BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 839 – 849
Original Article
nanomedjournal.com
Secretion of intestinal goblet cells: A novel excretion pathway of nanoparticles Baoquan Zhao, PhD a, 1 , Lan Sun, PhD a, 1 , Wuxu Zhang, MSc a, c , Yuxia Wang, MD, PhD a , Junjing Zhu b , Xiaoyu Zhu b , Liuzhong Yang, MM a , Chunqi Li, MD, PhD b , Zhenzhong Zhang c , Yingge Zhang, MD, PhD a,⁎ a
Institute of Pharmacology and Toxicology and Key Laboratory of Nanopharmacology and NanoToxicology, Beijing Academy of Medical Sciences, Beijing, China b Hangzhou Hunter Biotechnology Incorporation, Hangzhou, China c School of Pharmacy; and Nanotechnology Research Center for Drugs; Zhengzhou University, Zhengzhou, China Received 6 May 2013; accepted 18 October 2013
Abstract Understanding the excretion pathway is one of the most important prerequisites for the safe use of nanoparticles in biomedicine. However, the excretion of nanoparticles in animals remains largely unknown, except for some particles very small in size. Here we report a novel natural pathway for nanoparticle excretion, the intestinal goblet cell (GC) secretion pathway (IGCSP). Direct live observation of the behavior of 30-200 nm activated carbon nanoparticles (ACNP) demonstrated that ACNP microinjected into the yolk sac of zebrafish can be excreted directly through intestinal tract without involving the hepato-biliary (hap-bile) system. Histopathological examination in mice after ligation of the common bile duct (CBD) demonstrated that the intravenously-injected ACNP were excreted into the gut lumen through the secretion of intestinal GCs. ACNP in various secretion phases were revealed by histopathological examination and transmission electron microscopy (TEM). IGCSP, in combination with renal and hap-bile pathways, constitutes a complete nanoparticle excretion mechanism. From the Clinical Editor: Nanoparticle elimination pathways are in the forefront of interest in an effort to optimize and enable nanomedicine applications. This team of authors reports a novel natural pathway for nanoparticle excretion, the intestinal goblet cell (GC) secretion pathway (IGCSP). Direct live observation of the behavior of activated carbon nanoparticles has shown excretion directly through the intestinal tract without involving the hepato-biliary (hap-bile) system in a zebrafish model. © 2014 Elsevier Inc. All rights reserved. Key words: Nanoparticle; Excretion; Pathway; Goblet cell; Aggregation
The understanding of the excretion pathway becomes more and more important with the development of nanotechnology and the application of nanomaterials in biomedicine. It is important not only for the biosafety issue of engineered nanoparticles but also for the practical use of nanoparticles as diagnostic and therapeutic agents or as drug carriers, because excretion is the best way to cease the action of nanoparticles on tissues and cells. Based on the understanding of the excretion pathway, appropriate protocols can be worked out to deal with the nanoparticles once they entered the animal body. Unfortu-
This work was supported by the National Natural Science Foundation of China (No. 90406024), the National Basic Research Program of China (No. 2010CB933904) and Major New Drug Creations (No. 2011ZX09102-001-15). ⁎Corresponding author at: Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, PR China. E-mail address: [email protected] (Y. Zhang). 1 Equally contributed to the work.
nately, such pathway remains poorly understood, though there are many literatures on the clearance of nanoparticles from blood 1,2 or tissues such as lung 3,4 and liver. 5 These experiments provide information concerning the clearance mechanism to remove particles from local tissues rather than information on systemic excretion of nanoparticles. 6 Few studies have reported two main excretion pathways of intravenously-injected nanoparticles, the kidney–urine pathway and hepatobiliary system (HBS)–feces pathway. Kidney excretion of nanoparticles is limited to very small ones, such as quantum dots 7 and fullerines. 8 HBS excretes some larger nanoparticles, but the clearance rate is no more than 1% within 24 h, and there is an inverse relation between HBS excretion and sizes. 9 However, Manabe et al found that 500 nm latex particles can be cleared from medaca embryos, 10 suggesting that there are some other excretion pathways for nanoparticle excretion without involving the HBS. Souris et al reported that the concentration of intravenously-injected 50-100 nm silica nanoparticles in liver
1549-9634/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2013.10.004 Please cite this article as: Zhao B, et al, Secretion of intestinal goblet cells: A novel excretion pathway of nanoparticles. Nanomedicine: NBM 2014;10:839-849, http://dx.doi.org/10.1016/j.nano.2013.10.004
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was much lower than that in intestinal wall and feces. 11 This inconsistence in quantity does not support the hypothesis that HBS plays main roles in the intestinal excretion of nanoparticles. On the contrary, the high concentration of nanoparticles in intestinal wall 11 suggested that the intestinal wall may play important roles in the excretion of nanoparticles through intestinal tract. GCs are one of the four main cell types 12 in the intestinal membrane and have the ability to entrap nanoparticles. 13 Recently, there have been several studies on the interactions between GCs and orally-administered nanoparticles. 13–16 GCs can uptake nanoparticle, 17 which makes GCs the most possible candidate cells playing roles in the intestinal excretion of nanoparticles. Our earlier studies found that intravenously-injected nanoparticles had distribution in intestinal GCs 18 and hypothesized that intestinal wall may play roles in the excretion of nanoparticles but did not investigate in detail. 18,19 The present work carefully examined the mechanism for intestinal excretion of nanoparticles and revealed a novel pathway, IGCSP, for nanoparticle excretion. Methods Preparation and characterization of ACNP ACNP were prepared from medicinal activated carbon (MAC; Haichangqing Co. Ltd, Beijing, China) by a top-down method (supplemental materials). The diameters of ACNP were determined by atomic force microscope (AFM), scanning electron microscope (SEM) and Laser Particle Size and Zeta Potential Analyzer. The internal crystal structures were detected by X-ray defraction (XRD). For injection, 1 mg ACNP was added into 100 mL normal saline and suspended in an ultrasound field of 40 Hz, 180 W for 20 min. Handling of the animals Zebrafish eggs were collected, cleaned, and washed with eggwater 20 within one hour after fertilization. Eggs from different females were pooled, placed in 7 cm sterile Petri dishes containing eggwater, and incubated at 28.0 to 28.5 °C. Embryonic fish were stocked in an Aquatic Habitat re-circulating tank system at 28.5 °C with a 14 h light/10 h dark cycle. The water was purified by reverse osmosis and adjusted to pH 7 and conductivity of 350 μS. The mice were handled as described previously. 21 Briefly, 20 Kunming mice (20-25 g in body weight) were constantly monitored and fed fluid nutritional diet free from pathogens and particulate materials for 10 days before ACNP treatment to avoid the influences of ingested particles on the observation of ACNP. All the animal procedures were approved by the Animal Subject Review Committee of the Beijing Academy of Medical Science.
