Destroying Deep Lung Tumor Tissue through Lung-Selective

Subscriber access provided by READING UNIV

Article

Destroying deep lung tumor tissue through lung-selective accumulation and by activation of caveolin uptake channels using a specific width of carbon nanodrug Sang-Woo Kim, Jun-Young Park, Soyoung Lee, Sang-Hyun Kim, and Dongwoo Khang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16153 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 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

ACS Applied Materials & Interfaces 1

Destroying Deep Lung Tumor Tissue through LungSelective Accumulation and by Activation of Caveolin Uptake Channels Using a Specific Width of Carbon Nanodrug Sang-Woo Kim1,†, Jun-Young Park1,†, Soyoung Lee2, Sang-Hyun Kim3,*, and Dongwoo Khang1,4,*

1

Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon 21999, South Korea

2

Immunoregulatory Materials Research Center, Korea Research Institute of Bioscience and

Biotechnology, Jeonbuk 56212, South Korea 3

Department of Pharmacology, Kyungpook National University, Daegu 41566, South Korea.

4

Department of Physiology, Gachon University, Incheon 21999, South Korea

†

These authors contributed equally

*Corresponding Authors: Prof. Dongwoo Khang Department of Physiology, College of Medicine, Gachon University, Incheon 21999, South Korea, Tel: +82 32 899 6515, E-mail: [email protected]

Prof. Sang-Hyun Kim Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 41566, South Korea, Tel: +82 53 420 4838, E-mail: [email protected]

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 2 of 36 2

Abstract

The main difficulty with current anticancer nanotherapeutics comes from the low efficiency of tumor targeting. Although many strategies have been investigated, including cancerspecific antibody conjugation, lung tumors remain one of the invulnerable types of cancer that must be overcome in the near future. Meanwhile, despite their advantageous physiochemical properties, carbon nanotube structures are not considered safe medical drug delivery agents, but are considered a hazardous source that may cause pulmonary toxicity. However, high-aspect-ratio (width vs. length) nanostructures can be used as very efficient drug delivery agents due to its lung tissue accumulation property. Furthermore, selection of a specific width of the carbon nanostructures can activate additional caveolin uptake channels in cancer cells, thereby maximizing internalization of the nanodrug. The present study aimed to evaluate the therapeutic potential of carbon nanotube-based nanodrugs having various widths (10–30 nm, 60–100 nm, and 125–150 nm) as a delivery agent to treat lung tumors. The results of the present study provided evidence that both lung tissue accumulation (passive targeting) and caveolin-assisted uptake (active targeting) can simultaneously contribute to the destruction of lung tumor tissues of carbon nanotube.

Keywords: Lung tumor, carbon nanotubes, caveolin uptake, lung accumulation, anticancer efficacy


Page 3 of 36 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


Introduction

Although many previous studies have emphasized the dimensions (including width and length) and shapes of nanomaterials and their associated bio-distribution, there have been relatively few applications with respect to anticancer therapeutics for deep-tissue tumor model that have benefited from optimizing the shape and size of nanodrugs.1-4 Historically, the shape and size of nanodrugs were considered as not only independent factors in organ-specific accumulation after blood circulation (through intravenous injection), but also were regarded as important contributors to cellular uptake and subsequent intracellular trafficking.5-6 Different shapes with similar sizes exhibited different orders of deep tissue tumor accumulation.7-8 For exmple, gold (Au) nanostructures with four different shapes (nanosphere, nanodisks, nanorods, and nanocages) exhibited different bio-distribution and differential orders of tumor targeting efficiency.8 In other studies, cylindrical nanomaterials were highly accumulated in the lung endothelium because of the increased surface tension between rod-like nanoparticles, which tended to strongly adhere to the inner surface of the abundant micro-vessels in lung tissues.6,

9-10

Thus, long cylindrical nanostructures are

considered more advantageous materials in terms of tumor accumulation than spherical nanoparticles.11 Meanwhile, cylindrical nanostructures, including carbon nanotubes (CNTs), were considered a source of pulmonary aerosols and considered to have detrimental effects.12 For example, the 'needle-like' shape of CNTs, combined with their high aspect ratio (ratio of length and width), hydrophobicity, and biopersistence, triggered concerns regarding pulmonary toxicity.3,

12

Indeed, a number of in vivo studies have demonstrated that CNTs are a potential source of



Page 4 of 36 4

inflammation, fibrosis (scarring of the lungs), and granuloma (small nodule) formation when instilled into the lungs.12-13 However, in other aspects, this type of nanodrug can be a very effective anti-lung cancer agent that can specifically target lung tumors because of their selective accumulation in lung tumor tissue.14 Additionally, depending on the shape and size of the nanomaterials, the ATP-assisted uptake pathway is significantly influenced.15-18 For example, clathrin-mediated endocytosis occurs very commonly for nanoparticles around 100 nm in size.1 In contrast, a sub-100 nm encapsulated lipid nanodrug showed a superior anticancer efficacy through an increased uptake in cancer cells via the caveolin-mediated endocytosis pathway.19-20 Although there was no detailed information on the uptake channels, 40 to 50-nm sized Au nanoparticles exhibited a critical cutoff size for the receptor-mediated endocytosis pathway and efficiently triggered apoptotic signaling in breast cancer cells.15 Furthermore, antibodies conjugated with different shaped nanomaterials induced efficient selective cellular uptake.21 Thus, previous studies supported the evidence that the optimization of the shape and size of nanodrugs can maximize the anticancer efficacy to treat deep tumor tissue (specific organ-originated tumor tissues). In this line, the present study aimed to evaluate the therapeutic potential of various widths (10–30 nm, 60–100 nm, and 125–150 nm) of CNT-based nanodrugs as a delivery agent to treat lung tumors (Fig. 1a). The selective lung biodistribution of the various widths of cylindrical nanodrugs, the activation multiple cellular uptake pathways, and their associated anticancer efficacy were investigated.


Page 5 of 36 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


Results and Discussion

Characterization of nanodrugs and drug stability in physiological environment The morphology and size of the covalently conjugated CNTs (three different diameters) and doxorubicin (DOX), were confirmed by transmission electron microscopy (TEM) and particle size analysis. The TEM images showed that the attached DOX was directly exposed on the covalently linked nanotubes (Fig. 1b). The corresponding length of the covalently conjugated nanodrugs was 360, 375, and 420 nm for the CNTs with widths of 10–30 nm, 60– 100 nm, and 125–150 nm, respectively (Fig. 1c-d). Electrical potential analysis also identified that all the tested drugs possessed negative charges in neutral buffer (phosphate-buffered saline (PBS), pH 7.2) because of the negative charges in the CNTs (Fig. 1d). Most of the covalently conjugated nanodrugs exhibited an average charge between DOX (red) and the covalently conjugated CNTs (Fig. 1d). The amount of drug loaded onto the covalently conjugated nanodrugs was quantified by analyzing the absorption spectrum. The differences in absorbance peaks between the loaded drug (DOX) and the base nanomaterials (10–30 nm, 60–100 nm, and 125–150 nm CNTs) at a specific wavelength (at 490 nm) corresponded to the amount of loaded drug.22 The weight ratio of loaded drug for the different widths of nanodrugs was approximately 43 %, 101.4 % and 137.4 % for 10–30 nm, 60–100 nm, and 125–150 nm nanodrugs, respectively (Fig. 1e). To verify all the analytical calculations, the weight differences between the drug-loaded nanomaterials and the base nanomaterials were directly measured, and the reciprocal coincidences of weight (ultraviolet-visual (UV-vis) and balance) were verified (data not shown). In conclusion, TEM, particle size, and electric potential assessments demonstrated



