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Macrophage-Mediated Exocytosis of Elongated Nanoparticles Improves Hepatic Excretion and Cancer Phototherapy Nuri Oh, Yongjoo Kim, Hee-Seok Kweon, Wang-Yuhl Oh, and Ji-Ho Park ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10302 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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ACS Applied Materials & Interfaces

Macrophage-Mediated Exocytosis of Elongated Nanoparticles Improves Hepatic Excretion and Cancer Phototherapy

Nuri Oh,a,c Yongjoo Kimb,c, Hee-Seok Kweond, Wang-Yuhl Ohb,c, and Ji-Ho Parka,c,*

a

c

Department of Bio and Brain Engineering, bDepartment of Mechanical Engineering, and

KAIST Institute for Health Science and Technology, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea, and dElectron Microscopy Research Center, Korea Basic Science Institute, Daejeon 34133, Republic of Korea

*Address correspondence to [email protected]

Keywords: cancer, exocytosis, gold nanoparticle, macrophage, phototherapy

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Abstract The introduction of nanoparticle-mediated delivery and therapy has revolutionized cancer treatment approaches. However, there has been limited success in clinical trials because current approaches have not simultaneously satisfied therapeutic efficacy and biosafety criteria to an adequate degree. Here we employ efficient macrophage-mediated exocytosis of elongated nanoparticles to facilitate their localization in tumor cells for cancer therapy and their transport to hepatocytes for hepatobiliary excretion. In vitro studies show that PEGylated high-aspect ratio gold nanoparticles exit macrophages more rapidly and remain in tumor cells longer, compared with low-aspect ratio and spherical nanoparticles. In tumors, high-aspect ratio nanoparticles tend to stay in tumor cells and escape from tumorassociated macrophages when they are taken up by those cells. In the liver, high-aspect ratio nanoparticles cleared by Kupffer cells mostly take the hepatobiliary excretion pathway through efficient Kupffer cell-hepatocyte transfer. Furthermore, we demonstrate that timedependent localization of elongated gold nanoparticles towards tumor cells in tumor tissues enhances the overall phototherapeutic outcome. Engineering nanoparticles to modulate their exocytosis provides a new approach to improve cancer nanomedicine and pave the way towards clinical translation.


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Introduction The application of nanoparticles to medicine has provided new approaches for cancer treatments.1-2 Systemically administered nanoparticles travel through the entire body and accumulate mainly in tumors and organs in the mononuclear phagocytic system (MPS), such as the liver and spleen.3-5 Tumor-specific leaky vasculature allows nanoparticles to accumulate within the tumor microenvironment and subsequently encounter various types of cells, including tumor and endothelial cells, fibroblasts, and macrophages. The size, shape, and surface chemistry of nanoparticles affect their interaction with certain type of cells, and thus affects their ability to deliver therapeutic functions.6-11 Although most of therapeutic nanoparticles have been engineered to target tumor cells directly, recent studies have reported that significant quantities of nanoparticles are non-specifically cleared by intratumoral macrophages due to intrinsic phagocytic properties,3, 11 which could affect the overall therapeutic outcome. In addition, this macrophage-mediated clearance occurs largely in the liver and spleen of the MPS,12-13 reducing the delivery efficiency of nanoparticles to the tumor region. Particularly, solid nanoparticles that are cleared by Kupffer cells (liver-resident macrophages) and retained long-term could induce inflammation and apoptosis in the liver.14 Thus, engineering of therapeutic nanoparticles to facilitate their exocytosis in intratumoral and liver-resident macrophages and relocate them in tumor cells and hepatocytes is needed to simultaneously improve the therapeutic outcome and biosafety. We previously demonstrated that a polyethylene glycol (PEG) coating facilitates exocytosis of gold nanoparticles in macrophages in vitro, regardless of size.15 However, there have been limited studies examining how nanoparticle shape influences exocytosis although it has been known that elongated nanoparticles are more efficient in terms of transport in tumor microenvironments and targeting of tumor cells compared to spherical nanoparticles.6,



8

Here, we engineer the aspect ratio of PEGylated gold nanoparticles to facilitate their

exocytosis in tumor-associated and liver-resident macrophages. We also investigate the influence of aspect ratio on exocytosis of the nanoparticles in tumor cells. We then examine the influence of aspect ratio on time-dependent cellular distribution of the nanoparticles in liver and tumor tissues in vivo. Furthermore, we investigate how cancer phototherapy can benefit from macrophage-mediated exocytosis of the nanoparticles in tumor tissues.

Experimental Section Preparation of PEGylated Gold Nanoparticles with Various Aspect Ratios. Citrate- or cetyltrimethylammonium bromide (CTAB)-capped gold nanoparticles with average aspect ratios of 1, 4, and 7 were synthesized based on the methods of previously published papers.15-17 Gold nanoparticles with an aspect ratio of ~1 were synthesized with a previously published Turkevich method.16 The solution containing the synthetized nanoparticles was then dialyzed using a dialysis membrane (3.5 kDa MWCO, Spectrum Labs) to remove free citrate ions. Gold nanoparticles with an aspect ratio of ~ 4 were synthesize with a previously published seed-mediated growth method.15 Gold nanoparticles with an aspect ratio of ~ 7 were synthesized with a previously published seed-mediated growth method.17 For a longer aspect ratio, sodium oleate and hydrogen chloride were particularly added to the seed solution. The solution was vigorously stirred for 1 min and left undisturbed at 37°C for 12 h. The synthesized nanoparticles were washed by centrifugation at 6,500 rpm for 20 min, and re-suspended in ultra-pure water. For PEGylation, 900-µMAu gold nanoparticles were mixed with 25 mg/mL of mPEG (5K)-SH. The mixture was vortexed overnight at room temperature and then centrifuged at each optimal condition to remove free organic chemicals. For fluorescent labeling, 900-µMAu gold nanoparticles were mixed with