Microinjection and observation of ACNP in zebrafish Embryonic zebrafish at the age of 24 h were put into a 7-cm culture dish and anesthetized by addition of tricaine methanesulfonate into the dish at a final concentration of 0.64 mM. The anesthetized zebrafish were placed in the slanting grooves of a silica gel sheet. Excessive water around the fish was absorbed with filter papers to leave just enough water to bathe the fish body. Five μL suspension of ACNP at the concentration of 5 mg/mL was injected into the yolk sac of zebrafish in an IM 300 microinjection instrument (Narishige, Japan) (Figure S3). All ACNP suspensions were freshly dispersed by ultrasonication for 10 min before use. During the whole period of experiment, zebrafish that received microinjection of ACNP exhibited no significant differences in death rate, development, teratogenesis, and cardiovascular toxicity in comparison with control (Figure S4). Ligation of common bile duct and injection of ACNP into the mice The common bile duct was ligated with the method originally described by Cameron and Oakley 23 with modifications. Briefly, Kunming mice of 20-25 g were anaesthetized with pentobarbital (25 mg/kg) and fixed onto a wood surgical sheet. A midabdominal incision was made, and the abdominal tissues were separated carefully to clearly expose the CBD. Two sterile nylon medical surgical sutures (Unic Surgical Sutures, Mfg., Co., Ltd., Suzhou, China), 0.2 mm in diameter, were put through under the CBD, and two nodes were made at both ends of a segment of CBD (Figure S5), and the CBD was then cut off between the two ends. After closure of abdomen, ACNP were suspended in 0.9% NaCl to a final concentration of 5 mg/mL and injected through tail veins in a dose of 50 mg/kg (i.e., 10 mL/kg). All the suspensions of ACNP were freshly dispersed by sonication for 10 min before use. After the injection of ACNP, the animals' body weight, behavior, and number of blood cells were monitored (Figure S6). At the end of the experiments, these physical parameters showed no significant differences from those of control (Figure S6). Histopathological examination On the 4th day after injection of ACNP, the mice were anaesthetized and their internal organs were taken out and fixed in 40% formaldehyde. 5 μM thick sections were made on a Lecia RM2135 Rotary Microtome. The sections were stained in hematoxylin and eosin or alcian blue and observed under an Olympus BH2 phase contrast microscope (Japan). Quantitative evaluation of the efficiency of IGCSP for ACNP
Pigment inhibition in zebrafish Pigmentation genes were suppressed by adding 1-phenyl, 2-thiourea (PTU) (Sigma, USA) into fish water in a final concentration of 75 μM in the developmental stage of 28 somites. 22 This concentration produced enough pigment inhibition with no adverse effects on the hatching and survival.
The quantitative evaluation was carried out with rates occupied by ACNP-containing GCs in the total number of the GCs seen under a phase light microscope in 10 visions in 3 HEstained histopathological sections, which were calculated by a formula: Rates (%) = Number of ACNP containing GCs/total number of seen GCs×100%. 120 Mice without CBD ligation were randomly divided into 4 groups and 30, 60, 100 and
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Figure 1. ACNP characteristics. (A) The AFM images of the prepared ACNP of 30, 60, 100, and 200 nm. (B) The SEM image of 100 nm ACNP (left), which is spheric with many micropores in the sphere (right).
200 nm ACNP were intravenously injected respectively. The different organs of every 3 mice in each group were respectively taken out for quantitative histopathological examination on day 1-180 after ACNP injection. Results
Because HBS is a known excretion pathway of nanoparticles, we paid special attention to the distribution of ACNP in liver and gallbladder. Surprisingly, there were little ACNP found in liver and no ACNP in gallbladder (Figure 3, D), which does not support the hypothesis that HBS is the main pathway for in the excretion of ACNP.
Characteristics of ACNP
Excretion of ACNP through intestinal tract of zebrafish
Structurally, ACNP belong to a large family of carbonbased nanoparticles composed of graphene sheets 24–26. The prepared ACNP had diameters of 30, 60, 100, and 200 nm, respectively, as imaged with AFM (Figure 1, A) and SEM (Figure 1, B), which are spheres with mesopores (Figure 1, B). XRD revealed that the graphite microcrystals of ACNP have a thickness of 0.707 nm, a width of 2.007 nm and a layer distance of 0.372 nm. The ζ electropotentials are 0.65-6 mV in pH 7.4 saline. The measurements of some structures and properties of ACNP in different sizes are listed in Table S1. Withdrawing test demonstrated that the suspension was stable within 2 h.
Since there were little distribution of ACNP in liver and no ACNP found in gallbladder, there should be no ACNP in intestinal tract. Again to our surprise, a great amount of ACNP was observed in and excreted through intestinal tract with the peristalsis of the intestines as demonstrated in Figure 2. After microinjection, ACNP appeared as small aggregated particles (circles) in the yolk sac at the fourth hour, when the intestinal tract had not developed (Figure 2, A). On the third day, the intestinal tract (green arrow) had developed, and most yolk sac had been absorbed with a small part remaining (yellow arrow). No ACNP were in the intestinal tract; most ACNP still existed in the residual yolk sac, and some of them appeared in the tissues between intestinal wall (red arrow) and abdominal skin (Figure 2, B). On the fourth day, yolk sac was completely absorbed. ACNP began to appear in the intestinal wall (red arrows) and gut lumen (green arrow) (Figure 2, C). On the fifth day, more ACNP entered the intestinal tract (green arrow, Figure 2, D). On the sixth day, the quantity of ACNP in intestinal tract (green arrow) reached its peak (Figure 2, E). On the seventh day, ACNP had moved downward to the lower segment of the gut lumen with the intestinal peristalsis and were finally excreted through cloaca (yellow arrow, Figure 2, F). On the eighth day, while previously observed ACNP had been completely excreted out, ACNP continuously entered the intestinal tract from other tissues (Figure 2, G). These results suggested that
Tissue distribution of ACNP in zebrafish Under microscope, the black ACNP were well visible in the transparent embryonic zebrafish. The suspension of ACNP appeared in the yolk sac as cloud-like immediately after the injection (Figure S3, B). Within 3 h, most ACNP aggregated into larger recognizable particles (Figure S3, B). In 0-4 h after injection, ACNP mainly accumulated in the yolk sac (Figure 2, A). With the development of zebrafish, ACNP gradually distributed to intestinal wall (Figures 2 and 3, A), extracranial tissues (Figure 3, B), myocardium (Figure 3, C), kidney (Figure 3, D), and blood vessels (Figure 3, E).