Page 6 of 36 6

that DOX was covalently conjugated onto the CNTs (Fig. 1b-d). The time dependent drug release analysis in extracellular and intracellular environments is meaningful to examine the drug release from the CNTs. The drug release results showed that all covalently conjugated nanodrugs exhibited great drug stability over 288 h (less than 20 %) (Fig. 1f). In addition, analyzing drug stability in a hematological environment is also critical. This is because that the release of anticancer drugs from the CNTs in blood environment before reaching cancer cells will reduce anticancer efficacy of nanodrugs. To emulate the blood-like physiological conditions, fetal bovine serum (10% of FBS, pH = 7.2) was added in PBS. In FBS 10% condition, the 10-30 nm and 60-100 nm nanodrugs showed slow drug release; about 40 % of the conjugated DOX was released after 100 h (Fig. 1f). Slow release in FBS buffer considered that the enzymes in FBS contributed the detachment of covalently conjugated DOX from CNT. Both the 10–30 nm and 60–100 nm nanodrugs showed identical release patterns over 288 h (Fig. 1f) and exhibited the better stability compared with the 125– 150 nm nanodrugs (both in PBS and FBS). Two major biochemical parameters can mimic the acidic intracellular conditions in cancer cells: intracellular pH (pH 4~7) and high abundance of hydrolase enzymes. In this study, covalent conjugated DOX on various widths of CNTs was rapidly detached in acidic-lysozyme condition. In short, 40-60 % of the DOX was released from the tested nanodrugs within 5 h (Fig. 1f) and, thus, results supported the evidence that released DOX from CNTs by amide covalent bond cleavage transported into the cancer nucleus.23

Bio-distribution and in vivo toxicity evaluation Bio-distribution of the free DOX and covalently conjugated nanodrugs of different widths


Page 7 of 36 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


(10–30, 60–100, and 125–150 nm) was analyzed after intravenous (i.v.) injection of identical doses (2 mg/kg) in mice. Six hours after injection, the mice were sacrificed and the major organs (the heart, lungs, liver, stomach, intestine, spleen, kidneys, lymph node, and brain), and peripheral blood were collected into heparin-treated tubes. The samples were then analyzed for intrinsic fluorescence intensity of DOX (at 470 nm excitation/595 nm emission wavelengths). Six hours after injection, the covalently conjugated nanodrugs were highly accumulated in the lung (Fig. 2a). This selective lung accumulation showed that the nanodrugs could be a very effective anti-lung cancer agent, specifically targeting lung tumor tissues. The covalently conjugated nanodrugs with different lengths displayed differences in their distributions in other organs. Although the 10–30 nm nanodrugs mostly accumulated in the lung, compared to other organs (6 h), other types of covalent-conjugated nanodrugs (60– 100 nm and 125–150 nm) showed slight accumulation in the spleen, intestine, kidney, liver, and lymph nodes (Fig. 2a). Furthermore, fluorescence-activated cell sorting (FACS) analysis (after 24 h) also confirmed that the covalently conjugated nanodrugs (selected data from the 60–100 nm nanodrugs) showed significant accumulation (99.8 %) in the lung tumor tissues in A549 lung tumor nearing mice (Fig. 2a). The major biochemical indicators were investigated to evaluate the in vivo toxicity of covalent conjugated nanodrugs (Fig. 2b). Representative hematology markers, such as white blood cell (WBC) and red blood cell (RBC) of the tested nanodrugs were within normal ranges, while DOX alone showed elevated WBC and RBC compared with tested nanodrugs (Fig. 2b). Representative serum biochemical indicators for liver toxicity, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were analyzed. The ALT and AST levels of the 10–30 nm nanodrug-treated mice were above the normal range (Fig. 2b). This might be originated from increased CNT concentration (4.66 mg/kg) compared with



Page 8 of 36 8

other width of nanodrugs (1.97 mg/kg for 60-100 nm nanodrug and 1.46 mg/kg for 125-150 nm nanodrug) and suggested evidence that the 10–30 nm nanodrugs could induce liver toxicity compared with other widths of nanodrugs (60–100 nm and 125–150 nm). Representative serum biochemical indicators for kidney function, blood urea nitrogen (BUN) and creatinine (Crea) were also analyzed. The obtained results indicated that the level of BUN was significantly increased by the 125–150 nm nanodrugs (Fig. 2b). In contrast, treatment with DOX, 10–30 nm and 60–100 nm nanodrugs did not cause elevation of BUN or Crea compared with the control (Fig. 2b). In conclusion, the hematological toxicity evaluation suggested that only 60–100 nm nanodrug (2 mg/kg) did not induce any notable toxicity after 4 times of i.v. injection. Importantly, it has been reported that CNT may induce pulmonary and major organ toxicity. To identify the maximum tolerable dose of used CNTs (as well as CNT-DOX nanodrugs), single and repeated exposure toxicity evaluations (e.g., weight changes in the lung) on 10-30 nm nanodrug were performed (0.2, 1 and 5 mg/kg: based on DOX concentration). No notable weight changes in pulmonary (lung) and major organs (liver, heart and spleen) were identified by nanodrug with dose of 0.2 mg/kg and 1 mg/kg (based on DOX dose). However, notable weight changes in the lung, liver and heart and spleen were observed in 5 mg/kg of CNT-DOX conjugation (Figure S1-2). However, it is worth to note that weight changes in the lung and major organs stem from the toxicity of tested nanodrug (10-30 nm of nanodrugs) and not by CNTs alone. This is because that no notable weight changes in lung and major organs were observed for injection with 20 mg/kg of CNTs (which corresponded to the amount of 5 mg/kg of nanodrug) for both single and repeated exposure toxicity evaluations (Figure S1-2).


Page 9 of 36 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


In vivo anti-lung tumor efficacy To generate fluorescent lung tumors in vivo, A549Fluc-RFP cells (2 × 106) were i.v. injected into the tail vein of mice (Fig. 3a). Three weeks after the injection of A549Fluc-RFP cells, lung tumor-bearing mice were sorted based on similar tumor size and signal intensity using bioluminescence imaging (BLI) before the administration of the tested nanodrugs (DOX concentration set at 2 mg/kg) (Fig. 3a). BLI was conducted to analyze the changes in luciferase intensity and the size of the lung tumors (Fig. 3a-b). Among the tested nanodrugs, significant decreases in luciferase intensity from the lung tumors were found in the 10–30 nm and 60–100 nm nanodrug groups, whereas the free DOX and 125–150 nm nanodrug groups simply exhibited a delayed growth rate of lung tumors compared with the saline control group (PBS) (Fig. 3a-b). Notably, the longest survival of mice was observed for those receiving the 60–100 nm nanodrugs compared with the other groups at a same dosage (2 mg/kg) (Fig. 3c). Thus, the 60–100 nm nanodrugs provided a significantly improved survival time compared with the other treatment groups, with a median survival of 20 weeks, compared with the 8, 10, 18 and 15 weeks for the saline, free DOX, 10–30 nm and 125-150 nm nanodrugs, respectively (Fig. 3c). Hematoxylin and eosin (H&E) staining was consistent with the in vivo imaging system (IVIS) results (Fig. 3a-b). The H&E staining results of sliced lung sections clearly indicated that the 60–100 nm nanodrugs significantly eradicated lung tumor tissue (Fig. 3d). The magnified regions of both tumor tissues and lung epithelial vessels indicated significant suppression of the A549 tumor tissues in lungs by the 60–100 nm nanodrugs compared with the other groups (Fig. 4d). It is worth noting that the injected nanodrugs (CNT) accumulated in the lung tumor tissue sections (Fig. 3d) and coincide with obtained FACS results (Fig. 2a).