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20 mg/mL of mPEG (5K)-SH and 5 mg/mL of NH2- PEG (5K)-SH. The mixture was vortexed overnight at room temperature and then dialyzed against ultrapure water for 48 h to remove free organic chemicals. 900-µMAu amine-terminated PEGylated gold nanoparticles were mixed with 5 mg/mL of Alexa Fluor® 555 Succinimidyl Ester (AF555, Thermo Fisher Scientific, USA). The mixture was vortexed overnight at room temperature and centrifuged at each optimal condition two times to remove free dyes. Characterization of Various Aspect Ratio Nanoparticles. The absorption and fluorescence spectra were characterized with an ultraviolet-visible (UV-vis) spectrophotometer and spectrofluorometer (Molecular Devices, USA), respectively. The excitation wavelength for fluorescence measurements was 520 nm. The physical size and morphology were examined by using a field emission transmission electron microscope (FETEM, JEOL). The hydrodynamic size and surface charge were examined at 25°C by using a dynamic light scattering instrument (Malvern Panalytical, UK). The surface chemistry was analyzed with Fourier transform infrared (FTIR) spectroscopy (Thermo Fisher Scientific Instrument). Cell Culture. Murine breast cancer 4T1 cells (CRL-2539, ATCC) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 µg/mL penicillin and streptomycin in a 37°C humidified incubator (5% CO2, 95% air). Murine macrophage RAW264.7 cells (TIB-71, ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 100 µg/mL penicillin and streptomycin in a 37°C humidified incubator (5% CO2, 95% air). For classically activated M1-polarized macrophages, RAW264.7 cells were treated with 100 ng/mL lipopolysaccharide (LPS, Sigma) for 24 h. For alternatively activated M2polarized macrophages, RAW264.7 cells were treated with 10 ng/mL of interleukin-4 (IL4, Sigma) for 24 h. To confirm the macrophage polarization, the surface phenotype was ACS Paragon Plus Environment


analyzed by a flow cytometer (BD Bioscience, USA). Macrophages totaling 1 × 106 cells were incubated for 30 min with Alexa Fluor® 647-conjugated anti-mouse F4/80 Antibody (macrophage surface marker, 1:200 dilution in phosphate buffered saline (PBS) containing 1% FBS, Clone BM8, Biolegend, USA) and FITC-conjugated anti-mouse CD86 (M1 surface marker, 1:400 dilution in PBS containing 1% FBS, Clone GL1, TONBOBio, USA) or FITCconjugated anti-mouse CD206 (M2 surface marker, 1:400 dilution in PBS containing 1% FBS, Clone MR5D3, Thermo Fisher Scientific, USA). The cells were washed with fluorescence-activated cell sorting (FACS) buffer (PBS containing 1% FBS) to remove free antibodies and resuspended in the FACS buffer. In Vitro Endocytosis and Exocytosis Study. A total of 3 x 105 cells of M1/M2polarized RAW264.7 and 4T1 cells were seeded in 6-well plates with ∼90% confluence. The endocytosis and exocytosis studies were performed with previously published procedures.15 Cytokine Measurement. Raw 264.7 macrophages totaling 1 x 105 cells were seeded in 24-well plates and treated with LPS or IL4 for 24 h. After polarization, the macrophages were treated with 150-µMAu gold nanoparticles for 6 h, washed, and incubated in new fresh media for 18 h. The supernatants were collected and centrifuged at 10,000 g at 4°C to remove exocytosed nanoparticles and cell debris. The level of TNF-α and IL-6 in the supernatants was then measured by ELISA following the manufacturer’s protocols (Thermo Fisher Scientific, USA). β-hexosaminidase Assay. A total of 1 x 105 cells of Raw264.7 or 4T1 were seeded in 24-well plates with ∼90% confluence. Raw264.7 cells were treated with LPS or IL4 for 24 h for M1/M2 polarization. M1/M2 polarized Raw264.7 and 4T1 cells were treated with 150µMAu nanoparticles washed intensively, and further incubated for 48 h in fresh serumsupplemented media to induce the exocytosis of β-hexosaminidase enzymes. The


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supernatants were collected and centrifuged at 10,000 g at 4°C to remove exocytosed nanoparticles and cell debris. The level of β-hexosaminidase in the supernatants was then analyzed with a β-hexosaminidase assay ELISA kit (Elabscience, USA). Cytotoxicity Assay. A total of 1 x 104 cells of RAW264.7 or 4T1 were seeded in 96well plates with ∼90% confluence. Raw264.7 cells were differentiated to M1/M2 macrophages. M1/M2 polarized macrophages and 4T1 cells were treated with with 150µMAu nanoparticles for 6 h in serum-supplemented media at 37°C, washed intensively, and further incubated for 48 h in fresh serum-supplemented media. The cell viability was examined by the colorimetric thiazolyl blue tetrazolium bromide (MTT) assay (Sigma). Mouse Tumor Model. Seven-week-old female BALB/c wild-type mice were purchased from Koatech (South Korea); 1 × 106 green fluorescence protein (GFP)-tagged 4T1 cells were inoculated into the one of the fourth mammary fat pads of BALB/c mice. When the tumor volume reached an average size of ~ 50 mm3, the mice were randomized into groups for experiments. All animal procedures were performed in agreement with the guidelines and protocols for rodent research provided by the Institutional Animal Care and Use Committee of the Korea Advanced Institute of Science and Technology (IACUC approval number: KA2013-19). Blood Circulation and Organ Distribution. To measure the blood circulation, BALB/c wild-type mice were injected intravenously with fluorescently-labeled gold nanoparticles at an injection dose of 20 mgAu/kg. Blood (40 µL) was collected at 3 min, 2 h, 4 h, 8 h, and 24 h post-injection by using heparinized capillary tubes (Kimble Chase Life Science and Research Products), and immediately diluted with 40 µL of pH 7.4, 0.2% m/w ethylenediaminetetraacetic acid solution (EDTA, Junsei, 17385-0410) to prevent coagulation. The relative fluorescence intensity of the blood samples was measured with a spectrofluorometer. The blood half-life was calculated by fitting the fluorescence data to a



