UA-PSiNPs with significantly improved stability in physiological conditions were internalized into tumor cells by macropinocytosis-, caveolae-, and clathrin-mediated endocytosis and mainly colocalized with Golgi apparatus and lysosomes. Substantial evidence showed that UA-PSiNPs was excreted from cells via lysosomal exocytosis after cellular uptake. The exocytosed UA-PSiNPs induced a domino-like infection of adjacent cancer cells and allowed encapsulated doxorubicin (DOX) to deeply penetrate into both three-dimensional tumor spheroids and in vivo tumors. In addition, DOX-loaded UA-PSiNPs exhibited strong antitumor activity and few side effects in vivo. This study demonstrated that UA-PSiNPs as a drug carrier might be applied for deep tumor penetration, offering a new insight into the design of more efficient delivery systems of anticancer drugs. KEYWORDS: porous silicon, undecylenic acid conjugation, lysosomal exocytosis, intercellular delivery, tumor penetration

1. INTRODUCTION Nanomedicine-based therapeutics, including clinically approved Abraxane (albumin-bound paclitaxel) and Doxil (PEGylated liposomal doxorubicin), provide promising opportunities in cancer therapy.1−3 In spite of enhanced therapeutic efficacy and reduced side effects, nano drug delivery systems (NDDSs) were found to be majorly located around tumor vessels due to the aberrant vascular architecture, compact extracellular matrix, and elevated interstitial fluid pressure (IFP) in tumor tissues, thus reducing the overall therapeutic effects.4−6 Enhancing the penetration of anticancer agents into solid tumor tissue was becoming a crucial challenge in improving cancer treatment efficiency. Several strategies have been reported to enhance tumor penetration, including optimization of physical features (e.g., the surface charge and size of nanoparticles), 7−9 conjugation of targeting ligands,10,11 construction of tumor microenvironment responsive (e.g., pH and protease) intelligent nanocarriers,12,13 magnet-assisted penetration,14 and synergistic combination of chemotherapeutics with drugs overcoming physiological barriers of solid tumor (e.g., depleting tumor collagen, decreasing platelet aggregation, normalizing tumor vasculature).15−17 Although these means demonstrated effective tumor penetration to some extent, limited loading capacity, complicated formulations, and the unclarified intracellular transport of these nanocarriers need to be further improved. © XXXX American Chemical Society

Clarifying the intracellular fates of nanoparticles will contribute to understand their biological activities. Although the cellular internalization mechanisms of nanoparticles have been extensively explored, relatively little effort has been made to investigate their exocytosis from the cells. Several nanoparticles, including gold nanoparticles,18−21 quantum dots,22,23 carbon nanotubes,24,25 mesoporous silica nanoparticles,26,27 and polymeric nanoparticles,28,29 have been reported to be excreted from the cells after internalization. However, these studies mainly focused on the factors regulating their exocytosis, such as surface properties, size, shape of the nanoparticles, and cell type. The underlying exocytosis mechanism and potential exocytosisinduced biological effects of nanoparticles remained to be explored. Recent papers showed that some NDDSs, such as lipidcoated cisplatin and doxorubicin (DOX)-loaded nanogels,30,31 exhibited a domino-like effect in which NDDSs were released from cancer cells and then infected neighboring cells to enhance anticancer efficacy. Whether the domino-like effect of NDDSs was modulated by exocytosis and its application in anticancer drug delivery needs to be further elucidated. Received: September 3, 2016 Accepted: September 21, 2016

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DOI: 10.1021/acsami.6b11127 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. Schematic illustration of UA-PSiNPs as drug delivery carriers for deep tumor penetration. (I) Internalization of UA-PSiNPs into tumor cells. (II) Transport into endosome. (III) Translocation from endosome to lysosome or Golgi apparatus. (IV) Excretion via lysosome exocytosis. (V) Domino-like reinternalization to the adjacent cells. Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These cells were maintained in RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in the presence of 5% CO2. 2.3. Preparation of PSiNPs and UA-PSiNPs. The boron-doped ptype silicon wafers (0.8−1.2 mΩ/cm resistivity, ⟨100⟩ orientation) from Virginia Semiconductor, Inc. (Fredericksburg, VA, USA) were electrochemically etched in aqueous hydrofluoric acid (HF)/ethanol (4:1, v/v) solution at a constant current density of 165 mA/cm2 for 300 s using a Teflon etch cell (Wuhan, China). The porous silicon film was subsequently removed from the substrate using a constant current of 4.5 mA/cm2 for 90 s in 3.3% aqueous HF solution in ethanol, fractured in ultrapure water by ultrasonication overnight, and centrifuged at 8000 rpm for 20 min. PSiNPs were further heated at 60 °C for 3 h to activate the photoluminescence.35,36 UA-PSiNPs were synthesized by thermal hydrosilylation.37,38 Briefly, 10 mg of PSiNPs were submerged in 1 mL of undecylenic acid and reacted at 110 °C overnight in an Ar(g) environment. The resulting nanoparticles were then washed with n-hexane and ethanol to remove excess undecylenic acid. Both luminescent PSiNPs and UA-PSiNPs were stored in dimethyl sulfoxide (DMSO). 2.4. Characterization of PSiNPs and UA-PSiNPs. The chemical compositions of PSiNPs and UA-PSiNPs were examined by Fourier transform infrared (FTIR, Bruker Optics, Ettlingen, Germany) and Xray photoelectron spectroscopy (XPS, Kratos Analytical Ltd., Manchester, UK) analysis. The mean zeta potential and hydrodynamic diameter of PSiNPs and UA-PSiNPs were determined by dynamic light scattering (DLS, ZetaSizer Nano-ZS 90, Malvern Instruments Ltd., Worcestershire, UK). The fluorescence spectra of PSiNPs and UAPSiNPs were measured at λex = 480 nm and λem = 600−850 nm using a Fluorolog-3 spectrofluorometer (Horiba Jobin Yvon, Longjumeau, France). The photostability of PSiNPs and UA-PSiNPs was evaluated in ultrapure water on a Fluorolog-3 spectrofluorometer when PSiNPs and UA-PSiNPs were illuminated with a 100 W mercury lamp for different time courses. The degradation property of PSiNPs and UA-PSiNPs was investigated by inductively coupled plasma optical emission spectroscopy (ICP-OES, model Optima 4300 DV, PerkinElmer, Norwalk, CT, USA). Briefly, 100 μg/mL PSiNPs and UA-PSiNPs were incubated in