Page 10 of 36 10

Taken together, the in vivo results demonstrated that the 60–100 nm nanodrugs exhibited the best anti-lung tumor efficacy compared with other diameters of nanodrugs, with negligible in vivo toxicity (Fig. 2b). However, obtained results suggested the evidence that the 60–100 nm nanodrugs effects were mediated not only by lung-accumulation but also by an additional mechanism (activating additional uptake channels, which is discussed in the following section).

Increased anticancer efficacy via caveolin-mediated endocytosis It has been widely believed that nanodrug uptake occurs via ATP-assisted endocytosis (i.e., dynamin-dependent) when being internalized; for example, by clathrin-mediated endocytosis, caveolae-mediated endocytosis, and micropinocytosis.18 Increasing cellular internalization of nanodrugs via enhanced endocytosis is critical in terms of increasing nanodrug efficacy.22 To examine the selective nanodrug internalization and anticancer efficacy, intracellular (by FACS) and nuclear entry (by confocal microscopy) were examined after 2 h of incubation (Fig. 4a-b). The confocal microscopic images showed that nuclear DOX intensity (nuclear intake) exhibited the highest fluorescent signal for the 60–100 nm nanodrugs after 2 h (Fig. 4a). To understand what type of endocytic pathways were involved in the cellular uptake of the different widths of nanodrugs, 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) (an inhibitor of a Na+/H+ exchange protein), chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) and genistein (GEN, an inhibitor that disrupts caveolae function) were used. The results demonstrated that macropinocytosis and clathrin-mediated endocytosis are the major


Page 11 of 36 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


uptake channels for the 10–30 nm and 60–100 nm nanodrugs (Fig. 4b). However, caveolae-mediated endocytosis was additionally observed for the 60–100 nm nanodrugs (Fig. 4b). In contrast, the 125–150 nm nanodrugs exhibited negligible uptake related to the three endocytic pathways (Fig. 4b). As expected by previous reports, free DOX showed no ATPdependent uptake into A549 lung cancer cells (Fig. 4b) and suggested the evidence that DOX is uptake by non-ATP assisted diffusive intake.24-25 To verify the identified uptake channels in A549 cells for the tested nanodrugs, the endocytic inhibitors (EIPA, CPZ, GEN) were treated to examine the alteration of nuclear DOX intensity. Confocal images (Fig. 4c-d) clearly confirmed previous uptake analysis obtained by FACS (Fig. 4b). In particular, as shown in Figure 4c-d, treatment with GEN significantly reduced the cellular uptake of the 60–100 nm nanodrug by about 70%, while EIPA and CPZ slightly inhibited the uptake of the 60–100 nm nanodrugs by about 40–45 % (Fig. 4c-d). This showed that caveolin uptake is the main uptake channel for the 60–100 nm nanodrugs, compared with clathrin and micropinocytosis-mediated endocytosis and, caveolin channel significantly increased the nuclear and intracellular drug intake. Note that there was no inhibition of the uptake of the 10–30 nm nanodrugs when pretreated with GEN (Fig. 4c). In addition, there was no significant difference in the nuclear DOX intensity after blocking with EIPA, CPZ, and GEN for the 125–150 nm nanodrugs (Fig. 4c-d). To examine the anticancer efficacy influenced by the uptake channels, the cytotoxicity and apoptosis-inducing ability of the covalently conjugated nanodrugs were evaluated in vitro in A549 lung cancer cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and FACS analysis (Fig. 4e-f). The cytotoxicity and apoptotic percentage of A549 lung cancer cells treated with the 60–100 nm nanodrugs was greater than those of the other sized nanodrugs (Fig. 4e-f). This might be interpreted as the 60–100 nm nanodrugs being specifically effective



Page 12 of 36 12

for DOX delivery to the A549 nucleus by activating caveolin channels (Fig. 4a-d). Reciprocally, the cytotoxicity of the 60–100 nm nanodrugs after GEN treatment was reduced by about 30% (Fig. 4e). Furthermore, the apoptosis percentage of A549 lung cancer cells treated with the 60–100 nm nanodrug after GEN treatment (63.6 %) was reduced compared with the nanodrug treatment alone (91.6 %) (Fig. 4f). Taken together, obtained in vitro results clearly demonstrated that the 60–100 nm nanodrugs showed elevated overall anticancer efficacy for lung cancer cells via increased caveolin channel activity and confirmed obtained in vivo results (Fig. 3a-d).


Page 13 of 36 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


Conclusions

Understanding materials and biological interactions at the nanoscale has great advantages in therapeutic applications, especially for incurable diseases, including deep tumor tissues. Ironically, the detrimental and undesirable toxicological aspects, such as lung accumulation, of cylindrical CNTs-based anticancer drugs can make them a source of effective lung targeting agents, without triggering notable toxicity, contingent on the limited concentration of the nanomaterials used. Importantly, the tailored shape and size of nanodrugs can provide a unique solution to overcome the current limitations of treatment, especially in deep tumor target-oriented drug delivery strategies. The role of the size and shape of nanodrugs in cellular internalization is also a critical factor in terms of measuring anticancer efficacy. From this point of view, the present study provided both passive and active drug delivery strategies, and evaluated the in vivo anti-tumor efficacy using a non-small cell lung cancer bearing mouse model. Specifically, present study clearly demonstrated that covalently-conjugated nanodrug with a width of 60–100 nm has potential as an anti-lung tumor nanodrug through lung-specific accumulation and by increased activation of caveolin uptake channels, which simultaneously contributed to the destruction of lung tumors. A comprehensive understanding of the uptake, intracellular trafficking, and drug efficacy by controlling the shape and size of a nanodrug can provide greater organ targeting specificity, as well as optimal internalization by activating uptake channels. Furthermore, selection of biodegradable or biocompatible nanomaterials with optimized shape and size of nanodrugs can increase the chance of clinical success. Ultimately, suggested strategy suggested a platform technology for the treatment of inaccessible deep tumor tissues (with current



Page 14 of 36 14

chemotherapy) by selective accumulation to organ originated tumor tissues and by activated uptake channels.