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Figure 2. The dynamic changes of ACNP in gut lumen of zebrafish after ACNP microinjection in yolk sac. (A) At the fourth hour. (B) On the third day. (C) On the fourth day. (D) On the fifth day. (E) On the sixth day. (F) On the seventh day. (G) On the eighth day.
the yolk sac injected ACNP were effectively excreted through intestinal tract. Blood transportation of ACNP in zebrafish The above results gave rise to the question of how ACNP got to the intestinal tract. To answer this question, we first need to know the transportation of ACNP in animal body. The observation of the behavior of ACNP in blood vessels demonstrated that yolk sac-injected ACNP was transported through blood circulation. With the development of cardiovascular system in zebrafish, ACNP were first found to migrate from tissues to the wall of blood vessels. Figure 3, E demonstrated the case of ACNP in the wall of zebrafish tail veins. The yolk sac microinjected ACNP first migrated to
and then temporarily accumulated in the walls of blood vessels, where they were finally released into the blood stream. After injection, ACNP in the walls of blood vessels gradually decreased with their release into the blood stream. These results indicated that the ACNP were transported by the blood stream. Elimination of ACNP from various tissues To obtain the data supporting the excretion of ACNP from various tissues through intestinal tract, we investigated the relations between the elimination and intestinal excretion of ACNP. Results demonstrated that the intestinal excretion of ACNP was accompanied by their elimination from various tissues. ACNP in the tissues around the upper end of intestinal
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Figure 3. Dynamic changes of ACNP in the tissues outside of intestinal tract and the relation between ACNP and the mucous of GCs in the intestines of zebrafish. (A) ACNP in TAUEI (red circles). (B) ACNP (arrows) in the extracranial tissues. (C) ACNP (arrows) in the myocardium. (D) ACNP in the kidney, liver, and gallbladder. Blue circle: kidney; red circle: gallbladder; green circle: liver. (E) ACNP in the wall of tail veins. hpt: hours post treatment; dpt: days post treatment. (F) Alcian blue staining of the control intestines of zebrafish. (G) Alcian blue staining of the intestinal wall of zebrafish with yolk sac injection of ACNP.
tract (UEIT) had the fastest and most complete elimination, though there was a temporary increment on the fifth day (Figure 3, A). Vast majority of the ACNP in UEIT was eliminated on the sixth day. On the eighth day after injection, the ACNP in UEIT were completely eliminated. In extracranial tissues, ACNP gradually decreased, and there were significantly less ACNP on the eighth day (Figure 3, B). The yolk sacmicroinjected ACNP also distributed in myocardial muscles and were eliminated slowly too. On the eighth day, ACNP in the myocardium significantly decreased (Figure 3, C). In the kidney, there was little distribution of ACNP, which were eliminated slowly. On the eighth day after ACNP microinjection in yolk sac, there were still some ACNP in the kidney. The ACNP in the wall
of blood vessels had the slowest elimination. On the eighth day, there were still some ACNP in the vascular walls (Figure 3, E), which may be explained by the function of blood vessel walls as a relay station in the process of ACNP transportation from tissues to blood stream; therefore, a complete elimination from the walls of blood vessels is possible only after the complete elimination of ACNP from other tissues over the whole body. The relation between ACNP and the mucus in the intestinal membrane of zebrafish In the HBS pathway, nanoparticles cross the intestinal wall with the bile through CBD. To understand how ACNP got into intestinal tract through the intestinal wall without involving of
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Figure 4. ACNP in the intestinal contents of the mice with CBD ligation. (A) The schematic illustration of the relations of HBS with intestinal tract. (B) The ligation of common bile duct. (C) ACNP (arrows) in the intestinal contents of mice injected with ACNP through tail vein.
HBS, we observed the cellular distribution of ACNP in intestinal wall of zebrafish. In alcian blue-stained histopathological sections the mucus was stained into blue color (Figure 3, F) and ACNP appeared as dense black particles (Figure 3, G) in the mucous, suggesting that ACNP may be secreted by GC in the intestinal mucous membrane and enter into gut lumen with mucus.
transported only by the blood stream. In these experiments, ACNP were again observed in the intestinal contents of mice fed particle-free foods 21 (Figure 4, C) and injected with 30-200 nm ACNP while there were no particles seen in those of the control mice (Figure 4, C), which provided a conclusive evidence for non-HBS intestinal pathway of excreting ACNP.