Porous silicon nanoparticles (PSiNPs) have exhibited huge potentiality as drug carriers owing to their high surface area, adjustable pore size with a large drug loading capacity, strong intrinsic fluorescence for in vivo real-time tracking their accumulation, and high biocompatibility and biodegradability.30−32 However, the rapid degradation of PSiNPs in physiological conditions limited their application in anticancer drug delivery.33,34 Here, undecylenic acid-conjugated porous silicon nanoparticles (UA-PSiNPs) were constructed with relatively higher stability in aqueous media compared with PSiNPs. UA-PSiNPs were excreted from cancer cells via lysosomal exocytosis after internalization. The exocytosed UAPSiNPs could infect adjacent cancer cells efficiently, allowing the delivery of anticancer drugs deep inside the tumor and improving their anticancer efficiency (Figure 1). This strategy on lysosomal exocytosis-mediated domino-like intercellular delivery to enhance tumor penetration might contribute to the development of novel NDDSs in cancer treatment.

2. EXPERIMENTAL SECTION 2.1. Materials. Undecylenic acid was obtained from Aladdin Industrial Corporation (Shanghai, China). Sulforhodamine B sodium salt (SRB), amiloride, chlorpromazine (CPZ), 5-(N-ethyl-N-isopropyl) (EIPA), dynasore, nystatin, Brefeldin A, monensin, cytochalasin D, and nocodazole were obtained from Sigma-Aldrich (St Louis, MO, USA). LysoTracker Green, DAPI, DiO, Dil, and ionomycin were obtained from Beyotime Institute of Biotechnology (Shanghai, China). GolgiTracker Green was obtained from KeyGEN Biotechnology (Nanjing, China). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco BRL/Life Technologies (Grand Island, NY, USA). Doxorubicin hydrochloride (DOX·HCl, with purity above 98.0%) was obtained from Beijing HuaFeng United Technology CO., Ltd. (Beijing, China). 2.2. Cell Culture. Human hepatocarcinoma HepG2 and Bel7402 cells and mouse hepatocarcinoma H22 cells were purchased from Type B