Page 15 of 36 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


Materials and methods

Preparation of DOX-conjugated nanodrugs Purified multi-walled carbon nanotubes (mwCNT), (with a diameter of 10–30 nm, 900-1260, SES, USA), mwCNTs (diameter; 60–100 nm, 900-1280, SES, USA) and mwCNTs (diameter; 125–150 nm, 719811, Pyrograf, USA) were carboxylated according to previously described methods.23

N-ethyl-N′-(3-dimethylaminopropyl)

carbodiimide

hydrochloride-linked

mwCNTs were prepared. DOX was then added (44583, Sigma) at a weight ratio of 1:1 (DOX:CNT) in MES buffer (pH 6.0). The mixture was rotated at 4 °C overnight. The CNT-DOX suspension in MES was centrifuged and filtered (Amicon YM-100K, 4000 rpm) at least three times to remove the unconjugated DOX. Finally, CNT-DOX were stored in PBS (10010-023, Gibco). To measure the concentration of DOX on the CNTs, UV-vis adsorption spectroscopy (Libra S50, Biochrom) was analyzed at a wavelength of 490 nm. The weight percentage of covalently linked DOX on CNTs was determined by measuring the difference in absorbance signal intensities at 490 nm and extrapolation based on linear standard curves from the concentrations of DOX and CNTs. Drug loading was determined using the following formula: Drug loading (%) = (Weight of covalently linked DOX onto CNTs /Weight of CNTs) × 100.

Characterization of DOX-conjugated nanodrugs The particle size and electric potential were analyzed (Zetasizer Nano, Malvern, UK) to



Page 16 of 36 16

determine the size distribution profile and electric potential of the covalently conjugated nanodrugs in PBS. Transmission electron microscopy (TEM) was used to visualize the covalently conjugated DOX on the mwCNTs. The CNT-DOX dispersion was treated as prepared in a moderate ultrasonic bath and deposited onto a holey carbon TEM grid (plasma etched before dipping into each nanodrug solution). All TEM images were obtained on a Titan TEM instrument (FEI, USA) at 300 kV.

Tissue distribution studies BALB/c nude mice were randomly divided into two groups. Nanodrugs (widths of 10–30, 60–100 and 125–150 nm) and DOX solution were intravenously administrated via the tail vein at a dose of 2 mg/kg. After 6 h, the blood of mice was collected from the celiac artery into heparinized tubes and was centrifuged to obtain plasma samples. The mice were sacrificed and tissues of interest (brain, intestine, stomach, heart, liver, spleen, lung, lymph node, and kidney) were collected immediately after and lightly rinsed with normal saline and dried with tissue paper. The plasma and tissue samples were frozen at −20 °C until analysis. Tissue samples were weighed accurately and homogenized using a glass tissue homogenizer after the addition of 1 mL PBS buffer. Tissue homogenates were processed similarly to the plasma samples and the DOX fluorescence was measured in a multiplate reader (VICTOR X3, PerkinElmer, USA) at 470 nm excitation and 595 nm emission. To analyze relative lung tissue cell accumulation of the covalently conjugated nanodrugs, and to examine uptake into A549FLuc-RFP lung tumor tissues in vivo, tail vein injection of 60–100 nm of nanodrug (2 mg/kg) was performed. Twenty-four hours after tail vein injection, the lungs from tumor-bearing mice were homogenized individually in 7 mL of collagenase type


Page 17 of 36 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


II (17101015, Gibco) with 1 mg/mL in minimal essential medium (MEM) (Gibco) and incubated for 2 h in a CO2 incubator at 37 °C. Subsequently, the enzyme-digested lung tissues were washed in PBS and filtered through a 40-mm cell strainer (93070, SPL), centrifuged, washed with PBS supplemented with 2 % of FBS, and incubated for 30 min at room temperature with anti-CD54 antibodies (A549 positive marker) conjugated with Pacific blue dye in PBS containing 2 % FBS. After a final washing step, the cells were re-suspended in 500 mL PBS (supplemented with 10 % FBS) and analyzed for the CD54-positive cell population possessing DOX fluorescence by flow cytometry (BD LSRII, BD Biosciences). Ten thousand events were acquired per sample, and data were displayed on logarithmic scales. Forward and side light scatter signals were used to exclude dead cells and debris. Data were analyzed using the FlowJo software (Ver. 10.1, FlowJo, LLC, USA). A549 cells in the absence of the anti-CD54 antibodies were used to determine the background signal (0.1 %) and determine gate boundaries.

Peripheral blood sample analyses. To analyze peripheral circulating blood cells, blood samples were placed in a labeled vial containing heparin (5 units/mL) and transported on ice for hematological analysis. Blood cells were automatically counted (Sysmex F-820 Blood Counter, Toa Medical Electron, Japan). For the serum, the blood was allowed to clot by leaving it undisturbed at room temperature; the clot was removed and centrifuged at 2000 × g for 15 min at 4 °C, and the supernatant

was

retained.

Levels

of

alanine

aminotransferase

(ALT),

aspartate

aminotransferase (AST), and creatinine or blood urea nitrogen (BUN) were measured using clinical chemistry reagent kits (HUMAN, Germany).



Page 18 of 36 18

Single and repeated exposure toxicity test. Male and female Imprinting Control Region (ICR) mice (6 weeks old) were purchased from Dae-Han Experimental Animal Center (Daejeon, Korea). Mice were randomly assigned to treatment cages and acclimated for 1 week. Mice had ad libitum access to standard rodent chow and filtered water. Throughout the study, the mice were housed in a laminar air flow room in a controlled environment (temperature, 22 ± 2°C; relative humidity, 55 ± 5%; 12 h/12 h light/dark cycle). The care and treatment of the animals were in accordance with the guidelines established by the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IRB #2016-0151). Mice (n=10/group) were administered by intravenous injection with 200 μL of saline, DOX (5 mg/kg), 10-30 nm of CNT (20 mg/kg), CNT-DOX (DOX: 0.2 mg/kg & 10-30 nm of CNT: 0.8 mg/kg), CNT-DOX (DOX: 1 mg/kg & 10-30 nm of CNT: 4 mg/kg), CNT-DOX (DOX: 5 mg/kg & 10-30 nm of CNT: 20 mg/kg) in the tail vein. For single exposure test, mice were injected with respective drugs single time and monitored during 2 weeks. For repeated exposure test, mice were injected with drugs twice a week (total 4 times) and monitored during 2 weeks. At the end of treatment period, mice were fasted overnight and euthanized by carbon dioxide. Trunk blood was collected, and various organs were aseptically excised and weighed.

Cell lines and cell culture A549 (non-small cell lung cancer, CCL-185™) cells supplemented with 10 % FBS (16000-


Page 19 of 36 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


044, Gibco) and 1 % antibiotics (Invitrogen, Waltham, MA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, 11995-065, Gibco) and were cultured in a humidified incubator containing 5 % CO2 at 37 °C.22

Uptake mechanism identification (FACS) A549 lung cancer cells were seeded in a six-well plate at a density of 4 × 105 cells/well and incubated overnight. The cells were pre-incubated with inhibitors (EIPA, CPZ, and GEN; all Sigma, at 25 μM, 20 μM, and 200 μM, respectively) for 30 min at 37 °C to examine the uptake of micropinocytosis, caveolin and clathrin. Before treatment with each endocytosis inhibitor, cell viability was checked using the MTT assay to detect any cytotoxic effect by treatment with uptake inhibitors.22 The tested nanodrugs were then incubated with cells for another 2 h. The DOX concentration of nanodrugs was set to 200 ng/mL. The cells were then washed twice in PBS and suspended in 500 mL of PBS supplemented with 1 % FBS. The alexa 488 fluorescence obtained from single cell suspensions was evaluated using a BD LSR II flow cytometer (Becton Dickinson Immunocytometry Systems) and analyzed using FlowJo software (Ver. 10.1, FlowJo, LLC). Single cell suspensions treated with nanodrugs without inhibitors served as controls.