single-exponential equation using a one-compartment open pharmacokinetic model 18. To observe the time-dependent distribution in liver and tumor tissues, BALB/c wild type mice bearing orthotopic GFP-tagged 4T1 tumors were injected intravenously with fluorescentlylabeled gold nanoparticles at an injection dose of 20 mgAu/kg. At 1, 3, and 7 days postinjection, liver and tumor tissues were harvested after euthanasia and were fixed in 2.5 % glutaraldehyde (Sigma) for 24 h. For confocal microscopy, the tissues were embedded in paraffin, and cut into 10-µm-thick sections. Then, the sections were stained with Alexa Fluor® 647 anti-mouse F4/80 Antibody (1:200 dilution in PBS) and Hoechst 33342 (1:2000 dilution in PBS, Thermo Fisher Scientific, USA), and imaged by confocal microscopy (Nikon). For TEM, the tissues were incubated in 1% osmium tetroxide (OsO4, Sigma) plus 1.5% potassium ferrocyanide in a 0.1-M phosphate buffer (pH 7.3) for 1 h at 4°C in the dark and then embedded in Epon 812 after the they were dehydrated in an ethanolpopylene oxide series. The tissues were polymerized using pure resin at 70°C for 2 days. Finally, the tissue sections were sliced to a thickness of 70 nm, stained with 2% uranyl acetate for 15 min and imaged by TEM (Tecnai G2 Spirit, Thermo Fisher Scientific). Cellular Distribution in Tumors. BALB/c wild type mice bearing orthotopic GFPtagged 4T1 tumors were injected intravenously with fluorescently labeled gold nanoparticles at an injection dose of 20 mgAu/kg. At 1, 3, and 7 days post-injection, the tumors were cut from the skin to remove most of the tissues surrounding the tumor and then stored in a 6-well plate containing 2 mL/well of Roswell Park Memorial Institute (RPMI) medium on ice prior to digestion. The harvested tumors were cut into small pieces using scissors, then 1 mL of digestion medium [10 U/mL Collagenase I, 400 U/mL Collagenase IV, 30 U/mL DNAse I in Hanks’ Balanced Salt Solution (HBSS, Welgene, Korea)] was added to the same well, and the samples were incubated at 37°C for 25 min. The tumors were crushed with a syringe plunger and mixed with 5 mL of RPMI medium thoroughly by pipetting. The tumor


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suspension was filtered through a 70-µm sterile nylon mesh and centrifuged at 450 × g for 6 min at 4°C. After discarding the supernatant, the pellet was resuspended in 4 mL of erythrocyte lysis buffer and incubated for 2 min. After discarding the red blood cells, the suspension was added to 12 mL of RPMI medium for neutralization and filtered through a 70 µm sterile nylon mesh. The suspension was centrifuged at 450 × g for 6 min at 4°C and the supernatant was discarded. To isolate tumor-associated macrophages (TAMs), the pellet was resuspended in lymphoprep (Axis-shield, United Kingdom) at an approximate concentration of 1-2 × 107 cells/mL. The cell suspension was covered cautiously with 6 mL of RPMI medicum to obtain a two-phase gradient and then centrifuged at 800 g for 30 min at room temperature without acceleration or deceleration. The inter- and upper-phases containing RPMI medium were collected carefully. The inter-phase contained the living cells that were enriched with myeloid cells. The suspension was washed with a magnetic activated cell sorting (MACS) buffer and centrifuged at 800 × g for 5 min at 4°C. After discarding the supernatant and resuspending the pellet, the suspension was added with 5 µl aliquot of antiF4/80 magnetic microbeads (Miltenyi Biotec. Germany) and incubated for 20 min at 4°C on an orbital shaker at 50 rpm. The suspension was washed with MACS buffer and centrifuged at 450 × g for 6 min at 4°C. After discarding the supernatant and resuspending the pellet with 1 mL of MACS buffer, the suspension was placed on LS column in a MidiMACS separator attached to the MultiStand (Miltenyi Biotec. Germany). The macrophages were purified by eluting with 1 mL of MACS buffer. Intracellular concentration of gold nanoparticles in isolated macrophages was quantified by FACS and ICP-MS. For the FACS analysis, isolated macrophages were incubated with Alexa Fluor® 647 anti-mouse F4/80 Antibody (1:200) in 100 µL of FACS buffer (PBS containing 1% FBS) for 30 min. After attaching antibodies to the cells, the mixture was centrifuged at 1500 rpm for 3 min to remove free antibodies and resuspended in 300 µL of FACS buffer. The suspension was analyzed by flow cytometer (BD



Bioscience, USA). After isolation of the macrophages, a proportion of GFP-tagged tumor cells in the remaining cell pellet was analyzed by flow cytometry (Figure S1). This FACS result was used to quantify the intracellular concentration of gold nanoparticles in tumor cells by ICP-MS. For the FACS analysis, the cell suspension excluding macrophages was divided into GFP positive and GFP negative populations, and the intracellular concentration of gold nanoparticles in tumor cells was quantified with Alexa Fluor® 555 fluorescent intensity for the GFP-positive cell population. Cellular Distribution in the Liver. BALB/c wild-type mice bearing orthotopic 4T1 tumors were injected intravenously with fluorescently labeled gold nanoparticles at an injection dose of 20 mgAu/kg. At 1, 3, and 7 days post-injection, the livers were harvested after perfusion with HBSS (Welgene, Korea) containing 0.075% collagenase type І (Worthington Biochemical Co., USA) via the portal vein. The digested liver tissues were shaken for 5–10 min at 100 rpm at 37°C and then filtered with a 70-µm nylon mesh into a 50mL tube. The suspension was added with HBSS up to 50 mL and centrifuged 50 × g for 5 min at room temperature. The cell pellet was used for hepatocytes and the supernatant was used for additional isolation of Kupffer cells. Kupffer cells were purified using anti-F4/80 immunomagnetic beads as described in the purification of TAMs. After purification, intracellular concentrations of gold nanoparticles in isolated hepatocytes and Kupffer cells were quantified by FACS and ICP-MS. For the FACS analysis, isolated macrophages were labeled with Alexa Fluor® 647 anti-mouse F4/80 Antibody. In Vivo Phototherapy. A total of 1 × 106 GFP-tagged 4T1 cells were inoculated into the one of the fourth mammary fat pads of BALB/c mice. To prepare tumors with similar sizes when laser irradiation was performed, for 1 day post-injection, nanoparticles (NP7) were injected intravenously at an injection dose of 20 mgAu/kg 13 days after tumor inoculation when the 4T1 tumors reached an average size of ~ 100 mm3 (n = 6). For 7 days