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2.12. Intercellular Delivery of UA-PSiNPs. Intercellular delivery of UA-PSiNPs in Bel7402 cells and HepG2 cells was performed as described.31 Briefly, the cells seeded on the first coverslip were treated with 75 μg/mL UA-PSiNPs for 2 h. The treated cells were rinsed by PBS and then coincubated with the new cells on the second glass slide for 10 h in fresh medium. Afterward, the cells on the second glass slide were taken out and coincubated with the new cells on the third glass slide for 10 h in fresh medium. The cells were rinsed by PBS, and the intracellular UA-PSiNPs were detected by confocal microscopy with a 488/680 nm excitation/emission filter. On the other hand, the cells were collected to measure the intracellular concentration of UA-PSiNPs in cells on the glass slides quantitatively by flow cytometry analysis with argon laser excitation at 488 nm and fluorescence (FL4) detection. 2.13. Drug Loading into UA-PSiNPs. Loading of the model anticancer drug DOX into UA-PSiNPs was performed by putting the nanoparticles in DOX solution at a weight ratio of 10:3 and stirring at room temperature for 12 h. DOX-loaded nanoparticles (DOX/UAPSiNPs) were collected by centrifugation at 8000 rpm for 10 min and then gently rinsed twice with ultrapure water to remove free DOX. The amount of DOX loaded into UA-PSiNPs was measured by completely dissolving DOX/UA-PSiNPs in 1 M KOH for 10 min, followed by neutralization with an equal volume of 1 M HCl and then reading at 480 nm using UV−vis absorption spectroscopy.35 2.14. Penetration of DOX/UA-PSiNPs into Three-Dimensional (3D) Tumor Spheroids. HepG2 tumor spheroids were constructed as described.39 Briefly, the fibrinogen/cell mixtures were obtained by blending 2 mg/mL fibrinogen with the same volume of cell solution (2 × 103 cells/mL). 250 μL mixtures were loaded into each well of the 24 well plates preadded with 5 μL of thrombin (0.1 U/μL). The tumor spheroids were left to grow until they achieved a diameter of about 150− 200 μm, which took 5 days. To detect the migration of DOX/UAPSiNPs in tumor spheroids, the spheroids were treated with free DOX or DOX/UA-PSiNPs at the final DOX concentration of 10 μg/mL for 24 h, respectively. The spheroids were rinsed with PBS, fixed with 4% paraformaldehyde for 30 min, and then transferred to confocal dishes. DOX fluorescence was detected using Z-stack imaging with 5 μm intervals from the top of the spheroids by confocal microscopy with a 488/560 nm excitation/emission filter. 2.15. Tumor Penetration of Intratumorally Administered DOX/UA-PSiNPs. Male BALB/c mice (18−20 g) were obtained from the Center for Disease Control and Prevention in Hubei Province, China. All animal experiments were performed under the guidance approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). H22 cells (2 × 106 cells/mouse) were injected subcutaneously into the flanks of BALB/c mice to construct the tumor burden mice. When tumor volume reached around 250 mm3, the mice were injected intratumorally with free DOX or DOX/UA-PSiNPs at the final DOX concentration of 1 mg/kg at about 2 mm depth under the tumor surface, respectively. At 24 h after administration, the tumors were removed, rinsed with PBS, and then frozen-sectioned at different layers. The nuclei of tumor cells were labeled with 5 μg/mL Hoechst 33258 for 30 min. The fluorescence of DOX and Hoechst 33258 in the tumor sections was detected by confocal microscopy with 488/560 nm and 405/450 nm excitation/emission filters, respectively. The tumor section with the largest fluorescence was defined as the 0 μm location. 2.16. Tumor Penetration of Intravenously Administered DOX/UA-PSiNPs. When tumor volume reached around 250 mm3, the tumor burden mice were injected intravenously with free DOX or DOX/UA-PSiNPs at the final DOX concentration of 7.5 mg/kg, respectively. After 24 h administration, the mice were executed and the tumors were resected, washed by PBS, and then frozen-sectioned. The sections were labeled with FITC-CD31 antibody (Abcam, Cambridge, UK) at 37 °C for 30 min, rinsed with PBS, and then observed by confocal microscopy. The fluorescence of DOX and FITC-CD31 was observed using 559/600 nm and 488/520 nm excitation/emission filters, respectively. The distribution of DOX fluorescence from the blood vessel to the tumor tissues on the specified line was evaluated by ImageJ software. On the other hand, the distance between DOX and vessels was determined by the method based on simulated scatter

different media including PBS, RPMI 1640 medium, and ultrapure water for different time courses. The supernatants were filtered using a centrifugal filter (30 000 Da, BioSharp, Hefei, China) to remove undissolved PSiNPs and UA-PSiNPs, and then the Si concentration was measured. 2.5. Cellular Accumulation of UA-PSiNPs. Bel7402 cells were seeded in 6-well plates at a density of 2 × 105 cells per well overnight. The cells were incubated with different amounts of UA-PSiNPs for 2 h, then rinsed with PBS, and collected to analyze the intracellular fluorescence of UA-PSiNPs by flow cytometry (FC500, Beckman Coulter, Fullerton, CA, USA) with argon laser excitation at 488 nm and fluorescence (FL4) detection. 2.6. Endocytic Pathway of UA-PSiNPs. Bel7402 cells were pretreated with 10 μg/mL CPZ, 20 μM EIPA, 80 μM dynasore, or 50 μM nystatin for 1 h, respectively. The cells were then treated with 75 μg/ mL UA-PSiNPs with or without the above inhibitors for an additional 2 h. The cells were rinsed with PBS and collected for flow cytometric analysis with argon laser excitation at 488 nm and fluorescence (FL4) detection. 2.7. Intracellular Localization of UA-PSiNPs. Bel7402 cells were incubated in a serum-free RPMI 1640 medium containing 75 μg/mL UA-PSiNPs for 2 h. The cells were rinsed with PBS and then dyed with 50 nM LysoTracker Green, 5 μM Golgi-Tracker Green, 5 μg/mL DAPI, or 3 μM DiO for 10 min, respectively. The cells were detected by an Olympus FV1000 confocal microscope (Tokyo, Japan). The fluorescence of LysoTracker Green, Golgi-Tracker Green, and DiO was observed using a 488/520 nm excitation/emission filter, and the fluorescence of DAPI was observed using a 405/450 nm excitation/ emission filter, respectively. 2.8. Intracellular Retention of UA-PSiNPs. Bel7402 cells were incubated in serum-free RPMI 1640 medium containing 75 μg/mL UAPSiNPs for 2 h. The cells were rinsed by PBS and then maintained in fresh medium. At the designated time points, the cells were rinsed by PBS and harvested for flow cytometric analysis with argon laser excitation at 488 nm and fluorescence (FL4) detection. 2.9. β-Hexosaminidase Assay. Bel7402 cells were incubated in serum-free RPMI 1640 medium containing 75 μg/mL UA-PSiNPs for 2 h. After rinsing by PBS, the cells were incubated in fresh medium with or without chemical reagents (10 μg/mL cytochalasin D, 40 μM nocodazole, or 10 μM ionomycin) for 2 h. The cells were rinsed by cold PBS and lysed in an ice bath for 1 h. The β-hexosaminidase assay was carried out by blending 75 μL of cell lysates with 50 μL of 4nitrophenyl N-acetyl-β-D-galactosaminide (2 mg/mL, pH 4.5), followed by incubation for 1 h at 37 °C. Then, 100 μL of borate buffer (0.2 M, pH 9.8) was used to end the reaction. The absorbance at 405 nm was measured using a 318C plate reader (SANKO, China).27 2.10. Distribution of Lysosome Compartment and LysosomeAssociated Membrane Protein 1 (LAMP1) after Exocytosis. The cells were treated with 75 μg/mL UA-PSiNPs for 2 h. After rinsing by PBS, the cells were maintained in fresh RPMI 1640 medium for an additional 2 h. For observation of the distribution of lysosomes, the cells were labeled with 50 nM LysoTracker Green for 10 min and visualized using a confocal microscope. For observation of LAMP-1 distribution, the cells were fixed with 4% paraformaldehyde and then incubated with 6 μg/mL anti-LAMP1 antibody conjugated with Alexa Fluor-488 (Boster Biological Technology, Ltd., Wuhan, China) for 2 h. The cells were rinsed by PBS and then observed by confocal microscopy. The fluorescence of LysoTracker Green and Alexa Fluor-488 was observed using a 488/520 nm excitation/emission filter, and the fluorescence of UA-PSiNPs was observed using a 488/680 nm excitation/emission filter, respectively. 2.11. Real-Time Tracking of UA-PSiNPs during Exocytosis. Bel7402 cells were treated with 75 μg/mL UA-PSiNPs for 2 h. After rinsing by PBS, the cells were stained with 50 nM LysoTracker Green for 10 min. The cells were then maintained in fresh RPMI 1640 medium for additional time courses. The fluorescence images of UA-PSiNPs and lysosomes in Bel7402 cells were acquired using a confocal microscope every 60 s. The fluorescence of LysoTracker Green and UA-PSiNPs was observed using 488/520 and 488/680 nm excitation/emission filters, respectively. C