Intestinal excretion of ACNP in mice with CBD ligation
Cell distribution of ACNP in intestinal wall in mice with CBD ligation
To definitely prove that the intestinal excretion of ACNP without involving HBS, we carried out experiments in mice with CBD ligation (Figures 4, A and B, S5), which absolutely blocked the connection between HBS and intestinal tract (Figure 4, A and B). The intravenous injection made sure that ACNP was
As shown in Figure 5, in HE stained sections, the main cell populations of intestinal wall included the absorptive columnar epithelial cells (CECs) and excretory GCs. Each GC contained a large vacuole of mucus contents, which made them easily be recognized (Figures 5, A1 and 3). There were no black particles
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Figure 5. The distribution of ACNP in intestinal GCs of mice with CBD ligation. (A) The histopathological microsection (HE staining) of the jejunum and colon tissues of the mice treated with ACNP injection through tail veins at the dose of 50 mg/kg, once a day for 10 days. The GCs were easily identified for containing a large intracellular vacuole because of the mucins. (A1) The jejunum of control mice treated with saline injection through tail vein; (A2) The jejunum tissue of the mice treated with intravenous ACNP injection; (A3) The colon tissue of the control mice with intravenous saline injection; (A4) The colon tissue of the mice treated with intravenous ACNP injection. (B) The secretion of ACNP by intestinal GCs of the mice treated with intravenous ACNP injection. (B1) Some ACNP ready to be secreted by a GC (arrow); (B2) About half of ACNP had been expelled out from GC (arrows); (B3) Majority of ACNP had been expelled out from a GC (arrow); (B4) All of the ACNP had been completely expelled out of a GC (arrow). (C) The TEM images of different phases of the secretion of ACNP from GCs. C1: the ACNP in GC (yellow arrow); (C2) Some ACNP were being secreted from GC (green arrow) while some were still inside the cell (yellow arrow); (C3) Some ACNP had been secreted out from GC (red arrow) while some were being secreted (green arrow) and some were still in GC (yellow arrow). (C4) Some ACNP had entered into the gut lumen (red arrow) and some had just been secreted out (green arrow); and some were still inside the GC (yellow arrows).
in all intestinal tissue cells in control mice (Figures 5, A1 and 3), while many ACNP were distributed in GCs in mice intravenously injected with ACNP (Figures 5, A2 and 4). There were no ACNP found in CECs. With the increase of GCs, the ACNP in the intestinal wall increased (Figure 5, A3 and 4). These results were in coincidence with those obtained by the observation of alcian blue-stained sections of the intestines of zebrafish, suggesting that ACNP entered into gut lumen through GCs. Secretion phase of ACNP from GCs Histopathological examination further revealed the roles of GC in excretion of ACNP. In HE-stained histopathological sections, ACNP were seen being excreted by GCs. As shown in
Figure 5, B, ACNP were spotted both in the fused intracellular secretory vacuoles and in the vesicles undergoing exocytosis. Figure 5, B1 shows some ACNP located at the margin of a GC ready to secrete. Figure 5, B2 shows some ACNP passing through the membrane of a GC. Figure 5, B3 displays some ACNP that had been secreted out by a GC and some were still in it. Figure 5, B4 shows that some ACNP had been completely expelled out from a GC by exocytosis. TEM also revealed ACNP in various secretory phases in GCs (Figure 5, C). Figure 5, C1 shows the ACNP inside a secretory vesicle of GC, Figure 5, C2 demonstrates the scene of ACNP being secreted by a GC. In Figure 5, C3, some ACNP had already been expelled out of a GC, and Figure 5, C4 indicates that ACNP had been secreted into the gut lumen from GCs.
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Figure 6. The time course of IGCSP for ACNP excretion and its relations with sizes. (A) Histopathological observation of the changes in quantity of ACNP in some important internal organs of mice within 180 days. (B) The time course of intestinal ACNP excretion and its relations with sizes.
Time course and the effect-size relation of IGCSP The excretion of ACNP through IGCSP had a rather long time course. As shown in Figure 6, A, the intravenously-injected ACNP of 200 nm were widely distributed in various organs or tissues including liver, which was different from the case in zebrafish because of the difference in the way of injection. The ACNP in various tissues decreased obviously on day 30. The half elimination time was about 2 months. The complete excretion seemed to need 180 days (Figure 6). Taking the rates occupied by ACNP-containing GCs in total number of the observed GCs (RAC) as a parameter, the time course of the excretion of ACNP from mouse bodies may be studied quantitatively, which was shown in Figure 6, B. 30-200 nm ACNP appeared in the GCs of intestinal mucous membrane within 30 min, reached its highest value on day 10, began to decrease on day 30 and completely eliminated within 180 days. These results indicated that IGCSP was a highly efficient pathway for the excretion of nanoparticles, though it was very slow in speed. It can be observed that the height of RAC–time curves increased with the increment in sizes, indicating a positive correlation of the ability of IGCSP with sizes, which was identical with the results of Manabe et al. 3 RAC– time curves of 30-200 nm ACNP were quite similar, which indicate that IGCSP is the common mechanism for the excretion of ACNP with different sizes. Aggregation modes of ACNP in tissues and cells As shown in Figures 2-6, the ACNP existed in tissues and cells in clusters formed by aggregation. There were two modes
for ACNP aggregation. In tissues of zebrafish and the cells of liver, spleen, kidney and lung of mice, the aggregation of ACNP took Eden mode. 27 The clusters appeared as spots with a high density. In GCs, the aggregation of ACNP took DLA mode. 28 The clusters were dendritic with a loose configuration. These results indicated that NPs take different aggregation modes in different tissues or cells. Discussion To study the excretion of nanoparticles, we selected biologically inert ACNP as a model, because many nanoparticles used as diagnostic agents and drug carriers contain metal or nonmetabolizable cores such as gold, 29 iron, 30 silica, 31 and carbonaceous nanoparticles, 25,26 though their organic out-shells can be degraded by various enzymes. 32 ACNP, as carbonaceous mesoporous nanoparticles, have been used as drug carriers. 33,34 Because of their biological inertness, ACNP represent pure “particulate materials.” Although the excretion of nanoparticles is influenced by their chemical activities, their particulate properties are also an important affecting factor. The results of the present work are significant for the evaluation of the particulate effects of the non-metabolizable nanoparticles and the non-metabolizable cores of nanoparticles. From the inertness point of view, the results obtained from ACNP can be extrapolated to other non-metabolizable nanoparticles and the non-metabolizable cores of nanoparticles on their excretion. Of course, further investigations are required, since the present work is only a start point for the relative studies.