Nuclear DOX quantification To evaluate the intracellular uptake of nanodrugs into the nuclei of A549 cells, the uptake of free DOX and covalently conjugated nanodrugs (10–30 nm, 60–100 nm and 125–150 nm) by A549 lung cancer cells was tested. The cells were seeded on glass coverslips (at a density of



Page 20 of 36 20

3 × 104 cells) and incubated for 24 h. The cells were then incubated with a medium containing the tested drug at 200 ng/mL. After the 24 h, the medium was removed and the cells were washed twice using cold PBS. After each measurement, cells were mounted and visualized at the same setting using a confocal microscopy LSM700 (Carl Zeiss, Germany). For the inhibitor studies, A549 cells were treated with endocytic inhibitors (EIPA, CPZ, and GEN) in serum-free MEM for 1 h before incubation with the covalently conjugated nanodrugs. The laser wavelength of excitation was set to 488 nm and the detection wavelength was set to 550 nm. All fluorescence intensities were analyzed using image J software (Java 1.6.0_24, NIH, USA).

MTT cell viability and FACS apoptosis A549 lung cancer cells (seeded at 5 × 103 cells/well in 96-well plates) were treated with various concentrations of tested nanodrugs and incubated at 37 °C for 48 h. For inhibitor studies, A549 cells were treated with endocytic inhibitors (EIPA, CPZ, and GEN) in serumfree MEM for 1 h before incubation with covalently conjugated CNT-DOX. Cell viability was determined using the MTT assay (Sigma). After 48 h of treatment, 100 μL of MTT (1 mg/mL) was added to each sample well and the samples were incubated for 2 h. Dimethylsulfoxide was added to dissolve the formazan crystals. The absorbance of each sample was calculated relative to that of the control and expressed as a percentage increase. To investigate the effect of the covalently conjugated nanodrugs on cell apoptosis, A549 cells were seeded in 6-well plates (at a density of 5 × 105 cells/well) and treated with 500 ng/mL of the nanodrugs for 48 h. Untreated cells were used as controls. The apoptosis was analyzed by Annexin V. After 24 h, all treated cells were removed and prepared by the instruction of the


Page 21 of 36 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


Annexin V-Pacific Blue™ kit (A35122, Invitrogen, Carlsbad, CA, USA). In short, Annexin V- Pacific Blue™ (5 μL) was added to each sample and incubated for 15 min in the dark at room temperature (Add Nanotechnology Ref. here). Cells were then re-suspended in 400 μL of PBS (supplemented with 1 % FBS) and analyzed using a BD LSR II flow cytometer (Becton Dickinson, Germany) at an excitation wavelength of 410 nm and a 455 nm bandpass filter to detect Pacific Blue.26

Generation of A549Fluc-RFP cells A549 cells were seeded into 6-well plates at a density of 105 cells per well and allowed to attach overnight. The next day, the medium was changed to a mixture of 2 mL of complete medium and 1 mL lentiviral supernatant (105 viral particles/well) containing lentiviruses encoding the firefly luciferase (Fluc) and red fluorescence protein (RFP) (#GlowCell-14b-1, Biosettia). After 24 h, the culture medium containing the lentivirus was discarded, and the cells were cultured for another 72 h. Post transduction, the cells were cultured in medium supplemented with puromycin (1 mg/mL) to eliminate non-transduced cells. After initial selection, the cells were cultured in medium supplemented with 0.3 mg/mL puromycin for 1 month. Stably transfected A549Fluc-RFP cells were confirmed by bioluminescence imaging using 150 mg/mL D-luciferin and an in vivo imaging system (IVIS, IVIS-200, Xenogen).

Bioluminescence imaging Bioluminescence imaging (BLI) was used to measure the luciferase activity by IVIS imaging optimized for high sensitivity. BLI was performed weekly. Mice were anesthetized with



Page 22 of 36 22

isoflurane (approximately 3 % in air) and then intraperitoneally injected with luciferin (150 mg/kg, Xenogen) 40 min before imaging. The animals were then placed in the light-protected chamber of the IVIS and measured for 1 min. Regions of interest were captured using Living Image software (ver. 3.1, Xenogen). BLI was measured based on radiance as the average luminescence units detected on the surface of the animal per square centimeter per steradian (photons/s/cm²/sr).

A549 lung tumor model Female BALB/c nude mice (4–6 weeks old, weighing 16–20 g each) were purchased from Orient Bio (Seoul, Korea). All animal experiments were carried out under the regulation of the Care and Use of Laboratory Animals of Gachon University (IRB number: GCIRB201583). To establish lung tumor-bearing mice, 2 × 106 A549Fluc-RFP cells were suspended in 200 μL of PBS and intravenously injected into the tail vein of the mice. To confirm the establishment of the A549Fluc-RFP lung tumor model, mice were intraperitoneally injected with 150 mg/kg of D-luciferin and imaged after 40 min using Xenogen IVIS to analyze the size of the tumor.

In vivo antitumor efficacy Mice bearing lung tumors (abdominal bioluminescence greater than 1 × 107 photons/s) were randomly divided into five groups (n = 3). The tail vein of each mouse was injected intravenously with free DOX, nanodrugs (10–30 nm, 60–100 nm, and 125–150 nm) and saline (in 200 μL of PBS) every week, for 4 weeks. The tumor size was monitored by


Page 23 of 36 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


measuring the intensity using IVIS every week.

Histology Tissues isolated from mice were fixed in 10 % formaldehyde and embedded in paraffin, according to standard procedures. The paraffin-embedded samples were sectioned at 5-μm thickness using a rotary microtome (RM2255, Leica) and then stained with hematoxylin (MHS1, Sigma) and eosin (318906, Sigma) (H&E). The histology of the lung tissues was determined by light microscopy (Pannoramic MIDI and Pannoramic DESK, 3DHISTECH) and captured by the CaseViewer program (version 2.1, 3DHISTECH).

Statistical analysis The statistical differences between the mean values obtained from the two sample groups were analyzed using Student’s t-test. The differences were considered significant if the p value was less than or equal to 0.05. The statistical differences between several sample types were analyzed using analysis of variance (ANOVA, followed by the Newman–Keuls multiple comparison test. Asterisks (*, **, and ***) indicate the significance of p values (< 0.05, < 0.01, and < 0.001, respectively).



Page 24 of 36 24

Conflict of interest The authors declare no competing financial interest.

Acknowledgements This research was supported by grants from the National Research Foundation of Korea (2014R1A2A1A11052615, 2015R1A2A1A15056054 and 2016R1A2B4008513) and by Korea Health Technology R&D Project of the KHIDI, funded by the Ministry of Health & Welfare (HI14C1802).

Supporting Information The Supporting Information is available free of charge on the ACS Publications website.