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Figure 2. Synthesis and characterization of the luminescent PSiNPs and UA-PSiNPs. (A) The flowchart of preparation of the luminescent PSiNPs and UA-PSiNPs. (B) The FTIR spectra of PSiNPs and UA-PSiNPs. (C) The XPS spectra of PSiNPs and UA-PSiNPs. (D) The fluorescent spectra of PSiNPs and UA-PSiNPs at 488 nm excitation. (E) In vitro photostability of PSiNPs and UA-PSiNPs after exposure to a 100 W mercury lamp for different time courses. (F) The degradation behavior of PSiNPs and UA-PSiNPs in PBS, RPMI 1640 medium, and ultrapure water at 37 °C, respectively. diagrams.40 Briefly, the simulated scatter diagrams were created on the basis of the fluorescence images, in which the red dots represented DOX, the green dots represented the vessels, and the yellow dots represented the overlay of DOX and the vessels. The coordinate (X, Y) of each dot was addressed by the Rectangular Plane Coordinate System. The minimum range between each red dot and the closest green dot was gauged using the equation of

base (10 mM) was used to solubilize the bound stain. Absorbance at 540 nm was detected using a Labsystems iEMS microplate reader (Helsinki, Finland). 2.19. In Vivo Antitumor Effects of DOX/UA-PSiNPs. When tumor volume reached around 250 mm3, the tumor burden mice were administered intravenously with free DOX, DOX/PSiNPs, or DOX/ UA-PSiNPs at the dose of 7.5 mg/kg at days 1, 5, and 9, respectively. The size of the tumors was assessed every 2 days. On day 11, all the mice were executed, and the hearts and tumors were removed. The hearts were fixed with 4% paraformaldehyde, sectioned, and stained with hematoxylin and eosin (H&E). The tumors were weighed, fixed with 4% paraformaldehyde, sectioned, and stained using a TUNEL assay kit (Roche, Mannheim, Germany) according to the manufacturer’s protocol and imaged by an Olympus IX 71 optical microscope (Hamburg, Germany). The apoptotic rates were quantified using Image-pro plus 6.0 software. 2.20. Statistical Analysis. Experiments were repeated at least three times. Data were analyzed by Student’s t test, and P < 0.05 was regarded as statistically significant.

D = (X red − Xgreen)2 + (Yred − Ygreen)2 , and the mean value was defined as the distance of DOX and vessels. 2.17. Biodistribution of Intravenously Administered DOX/ UA-PSiNPs. When tumor volume reached around 250 mm3, the tumor burden mice were administered intravenously of free DOX or DOX/ UA-PSiNPs at the final DOX concentration of 7.5 mg/kg, respectively. After 24 h administration, the mice were executed and the tumors were removed. DOX content in the tumors was measured by lysing the tumor tissues and extracting free DOX by incubating 500 μL lysates in 500 μL KOH (1 M) for 10 min, followed by neutralization with the same volume of 1 M HCl. DOX concentration in the lysates was measured using a FlexStation3Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). 2.18. Cell Cytotoxicity Assay. Bel7402 cells were incubated with UA-PSiNPs or DOX/UA-PSiNPs at the indicated concentrations for 48 h. After rinsing with PBS, the cells were dyed with 200 μL of SRB (0.1%) for 15 min and then washed with 1% acetic acid to remove the unbound SRB. The plates were dried at room temperature, and 200 μL of TRIS