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post-injection, nanoparticles (NP7) were injected intravenously at a dose of 20 mgAu/kg 7 days after tumor inoculation when the 4T1 tumors reached an average size of ~ 50 mm3 (n = 6). Both tumors had similar sizes of around 100 mm3 when laser irradiation was performed. At 1 and 7 d post-injection, the tumor was irradiated with a custom-built wavelength swept laser 19 for 1 h while maintaining the average tumor surface temperature at ~ 46°C (monitored by IR thermographic camera). The center wavelength was tuned from 1000 to 1080 nm, depending on the absorption characteristics of the nanoparticles. The bandwidth of the light source was set to 50 nm and the measured output power was 140 mW. The laser output was delivered to an optical lens (AC254-030-B, Thorlabs, USA) where the illumination beam was collimated through a single-mode optical fiber. The diameter of the illumination beam (1/݁ ଶ width) was 5.2 mm, resulting in an irradiance of 0.65 Wcm-2. The tumor volume was measured at 3-day intervals over a period of 3 weeks (n = 6). A caliper was used for measuring the width (short) and length (long) of the tumor, and the volume was calculated via the following equation, V = (width) × (width) × (length) / 2. Liver Toxicity. BALB/c wild-type mice bearing 4T1 tumors were injected intravenously with fluorescently-labeled gold nanoparticles at an injection dose of 20 mgAu/kg (n = 6). Blood (500 µL) was collected at 1 and 7 days post-injection by using heparinized capillary tubes (Kimble Chase Life Science and Research Products). The blood samples were left at room temperature to coagulate. Plasma was prepared by centrifugation of the blood at 15 000 × rpm for 20 min at 4°C. The plasma was stored at −70°C prior to analysis. Plasma albumin, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total bilirubin (TBIL), and aspartate transaminase (AST) levels were measured with a chemistry analyzer (AU480 Beckman Coulter, Japan). The plasma separated from the blood of BALB/c wild-type mice without tumors was used as a control.



Statistical Analysis. Data represent the mean ± standard deviation (SD). Statistical differences were analyzed by one-way analysis of variance followed by Tukey’s test or analyzed by two-way analysis of variance followed by the Bonferroni test using GraphPad Prism 5.0 (GraphPad software). P values lower than 0.05 were considered to be statistically significant.

Results and Discussion Preparation of Various Aspect Ratio Nanoparticles. We prepared PEGylated gold nanoparticles with different aspect ratios: 1 to 1 (NP1), 1 to 4 (NP4), and 1 to 7 (NP7), using previously established methods (Figure 1a and Table 1).16-17 The nanoparticles exhibited distinct light absorption behavior depending on the aspect ratio (Figure 1b), which indicates the plasmonic property of gold nanoparticles.20 As the aspect ratio increased, the light absorption peak shifted towards longer wavelengths. The nanoparticles showed similar surface chemistry after PEG coating although the original nanoparticles were synthesized with different chemical ligands (Figure S2). The PEGylated nanoparticles were conjugated with fluorescent dyes, with similar surface charge and fluorescence per mass of gold ion (Figure S3 and Table 1). Endocytosis and Exocytosis of Nanoparticles in Macrophages and Tumor Cells. Nanoparticles that enter the tumor tissue can be preferentially taken up by intratumoral macrophages and tumor cells, whereas those left in circulation are eventually cleared by liverresident macrophages due to immune responses. We first examined how the aspect ratio of PEGylated nanoparticles affects the endocytosis and exocytosis in both macrophages and tumor cells in vitro. RAW 264.7 murine macrophages were chosen as a tissue-resident macrophage model. In the liver, Kupffer cells are known to comprise a heterogeneous


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population of M1 (proinflammatory) and M2 (anti-inflammatory) macrophages,21 while tumor-associated macrophages (TAMs) are largely polarized to M2 macrophages.22 To mimic Kupffer cells (M1/M2) and TAMs (M2), RAW 264.7 cells were activated to simulate M1and M2 macrophages via treatment with lipopolysaccharide (LPS) and interleukin 4 (IL4),23 respectively (Figure S4). 4T1 breast cancer cells were chosen as a tumor cell model because TAMs in breast cancer are closely linked to metastasis and tumor aggressiveness 24-25, and are abundant in the orthotopic model.26-27 For the endocytosis study, the cells were treated with nanoparticles for 6 h to saturate the endocytosis process. For the exocytosis study, the cells were treated with nanoparticles for 6 h, washed extensively to remove weakly bound nanoparticles on the cell surface, and incubated for 48 h in nanoparticle-free media for collection of exocytosed nanoparticles in supernatants. The quantities of nanoparticles internalized in and released from the cells were determined by measuring the amount of gold ions in the cells. Elemental analysis of the cells within the tumor tissue revealed that macrophages took up PEGylated nanoparticles more efficiently than tumor cells regardless of aspect ratio, and in both cell types, cellular uptake of NP7 was more dominant that for other aspect ratios (Figures 1c and 1d). The exocytosis of PEGylated nanoparticles differs depending on the aspect ratio and cell type. NP7 left the macrophages more efficiently than the other nanoparticles, regardless of phenotype, but NP7 remained longer in the tumor cells (Figures 1e-g). Furthermore, we found that the exocytosis patterns of nanoparticles were similar to those of β-hexosaminidase, an enzyme that resides inside lysosomes, indicating that these nanoparticles exit macrophages and tumor cells mainly via lysosomal exocytosis (Figure S5). No significant cytotoxicity was observed in the macrophages and tumor cells treated with nanoparticles (Figure S6). Collectively, these results demonstrate that macrophages export high-aspect ratio nanoparticles more efficiently via lysosomal exocytosis



compared to low-aspect ratio and spherical nanoparticles, while tumor cells tend to retain high-aspect ratio nanoparticles for longer compared to low-aspect ratio nanoparticles. Immunological Responses of Macrophages to Nanoparticle Entry and Residence. We studied whether the aspect ratio of nanoparticles affects the immunological responses of macrophages in vitro. The immunological responses of macrophages to nanoparticle entry and residence were examined using the secretion of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). M1/M2-polarized macrophages were treated with nanoparticles for 6 h, then washed and incubated in fresh media for 18 h. Enzyme-linked immunosorbent assay (ELISA) measurements of the supernatants revealed that NP7-treated macrophages released significantly larger quantities of proinflammatory cytokines, regardless of phenotype, compared to NP1- and NP4-treated cells (Figures 2a and 2b). This result indicates that nanoparticle shape affects the immune responses of macrophages, which may promote lysosomal exocytosis of nanoparticles.28 Time-dependent Residence of Nanoparticles in Liver and Tumor Tissues. Having observed efficient exocytosis of high-aspect ratio nanoparticles in macrophages, we next examined whether such exocytosis can alter the time-dependent residence of nanoparticles in liver and tumor tissues. Mice bearing orthotopic 4T1 tumors were injected intravenously with nanoparticles and sacrificed at 1 and 7 days post-injection to quantify time-dependent residence of nanoparticles in liver and tumor tissues. Elemental analysis of the liver tissues showed that accumulations of elongated nanoparticles (NP4 and NP7) at 1 day post-injection were slightly lower and their excretions over a 7-day period were significantly more efficient than that of spherical nanoparticles (Figure 3a). From the tumor analysis, the majority of NP7 remained in tumor tissues over the 7-day period (Figure 3b). However, the amounts of NP1 and NP4 accumulated in tumor tissues significantly diminished over the 7-day period. No significant liver toxicity was observed over the period of 7 days following nanoparticle ACS Paragon Plus Environment