Page 25 of 36 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


Figure legends

Figure 1. Physiochemical characteristics of covalent conjugated nanodrugs (a) Schematic diagram of covalent conjugated nanodrugs injection. Tested nanodrugs are injected intravenously into a mouse bearing an A549FLuc-RFP lung tumor. (b) TEM images (left: low resolution, right: high resolution) showing different widths of the tested nanodrugs. The TEM images showed that DOX is covalently attached on the CNTs. The scale bar is 100 nm. (c) Schematic illustration of covalently conjugated nanodrugs with the various widths (10–30 nm, 60–100 nm and 125–150 nm). (d) The length of the tested nanodrugs ranged from 350 to 400 nm (upper). The zeta potential (bottom) showed negative electrical potentials and the positive potential of DOX in neutral pH buffer (i.e., PBS pH 7.2). (e) The amount of attached DOX for each different width of covalent conjugated nanodrugs was calculated by UV-vis absorption. UV-vis spectra are depicted as follows: 10–30 nm (orange), 60–100 nm (blue), 125–150 nm nanodrugs (green) and free DOX (red). The corresponding nanomaterials were used as a base line to measure the amount of attached DOX. (f) Released DOX from different width of nanodrugs under the extra- and intracellular conditions. Each sample was analyzed in neutral (i.e., PBS, pH=7), acidic (i.e., ABS, pH=5), FBS (i.e., 10 % pH=7) and lysozymesupplemented (i.e., density= 1 mg/mL, both at pH 7 and pH 5) buffers up to 288 h. All data represent the mean ± SEM (n = 10). DOX, doxorubicin; TEM, transmission electron microscopy; CNT, carbon nanotube; PBS, phosphate-buffered saline; UV-vis, ultravioletvisual; FTIR, Fourier transform infrared (spectroscopy); ABS, acetate-buffered saline; FBS, fetal bovine serum.



Page 26 of 36 26

Figure 2. Bio-distribution and in vivo toxicity evaluation (a) Covalently conjugated nanodrugs and DOX biodistribution collected from lung tissues after intravenous injection (6 h). Although 10–30 nm nanodrug was mostly accumulated in the lung, compared with other organs, other types of covalent-conjugated nanodrugs (60–100 nm and 125–150 nm) showed slight accumulation in the spleen, intestine, kidney, liver, and lymph node. All data represent the mean ± SEM (n = 3). FACS histogram analysis (inset) shows selective lung tissue accumulation of the 60–100 nm nanodrug in A549 lung tumorbearing mice after 24 h of the intravenous injection. Covalently conjugated nanodrugs (60– 100 nm) showed significantly accumulation (99.8 %) in lung tumor tissues (CD54 positive A549-bearing mice) after 24 h. (b) in vivo toxicity evaluations on WBC, RBC, ALT, AST, BUN, and creatine in serum of mice after treatment with saline, free DOX, and the tested nanodrugs at the drug concentration of 2.0 mg/kg. Each set of data is represented as mean ± SEM (n = 10, *p < 0.001). DOX, doxorubicin; FACS, fluorescence activated cell sorting; WBC white blood cell; RBC, red blood cell; AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea nitrogen.

Figure 3. In vivo antitumor efficacy of nanodrugs (a) BLI of luciferase expression in A549 lung tumor-bearing mice after treatment with PBS, DOX (2 mg/kg), 10–30 nm (2 mg/kg), 60–100 nm (2 mg/kg), and 125–150 nm nanodrugs (2 mg/kg). (b) Time-dependent luciferase expression in A549 lung tumor-bearing mice after treatment with saline, DOX, 10–30 nm, 60–100 nm, and 125–150 nm nanodrugs. Data represent mean ± SEM (n = 3). Quantitative analysis was based on the product of luciferase intensity and tumor area, as determined by BLI. (c) Kaplan–Meier survival curve for mice


Page 27 of 36 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


with A549Fluc-RFP lung tumors after the administration of saline, DOX, 10–30 nm, 60–100 nm, and 125–150 nm nanodrugs (n = 3). Arrows indicate time points of intravenous injections with the tested drugs. (d) H&E staining (at 100 × magnification) of the lungs from A549 tumor-bearing mice after treatment with saline, DOX, 10–30 nm, 60–100 nm, and 125–150 nm nanodrugs. Representative H&E staining showing the presence of tumor tissues. A magnified view of the enclosed red and green boxes shows accumulated CNTs at the tumor tissue, as well as normal lung epithelial tissue. BLI, bioluminescence imaging; DOX, doxorubicin; CNT, carbon nanotube; PBS, phosphate-buffered saline; H&E, hematoxylin and eosin.

Figure 4. Increased anticancer efficacy by caveolin-mediated endocytosis (a) Confocal microscopy images (upper) for DOX from the nucleus of A549 cells after 2 h of treatment with free DOX and the tested nanodrugs. Uptake levels of DOX in the nucleus were compared with each other (bottom). The drug concentration of DOX was set to 200 ng/mL for the tested nanodrugs. (b) The uptake channel intensity of free DOX and tested nanodrugs

in

A549

cells

examined

by FACS

analysis.

Endocytosis

inhibitors

(macropinocytosis: EIPA; caveolin: GEN; clathrin: CPZ) were used to analyze the uptake response. Both macropinocytosis and clathrin-mediated endocytosis are the major uptake channels for the 10–30 nm and 60–100 nm nanodrugs. However, caveolae-mediated endocytosis was additionally observed for the 60–100 nm nanodrugs. In contrast, 125–150 nm mwCNT-DOX nanodrug exhibited negligible uptake via the three endocytic pathways (slight micropinocytosis). (c) Effect of endocytosis inhibitors on nuclear uptake. Confocal microscopy was used to observe the nuclear DOX intensity in A549 cells. (d) Quantitative



Page 28 of 36 28

analysis of nucleus DOX intensity in A549 cells treated with different widths of nanodrugs (200 ng/mL). Data represent mean ± SEM (n = 10). * and *** correspond to p < 0.05 and p < 0.001, respectively, as compared with the control (no inhibitor treatment). (e) MTT results of A549 cells after treatment with various concentrations of the nanodrugs. (f) FACS histogram corresponding to the control and drug-treated cells (10–30 nm, 60–100 nm, and 125–150 nm nanodrugs). A549 cells were stained with annexin V-Pacific blue. After 48 h of treatment with different widths of nanodrugs (500 ng/mL), samples were analyzed by FACS. The 60– 100 nm of nanodrug caused the most significant apoptosis compared with other nanodrugs. DOX, doxorubicin; FACS, fluorescence activated cell sorting; CNT, carbon nanotube; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


Page 29 of 36

29

a

b

High resolution

60-100 nm (W)

10-30 nm (W)

Low resolution

125-150 nm (W)

c 10-30 nm

o

d

20

Absorbance (a.u.)