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of the Luminescent UA-PSiNPs. PSiNPs were constructed by electrochemical etching of silicon wafers, stripping porous silicon film, ultrasonication, centrifugation, and finally activation of luminescence D


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ACS Applied Materials & Interfaces by heating in an aqueous solution.35,36 UA-PSiNPs were synthesized by covalently conjugating undecylenic acid to the activated PSiNPs via a thermal hydrosilylation reaction (Figure 2A).37,38 FTIR and XPS analysis confirmed that undecylenic acid was successfully conjugated to PSiNPs (Figure 2B,C). The mean hydrodynamic diameter of PSiNPs and UA-PSiNPs was about 120 and 200 nm, respectively, and their zeta potentials were about −6 and −12 mV, respectively. To clarify whether the conjugation of undecylenic acid affected the characters of PSiNPs, the photoluminescence property of PSiNPs and UAPSiNPs was first determined using photoluminescence spectroscopy. As shown in Figure 2D, conjugation with undecylenic acid resulted in a small blueshift of the luminescence emission spectrum and decreased the photoluminescence intensity of PSiNPs nearly to 80%. Both PSiNPs and UA-PSiNPs showed great photostability (Figure 2E). Furthermore, the degradation characteristics of PSiNPs and UA-PSiNPs were evaluated in different solutions (Figure 2F). PSiNPs were stable in ultrapure water but degraded quickly in PBS buffer and cell culture medium, which might impede their biological application. In contrast, conjugation with undecylenic acid effectively improved the degradation of PSiNPs in PBS and cell culture medium. 3.2. Cellular Uptake and Intracellular Trafficking of UAPSiNPs. To explore the possible advantages of conjugating PSiNPs with undecylenic acid in the biomedical fields including drug delivery systems, the intracellular behavior of UA-PSiNPs in tumor cells was investigated. Considering that the degradation of UA-PSiNPs was accompanied by the decline in luminescence (Figure S1), we used the intracellular photoluminescence intensity to represent the intracellular accumulation of UAPSiNPs. As shown in Figure 3A, the intracellular fluorescence of UA-PSiNPs increased dose-dependently in human hepatocarcinoma Bel7402 cells. Furthermore, the endocytic pathways of UA-PSiNPs were determined using several specific endocytic inhibitors (Figure 3B).41,42 EIPA (an inhibitor of micropinocytosis), nystatin (an inhibitor of caveolae-dependent endocytosis), and CPZ (an inhibitor of clathrin-mediated endocytosis) markedly reduced the cellular uptake of UAPSiNPs. Dynasore, inhibiting dynamin GTPase activity to control macropinocytosis and caveolae- and clathrin-mediated endocytosis, resulted in a significant decrease in the internalization of UA-PSiNPs. These results indicated that macropinocytosis-, caveolae-, and clathrin-mediated endocytosis played an important role in the internalization of UA-PSiNPs in Bel7402 cells. To clarify the intracellular trafficking of UA-PSiNPs in Bel7402 cells after internalization, the overlay of UA-PSiNPs with cellular compartments, which were labeled with organellestargeted fluorescent dyes, was evaluated by confocal microscopy (Figure 3C). UA-PSiNPs did not overlay with DiO (cell membrane marker) and DAPI (nucleus marker) after a 2 h incubation. UA-PSiNPs were found to almost completely overlay with LysoTracker Green-labeled lysosomes and Golgi-Tracker Green-labeled Golgi apparatus, revealing that the internalized UA-PSiNPs were majorly delivered to lysosome and Golgi apparatus. 3.3. Lysosomal Exocytosis of UA-PSiNPs after Internalization. To further investigate the intracellular fate of UAPSiNPs, the following exocytosis of UA-PSiNPs after internalization was determined. Bel7402 cells pretreated with UAPSiNPs were rinsed thoroughly with PBS to remove free nanoparticles and then incubated in fresh nanoparticle-free medium for different time courses. Flow cytometry was

Figure 3. Endocytic pathways and intracellular location of UA-PSiNPs in Bel7402 cells. (A) Relative fluorescence intensity of UA-PSiNPs in Bel7402 cells treated with various concentrations for 2 h by flow cytometry. (B) Relative fluorescence intensity of UA-PSiNPs in Bel7402 cells preincubated with 10 μg/mL CPZ, 20 μM EIPA, 80 μM dynasore, or 50 μM nystatin, followed by treatment with 75 μg/mL UA-PSiNPs for 2 h by flow cytometry. (C) Intracellular localization of UA-PSiNPs in Bel7402 cells after treatment with 75 μg/mL UA-PSiNPs for 2 h by confocal microscopy. The cells were labeled with Lysotracker Green, Golgi-Tracker Green, DAPI, or DiO, respectively. Scale bar was 20 μm. Data were represented as mean ± SD (n = 3). *P < 0.05, **P < 0.01.