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injection (Figure S7). These observations demonstrate that the dynamic clearance mechanisms of high-aspect ratio nanoparticles in liver and tumor tissues seem to be somewhat different from that of their spherical counterparts. Importantly, these results suggest that PEGylated high-aspect ratio nanoparticles are favorable for both rapid hepatic excretion and long-term tumor retention. Time-dependent Cellular Distribution of Nanoparticles in Liver and Tumor Tissues. To further elucidate nanoparticle clearance mechanisms, we next investigated the time-dependent cellular distribution of nanoparticles in liver and tumor tissues. Mice bearing orthotopic 4T1 tumors were injected intravenously with fluorescently tagged nanoparticles, and were sacrificed at 1, 3, and 7 days post-injection to isolate Kupffer cells and hepatocytes from the liver, and macrophages and tumor cells from the tumor. Time-dependent accumulation of nanoparticles in the isolated cells was analyzed using ICP-MS and flow cytometry. Nanoparticle subcellular distribution in the tissues was also observed using confocal microscopy and transmission electron microscopy (TEM). ICP-MS measurements and flow cytometry analysis of liver tissues showed that the intracellular concentration of NP7 decreased dramatically in both hepatocytes and Kupffer cells over the 7-day period following injection while NP1 remained for a relatively long time in both cells (Figures 4a-d). NP4 showed similar excretion patterns to NP7 in both cell types, although the NP4 excretion efficiency was slightly lower than that of NP7. Confocal microscopy and TEM images reflected an efficient release of NP7 from Kupffer cells (Figure 4i and Figure S8). ICP-MS measurements and flow cytometry analysis of tumor tissues also showed that the intracellular concentration of NP7 in TAMs decreased dramatically over the tumor growth period of 7 days post-injection, while TAMs released intracellular NP1 inefficiently (Figures 4e and 4g). Unlike Kupffer cells, TAMs released intracellular NP1 and NP4 at similar rates. Contrary to the TAM results, the amount of NP7 resident in tumor cells at 7 days post-injection was



significantly higher than that of NP1 and NP4 (Figures 4f and 4h), indicating that NP7 tends to remain in tumor cells longer than NP1 and NP4, as observed in vitro in Figure 1g. Furthermore, this result implies that significant quantities of NP7 exocytosed from TAMs could be transferred to tumor cells, if we consider the increase of tumor cells during this period (Figure S9). Confocal microscope images also confirmed poor accumulation of NP7 in TAMs, but efficient localization of NP7 in tumor cells at 7 days post-injection (Figures 4j and 4k). These observations support the time-dependent change in nanoparticle residence in liver and tumor tissues, as observed in Figures 3a and 3b. Collectively, these results suggest that cellular distribution of PEGylated high-aspect ratio nanoparticles in liver and tumor tissues can be re-arranged over time via macrophage-mediated exocytosis, thus leading to efficient hepatobiliary excretion and tumor cell localization of these nanoparticles. Therapeutic Benefits of Macrophage-mediated Exocytosis of Nanoparticles for Cancer Phototherapy. Elongated gold nanoparticles have been widely used as a photothermal agent for cancer therapy because they can generate efficient local heat upon tissue-penetrating near-infared (NIR) light irradiation.29 Thus, we investigated whether the overall phototherapeutic effects of gold nanoparticles could be improved by taking advantage of their exocytosis-mediated re-distribution in tumor tissues. NP7 were injected intravenously into mice bearing orthotopic 4T1 tumors. To prepare tumors of similar sizes (~ 100 mm3) when laser irradiation was performed 1 and 7 days post-injection, NP7 was injected 13 days and 7 days after tumor cell inoculation, respectively (Figure 5a). The tumors were irradiated for 1 h at 1 and 7 days post-injection while the temperature change at the tumor surface was measured using an IR thermographic camera. Tumor volumes were measured over a 3-week period following laser irradiation. Thermographic measurements revealed that laser irradiation induced a similar temperature increase (~46°C) in both tumors at 1 and 7 days post-injection (Figure 5b). This observation indicates that the intratumoral concentration of


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nanoparticles determining the photothermal performance did not change significantly over the 7-day period, reflecting the NP7 results shown in Figure 3b. Interestingly, in the phototherapeutic study, a single irradiation at 7 days post-injection significantly delayed tumor growth over 3 weeks compared with irradiation at 1 day post-injection (Figure 5c). Tumors that were not exposed to laser irradiation or were not injected with nanoparticles prior to laser irradiation continued to grow without significant inhibition. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis using histological samples revealed laser irradiation at 7 days post-injection induced significantly greater apoptosis in the tumor tissues compared with irradiation at 1 day post-injection (Figure 5d), supporting the tumor growth inhibition result shown in Figure 5c. We believe that substantial localization of NP7 in tumor cells at 7 days post-injection led to these strong phototherapeutic effects because tumor cells are more sensitive to hyperthermia than normal cells.30 Thus, the aspect ratio of PEGylated gold nanoparticles and the irradiation timing after nanoparticle injection seem to be important to determine the efficacy of photothermal therapy. Taken together, these results suggest that efficient macrophage-mediated exocytosis of elongated nanoparticles and their subsequent localization in tumor cells can enhance the overall phototherapeutic benefit.

Conclusion This work demonstrated that macrophage-mediated exocytosis (a nature transport process in tissues) can be harnessed to facilitate nanoparticle transport to hepatocytes in the liver for hepatobiliary excretion and to tumor cells in tumors for phototherapy. We believe that this exocytosis-mediated delivery approach could be further utilized to improve tissue distribution of nanoparticles towards a favorable therapeutic outcome, particularly in poorly permeable



tissues. This work provides new insights into overcoming the challenges associated with tissue distribution and excretion of solid nanoparticles, paving the way towards clinical translation.

Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:

Author Information Corresponding Author *E-mail: [email protected]. Author Contributions N.O. and J.-H.P. conceived and designed the research. N.O., H.-S.K., and Y.K. carried out the experiments. N.O. and Y.K., H.-S.K., Y.-W.O., and J.-H.P. analyzed the data. N.O. and J.-H.P. wrote the manuscript. Notes The authors declare no competing financial interest.

Acknowledgments This work was supported by the Basic Science Research Programs through the National Research Foundation funded (NRF-2017R1E1A1A01074847) and the National Research


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Council of Science & Technology (NST) grant (CAP-14-03-KRISS), funded by the Ministry of Science and ICT, Republic of Korea. Authors thank Dr. Hyo-Won Seo and Prof. Won-Il Jeong for their assistance on preparation of liver-associated cells.

References (1) Chow, E. K.-H.; Ho, D. Cancer Nanomedicine: From Drug Delivery to Imaging. Sci. Transl. Med. 2013, 5 (216), 216rv4. (2) Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Cancer Nanomedicine: Progress, Challenges and Opportunities. Nat. Rev. Cancer 2017, 17 (1), 20-37. (3) Miller, M. A.; Gadde, S.; Pfirschke, C.; Engblom, C.; Sprachman, M. M.; Kohler, R. H.; Yang, K. S.; Laughney, A. M.; Wojtkiewicz, G.; Kamaly, N.; Bhonagiri, S.; Pittet, M. J.; Farokhzad, O. C.; Weissleder, R. Predicting Therapeutic Nanomedicine Efficacy Using a Companion Magnetic Resonance Imaging Nanoparticle. Sci. Transl. Med. 2015, 7 (314), 314ra183. (4) Alidori, S.; Bowman, R. L.; Yarilin, D.; Romin, Y.; Barlas, A.; Mulvey, J. J.; Fujisawa, S.; Xu, K.; Ruggiero, A.; Riabov, V.; Thorek, D. L.; Ulmert, H. D.; Brea, E. J.; Behling, K.; Kzhyshkowska, J.; Manova-Todorova, K.; Scheinberg, D. A.; McDevitt, M. R. Deconvoluting Hepatic Processing of Carbon Nanotubes. Nat. Commun. 2016, 7, 12343. (5) Zhang, Y.-N.; Poon, W.; Tavares, A. J.; McGilvray, I. D.; Chan, W. C. W. Nanoparticle– Liver Interactions: Cellular Uptake and Hepatobiliary Elimination. J. Control. Release 2016, 240 (Supplement C), 332-348. (6) Park, J.-H.; von Maltzahn, G.; Zhang, L.; Schwartz, M. P.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. Magnetic Iron Oxide Nanoworms for Tumor Targeting and Imaging. Adv. Mater. 2008, 20 (9), 1630-1635.



(7) Choi, C. H.; Alabi, C. A.; Webster, P.; Davis, M. E. Mechanism of Active Targeting in Solid Tumors with Transferrin-Containing Gold Nanoparticles. Proc. Natl. Acad. Sci. USA 2010, 107 (3), 1235-1240. (8) Chauhan, V. P.; Popović, Z.; Chen, O.; Cui, J.; Fukumura, D.; Bawendi, M. G.; Jain, R. K. Fluorescent Nanorods and Nanospheres for Real-Time In Vivo Probing of Nanoparticle Shape-Dependent Tumor Penetration. Angew. Chem. Int. Ed. 2011, 50 (48), 11417-11420. (9) Cieslewicz, M.; Tang, J.; Yu, J. L.; Cao, H.; Zavaljevski, M.; Motoyama, K.; Lieber, A.; Raines, E. W.; Pun, S. H. Targeted Delivery of Proapoptotic Peptides to Tumor-Associated Macrophages Improves Survival. Proc. Natl. Acad. Sci. USA 2013, 110 (40), 15919-15924. (10) Sykes, E. A.; Chen, J.; Zheng, G.; Chan, W. C. Investigating the Impact of Nanoparticle Size on Active and Passive Tumor Targeting Efficiency. ACS Nano 2014, 8 (6), 5696-5706. (11) Miller, M. A.; Zheng, Y. R.; Gadde, S.; Pfirschke, C.; Zope, H.; Engblom, C.; Kohler, R. H.; Iwamoto, Y.; Yang, K. S.; Askevold, B.; Kolishetti, N.; Pittet, M.; Lippard, S. J.; Farokhzad, O. C.; Weissleder, R. Tumour-Associated Macrophages Act as a Slow-Release Reservoir of Nano-Therapeutic Pt(IV) Pro-Drug. Nat. Commun. 2015, 6, 8692. (12) Tsoi, K. M.; MacParland, S. A.; Ma, X. Z.; Spetzler, V. N.; Echeverri, J.; Ouyang, B.; Fadel, S. M.; Sykes, E. A.; Goldaracena, N.; Kaths, J. M.; Conneely, J. B.; Alman, B. A.; Selzner, M.; Ostrowski, M. A.; Adeyi, O. A.; Zilman, A.; McGilvray, I. D.; Chan, W. C. Mechanism of Hard-Nanomaterial Clearance by the Liver. Nat. Mater. 2016, 15 (11), 12121221. (13) MacParland, S. A.; Tsoi, K. M.; Ouyang, B.; Ma, X.-Z.; Manuel, J.; Fawaz, A.; Ostrowski, M. A.; Alman, B. A.; Zilman, A.; Chan, W. C. W.; McGilvray, I. D. Phenotype Determines Nanoparticle Uptake by Human Macrophages from Liver and Blood. ACS Nano 2017, 11 (3), 2428-2443.