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Aggregation is a common phenomenon for nanoparticles. To fulfill the needs of injection and blood transportation, aggregation must be prevented. Ultrasonication just before injection can make ACNP disperse in suspension. After they were injected into yolk sac or veins, the concentration of ACNP was greatly decreased by the dilutive effects of yolk sac fluid and blood, which prevent ACNP from aggregation. At the same time, the movement of animals and the flow of blood are also helpful for prevention of the aggregation. However, the case in tissues and cells was completely different from that in blood. The concentration of ACNP in tissues would be much higher than that in blood because of EPR effects, and the flow of tissue fluid was much slower than that of blood. The case in cells that can phagocytose ACNP was the same as that in tissues: high ACNP concentration and low flow rate, which explain the aggregation of ACNP in tissues and cells. There were two aggregation modes for ACNP in tissues and cells: the Eden mode 27 and the DLA mode, 28 which can be more clearly seen in hitopathological sections of mice, because the magnification of the direct observation of ACNP clusters in living zebrafish was too low to recognize the details. In tissue space and cells of liver, spleen, lungs and kidney of mice, the aggregation took Eden mode, in which the clusters were spots with high density. 27 In intestinal GCs, the aggregation took DLA mode, in which the clusters were dendritic with a loose configuration. 28 These results suggested that the aggregation of nanoparticles is a rather complex process and the growth of clusters can take different modes in different microenvironments. GCs had unique mucin contents, and, therefore, ACNP aggregation in them took unique DLA mode. Whether biological systems can remove or excrete clusters of nanoparticles is another question requiring answers for practical use of nanoparticles. The results of this study indicate that GCs have the capability to secrete clusters of nanoparticles. As for the clusters in tissues and cells, the removing process may be more complex. They must disaggregate into dispersed nanoparticles or clusters small enough for blood transportation, which is theoretically possible. The concentration of ACNP in the blood decreased gradually with time after the injection. When blood concentration was low enough, the ACNP in tissues began to enter into blood, and hence the concentration of ACNP in tissues began to decrease. When tissue concentration was low enough, some of ACNP at the edge of cluster tended to separate; leading to the decrease in size and finally disaggregation of the cluster. In spite of the difficulty, this process must happen, sooner or later, faster or slower. As discussed above, the aggregation only influences the speed but cannot prevent the excretion of nanoparticles. To understand how the ACNP cross the intestinal wall and are released into the gut lumen, HBS was firstly considered because it is a well-known excretion system. There have been many literatures that believe nanoparticles can be excreted through HBS based on the results that nanoparticles can be simultaneously seen in liver and intestinal tract. In the late 1990s, Renaud et al found that the low-density lipoprotein20 nm colloidal gold (CG) could be excreted through HBS but albumin- or polyvinylpyrrolidone-CG could not. 35 This selective excretion was rationally attributed to the function of
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the lipoprotein receptor of liver cells. In other words, the nanoparticles without low-density lipoproteins could not be excreted through HBS. Later studies found that negatively charged gold nanoparticles 9 and polystyrene carboxylated nanoparticles 36 could also be excreted through HBS, although it was limited to no larger than 20 nm and the excretion quantity was very low. Souris et al 11 reported that 80 nm nanoparticles can be excreted through intestinal tract and the concentration in feces was very high while the concentration in liver was very low. In comparison with liver, the nanoparticle concentration was much higher in intestinal wall. It seems easier to explain the intestinal excretion of nanoparticles by intestinal secretion than by HBS pathway. More recently, in one study by Manabe et al, it was found that that nanoparticles as large as 500 nm, far beyond the ability of HBS, can be eliminated from fish body and the elimination of 500 nm particles was faster and more efficient than 50 nm particles no matter whether modified or not, 10 which cannot be explained by HBS pathway. Obviously, there were paradoxes between the results of the experiments on HBS for nanoparticle excretion. These paradoxes are unexplainable based on the belief that HBS is the only pathway for excretion of nanoparticles through intestinal tract. Especially, the results of Manabe et al strongly suggest the existence of another pathway for intestinal excretion of nanoparticles without involving HBS, and the results of Souris et al suggest that intestinal secretion is a possible pathway for nanoparticle excretion, since the concentration of nanoparticles was more identical with that in feces than that in liver. 11 Based on previous reports, we also believed that HBS is the most possible pathway for nanoparticle intestinal excretion. However, surprisingly, there was only a little distribution of ACNP in the liver and no ACNP in gallbladder during the whole period of experiments in zebrafish. These observations do not support HBS pathway because gallbladder is the only organ to store bile and CBD is the only way for excretion of ACNP from liver to intestinal tract. Thus, we believe that there must be an alternative mechanism for ACNP to enter the intestinal tract, which does not involve HBS. To confirm the existence of this pathway, we carried out the experiments in mice with ligation of CBD in mice, which completely cut off the way that ACNP must pass through to get to gut lumen from liver. As it was anticipated, ACNP were still observed in the feces of these mice with CBD ligation. The histopathological and TEM examination revealed that the mechanism of such non-HBS pathway for nanoparticle excretion was through the secretion of intestinal GCs. The distribution of ACNP only in GCs suggested IGCSP, which was then proved by the secretion phase observed in histopathological sections and TEM ultrasections. IGCSP had a long time course, which is quite in favor of that nanoparticles are used as drug carriers for long lasting treatments of chronic diseases. 37–39 As demonstrated in zebrafish and mice, most intravenously-injected ACNP can be excreted through IGCSP, which greatly ameliorates our concern about the eternal existence of biologically inert nanoparticles in animal body. GC is a main cell type of the intestinal mucosal epithelium, which undergoes continuous cycles of renewal. 40
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The major function of intestinal GCs and their main secretory product, mucin, is the formation of mucus overlying layers on intestinal membranes, which promotes the elimination of gut contents and provides the first line of defense against physical and chemical injury caused by ingested food, microbes and the microbial products. 13,41 There have been no reports on the roles of GC in the intestinal excretion of nanoparticles, let alone the mechanism for ACNP to enter into GC. In our experiments with zebrafish, the yolk sac-injected ACNP were distributed in various tissues with the formation of organs and then was transported by blood flow to intestines and excreted through intestinal tract, which suggested that the primary tissue distribution was related to the development of tissues and ACNP may enter GC during the differentiation. Because GC continuously undergo cycles of renewal, it is considered that the mechanism for ACNP getting into GC may be related to the differentiation of GC. The ACNP in GC combined or mixed with mucus and then were excreted into gut lumen by GC along with the secretion of mucus. There are also studies on the interaction between intra-tracheal nanoparticles and GCs in the airway 42,43; however, we didn't examine the distribution of ACNP in airway GC because the airway is not a main excretion organ for solid substance. So, IGCSP was the main pathway for the excretion of nanoparticles. In the present work, zebrafish and mice were used as animal models. Zebrafish has been used for drug screening. 19,44,45 Mice have gene sequences 80% identical with human and are frequently used as animal models in basic life science of human and preclinical studies on therapeutic agents and methods. 46 Both of them are vertebrate animals with similar intestinal tissues composed of mainly columnar and goblet cells. Therefore, the results of the present study are of great significance for evaluating the biosafety of nanoparticles in human. To date, IGCSP for nanoparticles has been found not only in marine animals but also in mammals. This pathway is especially important for those larger nanoparticles without modification, because the kidney 7 and HBS are only able to excrete nanoparticles modified with various chemical structures in small sizes. 8,11 Considering that many nanoparticles used as diagnostic and therapeutic agents have metal cores and are relatively large, 47–49 in the sizes that are beyond the excretory capability of kidney and HBS, this novel IGCSP provided greater promise for future application of nanoparticles in biomedical fields by eliminating the concerns about the persistent effects of nanoparticles. Obviously, the present results are only a starting point for the studies on IGCSP. The details, such as how ACNP cross the cell membrane and get into the cells and how they are secreted by the intestinal GCs, warrant further investigations. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2013.10.004.