performed to measure the intracellular fluorescence of UAPSiNPs (Figure 4A). A remarkable reduction in intracellular fluorescence of UA-PSiNPs was found with increasing incubation time. Approximately half of the fluorescence intensity of UA-PSiNPs remained in cells after a 4 h incubation. To exclude that UA-PSiNPs were degraded inside Bel7402 cells, UA-PSiNPs were incubated in PBS at pH 4.5 (mimicking the physiological environment in lysosomes) or pH 7.4, and then, the degraded Si concentration was determined by ICP-OES. There was almost no degradation of UA-PSiNPs at pH 4.5 (Figure S2). These data suggested that the decline in intracellular fluorescence of UA-PSiNPs might be due to the exocytosis after internalization. It has been reported that, when the internalized nanoparticles colocalized with Golgi apparatus and lysosomes, they might either excrete via Golgi apparatus or go through lysosome exocytosis.27 To elucidate the exocytosis mechanisms of UAPSiNPs, the effects of Golgi apparatus blockers on the exocytosis of UA-PSiNPs were first determined.27 Treatment with Brefeldin A or monensin, which could collapse the Golgi apparatus to inhibit protein exocytosis, did not change the exocytosis of UAPSiNPs (Figure S3), suggesting that the Golgi apparatus was not involved in the exocytosis of UA-PSiNPs. To further investigate whether lysosomal exocytosis was involved in the excretion of UA-PSiNPs, the effects of lysosomal exocytosis promoters or inhibitors on intracellular accumulation of UA-PSiNPs were first E


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Figure 4. Exocytosis mechanism of UA-PSiNPs in Bel7402 cells. (A) Intracellular retention of UA-PSiNPs in Bel7402 cells treated with 75 μg/mL UAPSiNPs for 2 h, followed by incubation in fresh medium for various periods by flow cytometry. (B, C) The intracellular retention of UA-PSiNPs (B) and the percentages of released β-hexosaminidase (C) in Bel7402 cells treated with 75 μg/mL UA-PSiNPs for 2 h, followed by incubation in new medium with or without 10 μM ionomycin for 2 h. The intracellular fluorescence intensity of UA-PSiNPs and β-hexosaminidase activity in Bel7402 cells after treatment with 75 μg/mL UA-PSiNPs for 2 h was set at 100%, respectively. (D) The distribution of lysosomes in Bel7402 cells treated with 75 μg/mL UA-PSiNPs for 2 h. The right images are the magnification of the specified regions in the white box. Scale bars were 50 μm for the left images and 5 μm for the right images. (E) The distribution of LAMP1 protein in Bel7402 cells treated with 75 μg/mL UA-PSiNPs for 2 h. Scale bar was 50 μm. (F) The real-time intracellular tracking of UA-PSiNPs in Bel7402 cells treated with 75 μg/mL UA-PSiNPs for 2 h, followed by incubation in fresh medium for various periods. Scale bar was 10 μm. Data were represented as mean ± SD (n = 3). *P < 0.05, **P < 0.01.

determined in Bel7402 cells. Meanwhile, the release of βhexosaminidase into culture medium, a standard readout for lysosomal exocytosis, was further investigated.43,44 The exocytosis of UA-PSiNPs and the release of β-hexosaminidase were significantly enhanced in response to ionomycin, which transmits calcium into the cells to trigger the exocytosis process (Figure 4B,C).45 The polymerization of actin and microtubule is required to deliver lysosomes to the cell edge and fuse with cell membrane.46 Nocodazole and cytochalasin D, which inhibit the polymerization of microtubule and actin, respectively, were found to significantly decrease the exocytosis of UA-PSiNPs and the release of β-hexosaminidase (Figure S4A,B). These results indicated that lysosomal exocytosis might play an important role in the excretion of UA-PSiNPs. Lysosomal exocytosis undergoes two stages: lysosomes are transported to cell periphery followed by lysosomal fusion with cell membrane, during which LAMP1, a lysosomal membrane marker, is exposed on the cell surface. Therefore, the appearance of lysosomes and LAMP1 on the cell membrane is a hallmark of lysosomal exocytosis.43,44 To further confirm that UA-PSiNPs

were excreted through lysosomal exocytosis, the distribution of lysosomes and LAMP1 in Bel7402 cells treated with UA-PSiNPs was examined. As shown in Figure 4D, lysosomes were located in the cytoplasm in the control group. However, part of the lysosomes was translocated to the vicinity of the cells after treatment with UA-PSiNPs. Furthermore, using anti-LAMP1 antibody to identify LAMP1 and Dil to label cell membrane, LAMP1, usually existing on the inner edge of lysosomal membrane, was detected on the cell surface after treatment with UA-PSiNPs (Figure 4E). These data strongly confirmed that UA-PSiNPs underwent lysosomal exocytosis after internalization. The motions of endocytosed UA-PSiNPs within Bel7402 cells were traced by confocal microscopy after treatment with UAPSiNPs for 2 h, followed by incubation in nanoparticle-free fresh medium for different time courses. Lysosomes were labeled with LysoTracker Green to identify the localization of UA-PSiNPs. The overlay of UA-PSiNPs with lysosomes produced a yellow fluorescence in merged images. As shown in Figure 4F, the nanoparticles were first internalized into the cells and colocalized F


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Figure 5. Intercellular delivery of UA-PSiNPs in Bel7402 and HepG2 cells. (A) Confocal microscopy pictures of the successive transport of UA-PSiNPs from the infected Bel7402 cells or HepG2 cells to the untreated cells. Scale bar was 20 μm. (B) Relatvie intracellular fluorescence of UA-PSiNPs in the successively infected Bel7402 or HepG2 cells using flow cytometry. Data were represented as mean ± SD (n = 3).