Page 20 of 31

Page 21 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(14) Cho, W. S.; Cho, M.; Jeong, J.; Choi, M.; Cho, H. Y.; Han, B. S.; Kim, S. H.; Kim, H. O.; Lim, Y. T.; Chung, B. H.; Jeong, J. Acute Toxicity and Pharmacokinetics of 13 nm-Sized PEG-Coated Gold Nanoparticles. Toxicol. Appl. Pharmacol. 2009, 236 (1), 16-24. (15) Oh, N.; Park, J. H. Surface Chemistry of Gold Nanoparticles Mediates their Exocytosis in Macrophages. ACS Nano 2014, 8 (6), 6232-6241. (16) Kimling, J.; Maier, M.; Okenve, B.; Kotaidis, V.; Ballot, H.; Plech, A. Turkevich Method for Gold Nanoparticle Synthesis Revisited. J. Phys. Chem. B 2006, 110 (32), 1570015707. (17) Ye, X.; Zheng, C.; Chen, J.; Gao, Y.; Murray, C. B. Using Binary Surfactant Mixtures to Simultaneously Improve the Dimensional Tunability and Monodispersity in the Seeded Growth of Gold Nanorods. Nano Lett. 2013, 13 (2), 765-771. (18) Wunderbaldinger, P.; Josephson, L.; Weissleder, R. Tat Peptide Directs Enhanced Clearance and Hepatic Permeability of Magnetic Nanoparticles. Bioconjugate Chem. 2002, 13 (2), 264-268. (19) Park, J. R.; Choi, W.; Hong, H. K.; Kim, Y.; Park, S. J.; Hwang, Y.; Kim, P.; Woo, S. J.; Park, K. H.; Oh, W.-Y. Imaging Laser-Induced Choroidal Neovascularization in the Rodent Retina Using Optical Coherence Tomography Angiography. Invest. Ophthalmol. Vis. Sci. 2016, 57 (9), OCT331-40. (20) Dreaden, E. C.; Alkilany, A. M.; Huang, X.; Murphy, C. J.; El-Sayed, M. A. The Golden Age: Gold Nanoparticles for Biomedicine. Chem. Soc. Rev. 2012, 41 (7), 2740-2779. (21) Wan, J.; Benkdane, M.; Teixeira-Clerc, F.; Bonnafous, S.; Louvet, A.; Lafdil, F.; Pecker, F.; Tran, A.; Gual, P.; Mallat, A.; Lotersztajn, S.; Pavoine, C. M2 Kupffer Cells Promote M1 Kupffer Cell Apoptosis: A Protective Mechanism against Alcoholic and Nonalcoholic Fatty Liver Disease. Hepatology 2014, 59 (1), 130-142.



(22) De Palma, M.; Lewis, Claire E. Macrophage Regulation of Tumor Responses to Anticancer Therapies. Cancer Cell 2013, 23 (3), 277-286. (23) Wang, Y.-C.; He, F.; Feng, F.; Liu, X.-W.; Dong, G.-Y.; Qin, H.-Y.; Hu, X.-B.; Zheng, M.-H.; Liang, L.; Feng, L.; Liang, Y.-M.; Han, H. Notch Signaling Determines the M1 versus M2 Polarization of Macrophages in Antitumor Immune Responses. Cancer Res. 2010, 70 (12), 4840-4849. (24) Obeid, E.; Nanda, R.; Fu, Y.-X.; Olopade, O. I. The Role of Tumor-Associated Macrophages in Breast Cancer Progression. Int. J. Oncol. 2013, 43 (1), 5-12. (25) Yang, J.; Li, X.; Liu, X.; Liu, Y. The Role of Tumor-Associated Macrophages in Breast Carcinoma Invasion and Metastasis. Int. J. Clin. Exp. Pathol. 2015, 8 (6), 6656-6664. (26) Luo, Y.; Zhou, H.; Krueger, J.; xF; rg; Kaplan, C.; Lee, S.-H.; Dolman, C.; Markowitz, D.; Wu, W.; Liu, C.; Reisfeld, R. A.; Xiang, R. Targeting Tumor-Associated Macrophages as a Novel Strategy against Breast Cancer. J. Clin. Invest. 2006, 116 (8), 2132-2141. (27) Sun, X.; Gao, D.; Gao, L.; Zhang, C.; Yu, X.; Jia, B.; Wang, F.; Liu, Z. Molecular Imaging of Tumor-Infiltrating Macrophages in a Preclinical Mouse Model of Breast Cancer. Theranostics 2015, 5 (6), 597-608. (28) Blott, E. J.; Griffiths, G. M. Secretory Lysosomes. Nat. Rev. Mol. Cell Biol. 2002, 3, 122-131. (29) von Maltzahn, G.; Park, J.-H.; Agrawal, A.; Bandaru, N. K.; Das, S. K.; Sailor, M. J.; Bhatia, S. N. Computationally Guided Photothermal Tumor Therapy Using Long-Circulating Gold Nanorod Antennas. Cancer Res. 2009, 69 (9), 3892-3900. (30) Renato, C.; C., C. E.; C., G. B.; Charles, H.; O., J. R.; Mario, M.; Bruno, M.; Guido, M.; Alessandro, R.-F. Selective Heat Sensitivity of Cancer Cells. Biochemical and Clinical Studies. Cancer 1967, 20 (9), 1351-1381.


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Figure 1. Endocytosis and exocytosis of nanoparticles in macrophages and tumor cells. (a) Transmission electron microscopy images of PEGylated gold nanoparticles with various aspect ratios. NP1, NP4, and NP7 represent PEGylated gold nanoparticles with aspect ratios of 1, 4, and 7, respectively. The scale bar indicates 50 nm. (b) Absorption spectra of nanoparticles. (c and d) Quantity of nanoparticles taken up by macrophages (c) and tumor cells (d) after 6-h incubation. M1 and M2 indicate classically and alternatively activated macrophages, respectively. (e, f and g) Exocytosis rate of nanoparticles in M1 macrophages (e), M2 macrophages (f) and tumor cells (g) over a 48-h period. Data represent the mean ± standard deviation (SD) [n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001; one-way analysis of variance followed by Tukey’s test for (c) and (d); two-way analysis of variance followed by the Bonferroni test for (e)–(g)].


a

b 500

NPNP1 ***

NP4 NP7

***

*

250

0

M1

M2

TNF-α secretion (pg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

IL-6 secretion (pg/mL)


1500 1000

Page 24 of 31

NPNP1 ***

*

500 0

NP4 NP7

M1

M2

Figure 2. Immunological response of macrophages to nanoparticle entry and residence. (a and b) Secretion of interleukin 6 (IL-6) (a) and tumor necrosis factor-alpha (TNF-α) (b) from macrophages treated with nanoparticles for 6 h and incubated in fresh media for 18 h. Data represent the mean ± standard deviation (SD) (n = 3, *p < 0.05 and ***p < 0.001; oneway analysis followed by Tukey’s test).


a NP in liver (mgAu/g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


400

b NP1

NP4

NP7

***

300 200

**

100 0

1d

NP in tumor (mgAu/g)

Page 25 of 31

40

NP4

NP1

NP7

*

30

NS

**

20 10 0

7d

1d

7d

Figure 3. Time-dependent residence of nanoparticles in liver and tumor tissues. (a and b) Accumulation of nanoparticles in liver tissues (a) and tumor tissues (b) at 1 and 7 days postinjection. Data represent the mean ± standard deviation (SD) (n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001; one-way analysis followed by Tukey’s test).