References 1. Dreaden EC, Austin LA, Mackey MA, El-Sayed MA. Size matters: gold nanoparticles in targeted cancer drug delivery. Ther Deliv 2012;3(4):457-78. 2. Wahajuddin, Arora S. Superparamagnetic iron oxide nanoparticles: magnetic nanoplatforms as drug carriers. Int J Nanomedicine 2012;7:3445-71. 3. Zarogoulidis P, Chatzaki E, Porpodis K, Domvri K, Hohenforst-Schmidt W, Goldberg EP, Karamanos N, Zarogoulidis K. Inhaled chemotherapy in lung cancer: future concept of nanomedicine. Int J Nanomedicine 2012;7:1551-72. 4. Elder A, Gelein R, Finkelstein JN, Driscoll KE, Harkema J, Oberdorster G. Effects of subchronically inhaled carbon black in three species. I. Retention kinetics, lung inflammation, and histopathology. Toxicol Sci 2005;88:614-29. 5. Feng Y, Zong Y, Ke T, Jeong EK, Parker DL, Lu ZR. Pharmacokinetics, biodistribution and contrast enhanced MR blood pool imaging of GdDTPA cystine copolymers and Gd-DTPA cystine diethyl ester copolymers in a rat model. Pharm Res Aug. 2006;23:1736-42. 6. Hagens WI, Oomen AG, de Jong WH, Cassee FR, Sips AJ. What do we (need to) know about the kinetic properties of nanoparticles in the body? Regul Toxicol Pharmacol 2007;49:217-29. 7. Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, Bawendi MG, Frangioni JV. Renal clearance of quantum dots. Nat Biotecnol 2007;25:1165-70. 8. 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. Proc Natl Acad Sci U S A 2006;103(9):3357-62. 9. Hirn S, Semmler-Behnke M, Schleh C, Wenk A, Lipka J, Schäffler M, Takenaka S, Möller W, Schmid G, Simon U, Kreyling WG. Particle sizedependent and surface charge dependent biodistribution of gold nanoparticles after intravenous administration. Eur J Pharm and Biopharm 2011;77:407-16. 10. Manabe M, Tatarazako N, Kinoshita M. Uptake, excretion and toxicity of nano-sized latex particles on medaka (Oryzias latipes) embryos and larvae. Aquat Toxicol 2011:105576-81. 11. Souris JS, Lee CH, Cheng SH, Chen CT, Yang CS, Ho JA, Mou CY, Lo LW. Surface charge-mediated rapid hepatobiliary excretion of mesoporous silica nanoparticles. Biomaterials 2010;31:5564-74. 12. Kim YS, Ho SB. Intestinal goblet cells and mucins in health and disease: recent insights and progress. Curr Gastroenterol Rep 2010;12:319-30. 13. Behrens I, Pena AIV, Alonso MJ, Kissel T. Comparative uptake studies of bioadhesive and non-bioadhesive nanoparticles in human intestinal cell lines and rats: the effect of mucus on particle adsorption and transport. Pharm Res 2002;19:1185-93. 14. Jin Y, Song YP, Zhu X, Zhou D, Chen CH, Zhang ZR, Huang Y. Goblet cell-targeting nanoparticles for oral insulin delivery and the influence of mucus on insulin transport. Biomaterials 2012;33:1573-82. 15. des Rieux A, Fievez V, Garinot M, Schneider YJ, Préat V. Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach. J Control Releas 2006;116:1-27. 16. Dong YC, Feng SS. Poly(d, l-lactide-co-glycolide)/montmorillonite nanoparticles for oral delivery of anticancer drugs. Biomaterials 2005;26:6068-76. 17. Wang YY, Lai SK, Suk JS, Pace A, Cone R, Hanes J. Addressing the PEG mucoadhesivity paradox to engineer nanoparticles that “slip” through the human mucus barrier. Angew Chem 2008;47:9575-781. 18. Sun L, Yang LZ, Zhang YG. Study on the excretion pathway of nanoparticles. Chin Pharm J 2006;41:366-7. 19. Wang K, Ma JB, He M, Gao G, Xu H, Sang J, Wang YX, Zhao BQ, Cui DX. Toxicity assessments of near-infrared upconversion luminescent LaF3:Yb, Er in early development of zebrafish embryos. Theranostics 2013;3:258-66. 20. Westerfield M. The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio). Eugene, OR: University of Oregon Press; 1993.