Figure 6. Deep penetration of DOX/UA-PSiNPs in 3D tumor spheroids. (A) Z-stack images using confocal microscopy of DOX penetration into 3D HepG2 tumor spheroids treated with free DOX or DOX/UA-PSiNPs (eventual DOX concentration was 10 μg/mL) in the presence or absence of 10 μM ionomycin for 24 h, respectively. Scale bar was 75 μm. (B) Relative DOX fluorescence in each slice in the Z-stacks of 3D HepG2 tumor spheroids. Data were represented as mean ± SD (n = 3).

the size of UA-PSiNPs (Figure S5A) and the zeta potential of DOX/UA-PSiNPs changed to −6 mV. The DOX fluorescence property and intensity were not obviously influenced by UAPSiNPs (Figure S5B,C). Furthermore, the intercellular delivery of free DOX and DOX/UA-PSiNPs was evaluated in HepG2 cells and Bel7402 cells. Consistently, DOX/UA-PSiNPs were exocytosed to infect the adjacent cells more efficiently than free DOX (Figure S6A,B). Moreover, we found that the exocytosed DOX/UA-PSi nanoparticles could inhibit cell viability of the infected cells (Figure S6C). No cellular toxicity to Bel7402 cells was observed after treatment with UA-PSiNPs (Figure S7). These data indicated that UA-PSiNPs as drug carriers possessed strong domino-like intercellular delivery capability. 3.5. Deep Tumor Penetration of DOX/UA-PSiNPs. To clarify whether the intercellular delivery of UA-PSiNPs might result in enhanced tumor penetration, the tumor spheroids formed in 3D fibrin gel, as an in vivo-like tumor,47,48 were first employed to investigate the penetration of DOX/UA-PSiNPs. The tumor spheroids were treated with free DOX or DOX/UAPSiNPs for 24 h and then optically sectioned using confocal microscopy. A diagram of curves was made to show the relationship between the total DOX fluorescence in the Z-series of each slice and the distance from the top of the spheroids. As

with lysosomes. The nanoparticles/lysosomes complexes then moved toward the cell membrane and disappeared from cells. These results provided vivid evidence that UA-PSiNPs were excreted via lysosomal exocytosis after internalization. 3.4. Domino-Like Intercellular Delivery of UA-PSiNPs. To explore whether the exocytosis of UA-PSiNPs would affect the adjacent cells, the intercellular delivery of UA-PSiNPs was determined in Bel7402 cells and HepG2 cells. The cells on the first glass slide were pretreated with UA-PSiNPs for 2 h, rinsed with PBS, and then coincubated with new cells on the second glass slide for 10 h. The process was continued through coincubating cells on the second glass slide with the new cells on the third glass slide for 10 h. Confocal microscopy showed that UA-PSiNPs were internalized into the cells on the first coverslip. After the subsequent coincubation with the fresh cells, the intracellular fluorescence of UA-PSiNPs could also be detected in the infected cells on the second coverslip and even those on the third coverslip (Figure 5A). The quantitative outcomes from flow cytometry (Figure 5B) were in agreement with the qualitative one from confocal microscopy. DOX, as a model anticancer drug, was then loaded into UAPSiNPs (denonated as DOX/UA-PSiNPs, the loading efficiency of UA-PSiNPs was 7%). Loading of DOX did not markedly affect G


Research Article


Figure 7. Deep penetration capacity of DOX/UA-PSiNPs into tumor tissues of H22-bearing mice. (A) DOX penetration into the tumors of mice intratumorally injected with free DOX or DOX/UA-PSiNPs at a DOX concentration of 1 mg/kg for 24 h using confocal microscopy, respectively. Scale bar was 200 μm. (B) Relative DOX fluorescence in each tumor slice of H22-bearing mice as above. Data were represented as mean ± SD (n = 3). **P < 0.01. (C) DOX penetration into the tumors of mice intravenously injected with free DOX or DOX/UA-PSiNPs at DOX concentration of 7.5 mg/kg for 24 h using confocal microscopy, respectively. Scale bar was 200 μm. (D) Distribution profile of DOX from the blood vessel to the tumor tissues on the specified yellow line in H22-bearing mice as above.

shown in Figure 6A,B, more DOX/UA-PSiNPs were penetrated into the 3D tumor spheroids than free DOX. DOX/UA-PSiNPstreated spheroids exhibited a 2.5-fold increase in DOX fluorescence intensity compared with free DOX-treated spheroids with a maximum at 20 μm depth, respectively. Moreover, a clear DOX fluorescence was detected in the central region of DOX/UA-PSiNPs-treated spheroids even at 40 μm depth. In contrast, DOX fluorescence was only distributed at the edge of free DOX-treated tumor spheroids at 40 μm depth. In addition, ionomycin, which increased lysosomal exocytosis of UA-PSiNPs (Figure 4B), promoted their penetration in 3D tumor spheroids. These data suggested that UA-PSiNPs as drug carriers might exhibit strong penetration potential via lysosomal exocytosis. The deep tumor penetration effects of DOX/UA-PSiNPs were further investigated after intratumoral administration with free DOX or DOX/UA-PSiNPs into hepatocarcinoma H22-bearing mice at a fixed needle-insertion depth (Figure 7A,B). At 24 h after injection of DOX/UA-PSiNPs, the red DOX fluorescence was scattered in each tumor slice even at 700 μm depth under the injection point. However, only low DOX fluorescence was detected in free DOX-injected tumors at 300 μm depth. Furthermore, the tumor accumulation and penetration were determined by intravenous injection of free DOX or DOX/UAPSiNPs into H22-bearing mice. More DOX/UA-PSiNPs were accumulated in the tumor tissues than free DOX (Figure S8). Moreover, DOX/UA-PSiNPs were distributed further away from the tumor vessels than free DOX (Figure 7C) in view that their stronger merge with FITC-conjugated CD31, an endothelial cell marker. The distance-dependent DOX fluorescence intensity