Electric Potential (mV)

125-150 nm

o

60-100 nm

o

e

10 0 -10 -20

DOX conjugation -30 -40

DOX CNT-DOX (10-30nm) CNT-DOX (60-100nm) CNT-DOX (125-150nm)

CNT (10-30nm) CNT (60-100nm) CNT (125-150 nm)

3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2

f

CNT-DOX 10-30 nm CNT 10-30 nm

CNT-DOX 60-100 nm CNT 60-100 nm

CNT-DOX 125-150 nm CNT 125-150 nm

DOX 250 300 350 400 450 500 550 600 650 700

CNT-DOX 10-30 nm

80 60 40 20

100

Drug Release (%)

Wavelength (nm) 100

Drug Release (%)

Drug Release (%)

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

ACS Applied Materials & Interfaces

CNT-DOX 60-100 nm

80 60 40 20 0

0 0

5

10

100

200

300

0

5

PBS+Lyz (pH 7.2) ABS+Lyz (pH 5.0)

100

200

300

CNT-DOX 125-150 nm

80 60 40 20 0 0

5

Time (h)

Time (h) PBS (pH 7.2) ABS (pH 5.0) FBS (pH 7.2)

10

100

PBS (pH 7.2) ABS (pH 5.0) FBS (pH 7.2)


Figure 1.


10

100

200

300

Time (h) PBS (pH 7.2) ABS (pH 5.0) FBS (pH 7.2)



CNT-DOX(60-100nm) CNT-DOX(125-150nm)

DOX CNT-DOX(10-30nm)

a

Distribution of 60-100 nm CNT-DOX in lung tumor (25 hrs)

Lung tumor-bearing mice (A549FLuc-RFP)

400

300

% ID/g

DOX positive A549 99.8 %

DOX negative A549

200 50 25

**

10 5

ph

B

lo o

d

e no d

ea rt l

lin Te D C O D N X O T(1 X D 0C O 3 N T- X (6 0nm D 0O 10 ) X 0 (1 25 nm ) -1 50 nm )

C

C

N

N

TD

sa

ro l

*** 100

50

0.4 0.3 0.2 0.1

l sa lin e C O D N O T- X (1 X D 0C O 3 N T- X (6 0nm D 0O 10 ) X 0 (1 25 nm ) -1 50 nm )

ro

TD

N

C

Figure 2.


ro l sa l N in Te D C O D N O T- X (1 X D 0C O 3 N T- X (6 0nm D 0O 10 ) X 0 (1 25 nm ) -1 50 nm )

0.0

co nt

ro l sa l N i n Te D C O D N O T- X (1 X D 0C O 3 N T- X (6 0nm D 0O 10 ) X 0 (1 25 nm ) -1 50 nm )

***

10

0.5

0

0

20

C

*

ALT (U/L)

200

30

0

0

C

300

***

40

150

***

C

H

ne y id

*** 15

co nt

co nt

AST (U/L)

K

20

ro

0

10-30 nm : 4.66 mg/kg 60-100 nm : 1.97 mg/kg 125-150 nm : 1.46 mg/kg

Drug Sacrifice

BUN (mg/dL)

5

CNT concentration

co nt

10

4

Drug RBC (x 106 cells/uL)

15

100

ee n

Ly m

3

Drug

***

400

Sp l

ra in B

Li

2

ro l sa l N in Te D C O D N O T- X (1 X D 0C O 3 N T- X (6 0nm D 0O 10 ) X 0 (1 25 nm ) -1 50 nm )

WBC (x 10 3 cells/uL)

Drug 20

ve r

ac h m

1

Crea (mg/dL)

0

co nt

Weeks

sa lin e C O D N X O T(1 X D 0C O 3 N T- X (6 0nm D 0O 10 ) X 0 (1 25 nm ) -1 50 nm )

b

St o

In

Lu

te st in

ng

e

0

co nt

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 30 of 36

Page 31 of 36

31

a

b 2

4

3

DOX (2 mg/kg)

PBS

60-100 nm

125-150 nm

4 +++

3 2 1

###

*

0

10-30 nm

BLI1

Control PBS

c

BLI2

BLI3

DOX CNT-DOX (10-30)

BLI4

CNT-DOX (60-100) CNT-DOX (125-150)

100

Survival rate (%)

CNT-DOX (2 mg/kg)

10-30 nm

DOX

5

Tumor area  luciferase intensity (A.U.)

1

PBS

BLI

125-150 nm 60-100 nm

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

ACS Applied Materials & Interfaces

50

0 3

6

9

12

15

18

21

24

27

Weeks

d Control

Saline

2 mg/kg

2 mg/kg

DOX

CNT-DOX (10-30) CNT-DOX (60-100) CNT-DOX (125-150)

Figure 3.



a

10-30 nm Free DOX CNT-DOX

60-100 nm 125-150 nm CNT-DOX CNT-DOX

b Uptake intensity (a.u.)

Nucleus DOX intensity (a.u.)

80

60000

40000

Macropinocytosis Clathrin 60

Caveolin

40

20

20000

0

0 DOX

DOX

10~30 nm 60~100 nm 125-150 nm

10-30 nm

c

+EIPA

+CPZ

+GEN

125-150 nm 60-100 nm 10-30 nm CNT-DOX CNT-DOX CNT-DOX CNT 10-30nm CNT-DOX 10-30nm

CNT 60-100nm CNT-DOX 60-100nm CNT-DOX 60-100nm (+Cav inhibitor)

CNT 125-150nm CNT-DOX 125-150nm

+CPZ (Clathrin)

+GEN (Caveolin)

* 100

80

*** ***

60

40

20

0

f

100 80 60 40 20 0

125-150 nm

120

10~30nm DOX

e

+EIPA (Macropinocytosis)

CTL

d Nucleus DOX intensity (a.u.)

CTL

60-100 nm CNT-DOX

CNT-DOX

Cell viability (%)

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 32 of 36

0

200

400

600

800

1000 DOX: ng/ml

0

0.35

0.69

1.03

1.38

1.72 DOX: mol/l

Figure 4.