B. Zhao et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 839–849 21. Yang Z, Zhang Y, Yang Y, Sun L, Han D, Li H, Wang C. Pharmacological and toxicological target organelles and safe use of single-walled carbon nanotubes as drug carriers in treating Alzheimer disease. Nanomedicine:NBM 2010;6:427-41. 22. Karlsson J, von Hofsten J, Olsson P-E. Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development. Mar Biotechnol 2001;3:522-7. 23. Cameron GR, Oakley CL. Ligation of the common bile duct. J Pathol Bacteriol 1932;35:769-98. 24. Geim AK, Novoselov KS. The rise of graphene. Nat Mat 2007;6:183-91. 25. Buseck PR, Tsipursky SJ, Hettich R. Fullerenes from the geological environment. Science 1992;257:215-7. 26. Allen MJ, Tung VC, Kaner RB. Honeycomb carbon: a review of graphene. Chem Rev 2010;110:132-45. 27. Herrmann HJ. Geometrical cluster growth models and kinetic gelation. Phys Rep 1986;136:517-30. 28. Meakin P, Tolman S. Diffusion-limited aggregation. Proc R Soc Lond A 1989;423:620-5. 29. Sadauskas E, Danscher G, Stoltenberg M, Vogel U, Larsen A, Hide HW. Protracted elimination of gold nanoparticles from mouse liver. Nanomedicine: NBM 2009;5:162-9. 30. Kumar A, Jena PK, Behera S, Lockey RF, Mohapatra, Hide SM. Multifunctional magnetic nanoparticles for targeted delivery. Nanomedicine: NBM 2010;6:64-9. 31. Liu S, Chen G, Ohulchanskyy TY, Swihart MT, Prasad PN. Facile synthesis and potential bioimaging applications of hybrid upconverting and plasmonic NaGdF4: Yb(3 +), Er(3 +)/silica/gold nanoparticles. Theranostics 2013;3:275-81. 32. Stanishevsky AV, Styres C, Yockell-Lelievre H, Yusuf N. Nanostructured carbon beads—properties and biomedical applications. J Nanosci Nanotechnol 2011;11:8705-11. 33. Yang Z, Ma S, Zhang Y. Using activated carbon nanoparticles to decrease the genotoxicity and teratogenicity of anticancer therapeutic agents. J Nanosci Nanotechnol 2010;10:8603-9. 34. Hu B, Zhao Y, Zhu HZ, Yu SH. Selective chromogenic detection of thiol-containing biomolecules using carbonaceous nanospheres loaded with silver nanoparticles as carrier. ACS Nano 2011;5:3166-71. 35. Renaud G, Hamilton RL, Havel RJ. Hepatic metabolism of colloidal gold-low density lipoprotein complexes in the rat: evidence for bulk excretion of lysosomal contents into bile. Hepatology 1989;9: 380-92. 36. Johnston HJ, Semmler-Behnke M, Brown DM, Kreyling W, Tran L, Stone V. Evaluating the uptake and intracellular fate of polystyrene
37.
38.
39.
40.
41.
42.
43.
44.
45.
46. 47.
48. 49.
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nanoparticles by primary and hepatocyte cell lines in vitro. Toxicol Appl Pharmacol 2010;242:66-78. Dong J, Zhou G, Tang D, Chen Y, Cui B, Dai X, Zhang J, Lan Q, Huang Q. Local delivery of slow-releasing temozolomide microspheres inhibits intracranial xenograft glioma growth. J Cancer Res Clin Oncol 2012;138:2079-84. Ratanajiajaroen P, Ohshima M. Synthesis, release ability and bioactivity evaluation of chitin beads incorporated with curcumin for drug delivery applications. J Microencapsul 2012;29:549-58. Lee MJ, Jin SE, Kim CK, Choung HK, Kim HJ, Hwang JM. Effect of slow-releasing all-trans-retinoic acid in bioabsorbable polymer on delayed adjustable strabismus surgery in a rabbit model. Am J Ophthalmol 2009;148:566-72. Lievin-Le Moal V, Servin AL. The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota. Clin Microbiol Rev 2006;19:315-37. Dharmani P, Srivastava V, Kissoon-Singh V, et al. Role of intestinal mucins in innate host defense mechanisms against pathogens. J Innate Immun 2009;1:123-35. Tada Y, Yano N, Takahashi H, Yuzawa K, Ando H, Kubo Y, Nagasawa A, Ogata A, Nakae D. Acute phase pulmonary responses to a single intratracheal spray instillation of magnetite (fe(3)o(4)) nanoparticles in Fischer 344 rats. J Toxicol Pathol 2012;25:233-9. Jang S, Park JW, Cha HR, Jung SY, Lee JE, Jung SS, Kim JO, Kim SY, Lee CS, Park HS. Silver nanoparticles modify VEGF signaling pathway and mucus hypersecretion in allergic airway inflammation. Int J Nanomedicine 2012;7:1329-43. Vlasits AL, Simon JA, Raible DW, Rubel EW, Owens KN. Screen of FDA-approved drug library reveals compounds that protect hair cells from aminoglycosides and cisplatin. Hear Res 2012;294:153-65. Rovira M, Huang W, Yusuff S, Shim JS, Ferrante AA, Liu JO, Parsons MJ. Chemical screen identifies FDA-approved drugs and target pathways that induce precocious pancreatic endocrine differentiation. PNAS 2011;108:19264-9. Pennisi E. Sequence tells mouse, human genome secrets. Science 2002;298:1863-5. Alexis F, Pridgen E, Molnar LK, Farokhzad OC. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm 2008;5:505-15. Almeida JPM, Chen AL, Foster A, Drezek R. In vivo biodistribution of nanoparticles. Nanomedicine:NBM 2011;6:815-35. Cho K, Wang X, Nie S, Chen Z, Shin DM. Therapeutic nanoparticles for drug delivery in cancer. Clin Cancer Res 2008;14:1310-6.