*

1 2 3 4 5 6 7 Time after NP injection (day)

0.0020 0.0015 0.0010 0.0005 0.0000


NP1

NP4

NP7

0.008 0.006

0.006 0.004

0


h


1 day

j

40 20 0


k

7 days

20 10 0

* 1 2 3 4 5 6 7 Time after NP injection (day)

NP1 NP4 NP7

60 40 20 0


1 day

7 days

NP1

1 day

NP1 NP4 NP7

60

NP1 NP4 NP7

30

*


7 days

20

*

0.002

*

0.000

NP1 NP4 NP7

0.008

0.000

40

g

0.010

*

0.002

NP1 NP4 NP7

*

0.004

NP in tumor cells (ngAu/cell)

f 0.010

d 60

% of NP+ hepatocytes

0.000

NP1 NP4 NP7

% of NP+ TAM

NP in hepatocytes (ngAu/cell)

NP in Kupffer cells (ngAu/cell)

0.005

c 0.0025

*

0.010

*

NP in TAM (ngAu/cell)

0.015

*

i

NP1 NP4 NP7

***

e

0.020

Page 26 of 31

% of NP+ tumor cells

b

a

*

NP Kupffer cell

NP TAM

NP tumor cell

NP7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

% of NP+ Kupffer cells


Figure 4. Time-dependent cellular distribution of nanoparticles in liver and tumor tissues. (a and b) Intracellular concentrations of nanoparticles in Kupffer cells (a) and hepatocytes (b) in the liver over a 7-d period after intravenous injection. (c and d) Percentages of Kupffer cells (c) and hepatocytes (d) engulfing nanoparticles in liver over a 7day period after their intravenous injection. (e and f) Intracellular concentrations of nanoparticles in macrophages (e) and tumor cells (f) in the tumor over a 7-day period after intravenous injection. (g and h) Percentages of macrophages (g) and tumor cells (h) engulfing nanoparticles in the liver over a 7-day period after intravenous injection. (i) Confocal microscope images of NP1 and NP7 accumulated in Kupffer cells of liver tissues at 1 and 7 days post-injection. (j and k) Confocal microscope images of NP1 and NP7 accumulated in macrophages (j) and tumor cells (k) of tumor tissues at 1 and 7 days post-injection. Nanoparticles were labeled with fluorescent dye (red). Kupffer cells and macrophages were


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stained with fluorophore-labeled anti-F4/80 antibodies (green) and tumor cells were tagged with green fluorescent protein (GFP; green). The scale bars indicate 20 µm. Data represent the mean ± SD [n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001, two-way analysis of variance followed by the Bonferroni test].



a

NP7-L+ NP7(1d)+L+ NP7(7d)+L+

Tumor cell inoculation

NP7(7d) injection

c

50 45

NP7NP7(1d)+ NP7(7d)+

40 35 30

0

NP7-L+

10 20 Time (min)

30

NP7(1d)+L+

13

14

NP7(1d) Laser injection irradiation

600

NP7(7d)+ L+ NP7(1d)+ L+ NP7+ LNP7- L+ NP7- L-

400 200 0

*** 0

3

NP7(7d)+L+

6

9 12 15 18 21 Time (days) 15

***

10 5 0 NP NP7 7(1 -L+ NP d)+L 7(7 + d)+ L+

d

7

Relative fluorescence

b

0

Tumor volume (mm3)

day

Temperature (oC)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 31

Figure 5. Therapeutic benefits of macrophage-mediated exocytosis of elongated nanoparticles for cancer phototherapy. (a) Schematic of experimental procedure for cancer phototherapy. (b) Temperature profile of the tumors upon near-infrared (NIR) irradiation. (c) Tumor growth inhibition after single laser irradiation (arrowhead) of the tumors. (d) Histological observation and quantification of apoptotic cells in the tumor tissues collected 24 h after single laser irradiation. Tumor cells were labeled with GFP (green) and apoptotic cells were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL; red). Nuclei were stained with Hoechst (blue). The scale bar represents 20 µm. ‘1d’ and ‘7d’ indicate the number of days after NP7 injection and ‘L’ indicates NIR laser irradiation. ‘+’ and ‘-’ indicate presence and absence of NP7 injection or laser irradiation, respectively. Data


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represent the means ± SD [n = 6 in each group, ***p < 0.001, two-way analysis of variance followed by the Bonferroni test for (c); n = 3, ***p < 0.001, one-way analysis of variance followed by Tukey’s test for (d)].



Page 30 of 31

Table 1. Physicochemical properties and pharmacokinetics of nanoparticles used in this study. Zeta potential (mV)3 Absorbance

Aspect NP1

Blood 4

Size2

PEG

PEG-dye

coating

coating

ratio

Half-life5 (nm) (min)

NP1

1

18 ± 2.9

-13 ± 2.85

-5 ± 5.95

520

346.6 ± 32.5

NP4

4

(58 ± 3.4) x (14 ± 1.7)

-4 ± 7.56

-3 ± 3.18

810

231.0 ± 54.4

NP7

7

(110 ± 4.6) x (16 ± 1.2)

1 ± 1.21

-2 ± 2.32

1100

115 ± 24.5

1

The number after the NP (gold nanoparticle identifier) designates the aspect ratio.

2

The size was determined from the images obtained with TEM (n = 10).

3

The zeta potential was determined based on dynamic light scattering measurements (n = 3).

4

The absorbance represents the maximum peak of absorption spectrum of gold nanoparticles.

5

The blood half-life was calculated by fitting the fluorescence data to a single-exponential

equation using a one-compartment open pharmacokinetic model (n = 3). Data represent the means ± SD.


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Table of Contents (TOC)

PEGylated elongated nanoparticles

In tumors

In the liver

7 days after intravenous injection

NP tumor cell

Preferential localization in tumor cells


NP Kupffer cell

Efficient exocytosis from Kupffer cells

Macrophage-Mediated Exocytosis of Elongated ... - ACS Publications

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