60~100 nm CNT-DOX

125-150 nm

Page 33 of 36 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


References

(1) Petros, R. A.; DeSimone, J. M. Strategies in the design of nanoparticles for therapeutic applications. Nature reviews. Drug discovery 2010, 9, 615-627, DOI: 10.1038/nrd2591. (2) Ernsting, M. J.; Murakami, M.; Roy, A.; Li, S. D. Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles. Journal of controlled release : official journal of the Controlled Release Society 2013, 172, 782-794, DOI: 10.1016/j.jconrel.2013.09.013. (3) Nalwa, H. S. A special issue on reviews in nanomedicine, drug delivery and vaccine development. Journal of biomedical nanotechnology 2014, 10, 1635-1640. (4) Chaudhary, S.; Umar, A.; Bhasin, K. K.; Singh, S. Applications of Carbon Dots in Nanomedicine. Journal of biomedical nanotechnology 2017, 13, 591-637, DOI: 10.1166/jbn.2017.2390. (5) Veiseh, O.; Gunn, J. W.; Zhang, M. Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging. Advanced drug delivery reviews 2010, 62, 284-304, DOI: 10.1016/j.addr.2009.11.002. (6) Sanvicens, N.; Marco, M. P. Multifunctional nanoparticles – properties and prospects for their use in human medicine. Trends in Biotechnology 2008, 26, 425-433, DOI: https://doi.org/10.1016/j.tibtech.2008.04.005. (7) Toy, R.; Peiris, P. M.; Ghaghada, K. B.; Karathanasis, E. Shaping cancer nanomedicine: the effect of particle shape on the in vivo journey of nanoparticles. Nanomedicine 2014, 9, 121-134, DOI: 10.2217/nnm.13.191. (8) Black, K. C.; Wang, Y.; Luehmann, H. P.; Cai, X.; Xing, W.; Pang, B.; Zhao, Y.; Cutler, C. S.; Wang, L. V.; Liu, Y.; Xia, Y. Radioactive 198Au-doped nanostructures with different shapes for in vivo analyses of their biodistribution, tumor uptake, and intratumoral distribution. ACS nano 2014, 8, 4385-4394, DOI: 10.1021/nn406258m. (9) Singh, R.; Pantarotto, D.; Lacerda, L.; Pastorin, G.; Klumpp, C.; Prato, M.; Bianco, A.; Kostarelos, K. Tissue biodistribution and blood clearance rates of intravenously administered carbon nanotube radiotracers. Proceedings of the National Academy of Sciences of the United States of America 2006, 103, 3357-3362, DOI: 10.1073/pnas.0509009103. (10) Truong, N. P.; Whittaker, M. R.; Mak, C. W.; Davis, T. P. The importance of nanoparticle shape in cancer drug delivery. Expert opinion on drug delivery 2015, 12, 129-142, DOI: 10.1517/17425247.2014.950564. (11) Tan, A.; Yildirimer, L.; Rajadas, J.; De La Pena, H.; Pastorin, G.; Seifalian, A. Quantum dots and carbon nanotubes in oncology: a review on emerging theranostic applications in nanomedicine. Nanomedicine 2011, 6, 1101-1114, DOI: 10.2217/nnm.11.64. (12) Donaldson, K.; Poland, C. A.; Murphy, F. A.; MacFarlane, M.; Chernova, T.; Schinwald, A. Pulmonary toxicity of carbon nanotubes and asbestos - similarities and differences. Advanced drug delivery reviews 2013, 65, 2078-2086, DOI: 10.1016/j.addr.2013.07.014. (13) Yang, S. T.; Wang, X.; Jia, G.; Gu, Y.; Wang, T.; Nie, H.; Ge, C.; Wang, H.; Liu, Y. Longterm accumulation and low toxicity of single-walled carbon nanotubes in intravenously exposed mice. Toxicology letters 2008, 181, 182-189, DOI: 10.1016/j.toxlet.2008.07.020. (14) Liu, Z.; Cai, W.; He, L.; Nakayama, N.; Chen, K.; Sun, X.; Chen, X.; Dai, H. In vivo biodistribution and highly efficient tumour targeting of carbon nanotubes in mice. Nature



Page 34 of 36 34

nanotechnology 2007, 2, 47-52, DOI: 10.1038/nnano.2006.170. (15) Jiang, J., W.; Kim, B. Y.; Rutka, J. T.; Chan, W. C. Nanoparticle-mediated cellular response is size-dependent. Nature nanotechnology 2008, 3, 145-150, DOI: 10.1038/nnano.2008.30. (16) Gratton, S. E.; Ropp, P. A.; Pohlhaus, P. D.; Luft, J. C.; Madden, V. J.; Napier, M. E.; DeSimone, J. M. The effect of particle design on cellular internalization pathways. Proceedings of the National Academy of Sciences of the United States of America 2008, 105, 11613-11618, DOI: 10.1073/pnas.0801763105. (17) Kang, B.; Chang, S.; Dai, Y.; Yu, D.; Chen, D. Cell response to carbon nanotubes: sizedependent intracellular uptake mechanism and subcellular fate. Small 2010, 6, 2362-2366, DOI: 10.1002/smll.201001260. (18) Yameen, B.; Choi, W. I.; Vilos, C.; Swami, A.; Shi, J.; Farokhzad, O. C. Insight into nanoparticle cellular uptake and intracellular targeting. Journal of controlled release : official journal of the Controlled Release Society 2014, 190, 485-499, DOI: 10.1016/j.jconrel.2014.06.038. (19) Bawa, R.; Fung, S. Y.; Shiozaki, A.; Yang, H.; Zheng, G.; Keshavjee, S.; Liu, M. Selfassembling peptide-based nanoparticles enhance cellular delivery of the hydrophobic anticancer drug ellipticine through caveolae-dependent endocytosis. Nanomedicine 2012, 8, 647-654, DOI: 10.1016/j.nano.2011.08.007. (20) Sundaramoorthy, P.; Ramasamy, T.; Mishra, S. K.; Jeong, K. Y.; Yong, C. S.; Kim, J. O.; Kim, H. M. Engineering of caveolae-specific self-micellizing anticancer lipid nanoparticles to enhance the chemotherapeutic efficacy of oxaliplatin in colorectal cancer cells. Acta biomaterialia 2016, 42, 220-231, DOI: 10.1016/j.actbio.2016.07.006. (21) Barua, S.; Yoo, J. W.; Kolhar, P.; Wakankar, A.; Gokarn, Y. R.; Mitragotri, S. Particle shape enhances specificity of antibody-displaying nanoparticles. Proceedings of the National Academy of Sciences of the United States of America 2013, 110, 3270-3275, DOI: 10.1073/pnas.1216893110. (22) Kim, S. W.; Lee, Y. K.; Kim, S. H.; Park, J. Y.; Lee, D. U.; Choi, J.; Hong, J. H.; Kim, S.; Khang, D. Covalent, Non-Covalent, Encapsulated Nanodrug Regulate the Fate of Intra- and Extracellular Trafficking: Impact on Cancer and Normal Cells. Scientific reports 2017, 7, 6454, DOI: 10.1038/s41598-017-06796-7. (23) Lee, Y. K.; Choi, J.; Wang, W.; Lee, S.; Nam, T. H.; Choi, W. S.; Kim, C. J.; Lee, J. K.; Kim, S. H.; Kang, S. S.; Khang, D. Nullifying tumor efflux by prolonged endolysosome vesicles: development of low dose anticancer-carbon nanotube drug. ACS nano 2013, 7, 8484-8497, DOI: 10.1021/nn4041206. (24) Lai, P. S.; Lou, P. J.; Peng, C. L.; Pai, C. L.; Yen, W. N.; Huang, M. Y.; Young, T. H.; Shieh, M. J. Doxorubicin delivery by polyamidoamine dendrimer conjugation and photochemical internalization for cancer therapy. Journal of controlled release : official journal of the Controlled Release Society 2007, 122, 39-46, DOI: 10.1016/j.jconrel.2007.06.012. (25) Wong, H. L.; Bendayan, R.; Rauth, A. M.; Xue, H. Y.; Babakhanian, K.; Wu, X. Y. A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system. The Journal of pharmacology and experimental therapeutics 2006, 317, 1372-1381, DOI: 10.1124/jpet.106.101154. (26) Kim, S. W.; Kyung Lee, Y.; Yeon Lee, J.; Hee Hong, J.; Khang, D. PEGylated anticancer-carbon nanotubes complex targeting mitochondria of lung cancer cells. Nanotechnology 2017, 28, 465102, DOI: 10.1088/1361-6528/aa8c31.


Page 35 of 36


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



TOC image-ACS Applied Materials & Interfaces

60~100 nm

Clathrin

Caveolin

TOC image legend: Schematic image illustrates 60-100 nm width of anticancer carbon nanodrug those can eliminate deep lung tumor tissue through lung selective accumulation and by activation of caveolin channel.


Page 36 of 36

Destroying Deep Lung Tumor Tissue through Lung-Selective

Recommend Documents