also revealed the excellent tumor penetration of UA-PSiNPs. The penetration distance was roughly calculated by the method based on simulated scatter diagrams (Figure S9),40 and it was found that DOX/UA-PSiNPs penetrated outside the tumor vessels 4.1fold further than free DOX (Figure 7D). These data strongly revealed that UA-PSiNPs could penetrate deeply into tumor tissues, leading to higher DOX accumulation in the tumors. 3.6. Antitumor Activity of DOX/UA-PSiNPs. Considering the effective penetration of DOX/UA-PSiNPs into tumor tissues, the in vivo antitumor activity of DOX/UA-PSiNPs was determined (Figure 8A,B). As anticipated, DOX/UA-PSiNPs exhibited a significantly stronger inhibition in tumor growth compared with free DOX and DOX/PSiNPs. Furthermore, the cell apoptosis in tumor tissues was evaluated by TUNEL analysis (Figure 8C,D). Administration of DOX/UA-PSiNPs markedly augmented the number of TUNEL-positive tumor cells compared with free DOX and DOX/PSiNPs, indicating that the enhanced apoptosis in tumor cells might result in excellent antitumor efficiency of DOX/UA-PSiNPs. DOX/UA-PSiNPsinduced cardiotoxicity was evaluated on H&E-stained slides (Figure S10). Pronounced neutrophil gathering and myocardial necrosis were detected in heart sections of mice treated with free DOX, whereas DOX-evoked cardiotoxicity was significantly alleviated in DOX/UA-PSiNPs-treated mice.

4. CONCLUSIONS UA-PSiNPs exhibited good stability in physiological conditions. After internalization by macropinocytosis-, caveolae-, and clathrin-mediated endocytosis, UA-PSiNPs were excreted from cancer cells via lysosomal exocytosis pathway. The exocytosed H


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Figure 8. In vivo antitumor activity of DOX/UA-PSiNPs. (A) Tumor growth curves of H22-bearing mice after intravenous administration with free DOX, DOX/PSiNPs, or DOX/UA-PSiNPs at DOX concentration of 7.5 mg/kg, respectively. (B) Tumor weights of H22-bearing mice at the end of tumor growth inhibition experiments. (C) Representative photos of TUNEL staining assay for tumor tissues of H22-bearing mice at the end of tumor growth inhibition experiments. Scale bar was 200 μm. (D) Percentages of TUNEL-positive cells in tumor tissues of H22-bearing mice at the end of tumor growth inhibition experiments. Data were represented as mean ± SD (n = 6). *P < 0.05, **P < 0.01.

UA-PSiNPs could efficiently mediate domino-like intercellular migration of anticancer drugs, which resulted in deep penetration ability in both 3D tumor spheroids and in vivo tumors, thereby enhancing the therapeutic efficacy of anticancer drugs. This work indicated UA-PSiNPs as excellent anticancer drug carriers and provided new views to improve tumor penetration of anticancer drugs.

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in tumors of mice intravenously injected with free DOX or DOX/UA-PSiNPs; H&E stain of the heart in H22-bearing mice after intravenous injection with free DOX, DOX/ PSiNPs, or DOX/UA-PSiNPs (PDF)

AUTHOR INFORMATION

Corresponding Author

ASSOCIATED CONTENT

*E-mail: [email protected]. Tel: +86 27 87792147.

S Supporting Information *

Author Contributions

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b11127. The linear correction between the degradation and fluorescence intensity of UA-PSiNPs; the degradation behavior of UA-PSiNPs in PBS at different pH values; the effects of Brefeldin A or monensin on intracellular retention of UA-PSiNPs; the effects of cytochalasin D or nocodazole on intracellular retention of UA-PSiNPs and the percentages of released β-hexosaminidase; the characterization of DOX/UA-PSiNPs; the intercellular delivery of DOX/UA-PSiNPs; the cell viability of Bel7402 cells treated with different concentrations of UA-PSiNPs for 48 h; the biodistribution of DOX/UA-PSiNPs in tumors of mice; the localization of DOX and blood vessels

§

T.Y. and J.H. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

This work was supported by National Basic Research Program of China (973 Programs, 2012CB932500 and 2015CB931802) and the National Natural Science Foundation of China (81372400, 81672937, 81473171, and 81201193). We thank the Analytical and Testing Center of Huazhong University of Science and Technology for related analysis. I


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Domino-Like Intercellular Delivery of Undecylenic Acid-Conjugated

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