Original Article
nanomedjournal.com
Secretion of intestinal goblet cells: A novel excretion pathway of nanoparticles Baoquan Zhao, PhD a, 1 , Lan Sun, PhD a, 1 , Wuxu Zhang, MSc a, c , Yuxia Wang, MD, PhD a , Junjing Zhu b , Xiaoyu Zhu b , Liuzhong Yang, MM a , Chunqi Li, MD, PhD b , Zhenzhong Zhang c , Yingge Zhang, MD, PhD a,⁎ a
Institute of Pharmacology and Toxicology and Key Laboratory of Nanopharmacology and NanoToxicology, Beijing Academy of Medical Sciences, Beijing, China b Hangzhou Hunter Biotechnology Incorporation, Hangzhou, China c School of Pharmacy; and Nanotechnology Research Center for Drugs; Zhengzhou University, Zhengzhou, China Received 6 May 2013; accepted 18 October 2013
Abstract Understanding the excretion pathway is one of the most important prerequisites for the safe use of nanoparticles in biomedicine. However, the excretion of nanoparticles in animals remains largely unknown, except for some particles very small in size. Here we report a novel natural pathway for nanoparticle excretion, the intestinal goblet cell (GC) secretion pathway (IGCSP). Direct live observation of the behavior of 30-200 nm activated carbon nanoparticles (ACNP) demonstrated that ACNP microinjected into the yolk sac of zebrafish can be excreted directly through intestinal tract without involving the hepato-biliary (hap-bile) system. Histopathological examination in mice after ligation of the common bile duct (CBD) demonstrated that the intravenously-injected ACNP were excreted into the gut lumen through the secretion of intestinal GCs. ACNP in various secretion phases were revealed by histopathological examination and transmission electron microscopy (TEM). IGCSP, in combination with renal and hap-bile pathways, constitutes a complete nanoparticle excretion mechanism. From the Clinical Editor: Nanoparticle elimination pathways are in the forefront of interest in an effort to optimize and enable nanomedicine applications. This team of authors reports a novel natural pathway for nanoparticle excretion, the intestinal goblet cell (GC) secretion pathway (IGCSP). Direct live observation of the behavior of activated carbon nanoparticles has shown excretion directly through the intestinal tract without involving the hepato-biliary (hap-bile) system in a zebrafish model. © 2014 Elsevier Inc. All rights reserved. Key words: Nanoparticle; Excretion; Pathway; Goblet cell; Aggregation
The understanding of the excretion pathway becomes more and more important with the development of nanotechnology and the application of nanomaterials in biomedicine. It is important not only for the biosafety issue of engineered nanoparticles but also for the practical use of nanoparticles as diagnostic and therapeutic agents or as drug carriers, because excretion is the best way to cease the action of nanoparticles on tissues and cells. Based on the understanding of the excretion pathway, appropriate protocols can be worked out to deal with the nanoparticles once they entered the animal body. Unfortu-
This work was supported by the National Natural Science Foundation of China (No. 90406024), the National Basic Research Program of China (No. 2010CB933904) and Major New Drug Creations (No. 2011ZX09102-001-15). ⁎Corresponding author at: Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, PR China. E-mail address: [email protected] (Y. Zhang). 1 Equally contributed to the work.
nately, such pathway remains poorly understood, though there are many literatures on the clearance of nanoparticles from blood 1,2 or tissues such as lung 3,4 and liver. 5 These experiments provide information concerning the clearance mechanism to remove particles from local tissues rather than information on systemic excretion of nanoparticles. 6 Few studies have reported two main excretion pathways of intravenously-injected nanoparticles, the kidney–urine pathway and hepatobiliary system (HBS)–feces pathway. Kidney excretion of nanoparticles is limited to very small ones, such as quantum dots 7 and fullerines. 8 HBS excretes some larger nanoparticles, but the clearance rate is no more than 1% within 24 h, and there is an inverse relation between HBS excretion and sizes. 9 However, Manabe et al found that 500 nm latex particles can be cleared from medaca embryos, 10 suggesting that there are some other excretion pathways for nanoparticle excretion without involving the HBS. Souris et al reported that the concentration of intravenously-injected 50-100 nm silica nanoparticles in liver
1549-9634/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2013.10.004 Please cite this article as: Zhao B, et al, Secretion of intestinal goblet cells: A novel excretion pathway of nanoparticles. Nanomedicine: NBM 2014;10:839-849, http://dx.doi.org/10.1016/j.nano.2013.10.004
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was much lower than that in intestinal wall and feces. 11 This inconsistence in quantity does not support the hypothesis that HBS plays main roles in the intestinal excretion of nanoparticles. On the contrary, the high concentration of nanoparticles in intestinal wall 11 suggested that the intestinal wall may play important roles in the excretion of nanoparticles through intestinal tract. GCs are one of the four main cell types 12 in the intestinal membrane and have the ability to entrap nanoparticles. 13 Recently, there have been several studies on the interactions between GCs and orally-administered nanoparticles. 13–16 GCs can uptake nanoparticle, 17 which makes GCs the most possible candidate cells playing roles in the intestinal excretion of nanoparticles. Our earlier studies found that intravenously-injected nanoparticles had distribution in intestinal GCs 18 and hypothesized that intestinal wall may play roles in the excretion of nanoparticles but did not investigate in detail. 18,19 The present work carefully examined the mechanism for intestinal excretion of nanoparticles and revealed a novel pathway, IGCSP, for nanoparticle excretion. Methods Preparation and characterization of ACNP ACNP were prepared from medicinal activated carbon (MAC; Haichangqing Co. Ltd, Beijing, China) by a top-down method (supplemental materials). The diameters of ACNP were determined by atomic force microscope (AFM), scanning electron microscope (SEM) and Laser Particle Size and Zeta Potential Analyzer. The internal crystal structures were detected by X-ray defraction (XRD). For injection, 1 mg ACNP was added into 100 mL normal saline and suspended in an ultrasound field of 40 Hz, 180 W for 20 min. Handling of the animals Zebrafish eggs were collected, cleaned, and washed with eggwater 20 within one hour after fertilization. Eggs from different females were pooled, placed in 7 cm sterile Petri dishes containing eggwater, and incubated at 28.0 to 28.5 °C. Embryonic fish were stocked in an Aquatic Habitat re-circulating tank system at 28.5 °C with a 14 h light/10 h dark cycle. The water was purified by reverse osmosis and adjusted to pH 7 and conductivity of 350 μS. The mice were handled as described previously. 21 Briefly, 20 Kunming mice (20-25 g in body weight) were constantly monitored and fed fluid nutritional diet free from pathogens and particulate materials for 10 days before ACNP treatment to avoid the influences of ingested particles on the observation of ACNP. All the animal procedures were approved by the Animal Subject Review Committee of the Beijing Academy of Medical Science.
Microinjection and observation of ACNP in zebrafish Embryonic zebrafish at the age of 24 h were put into a 7-cm culture dish and anesthetized by addition of tricaine methanesulfonate into the dish at a final concentration of 0.64 mM. The anesthetized zebrafish were placed in the slanting grooves of a silica gel sheet. Excessive water around the fish was absorbed with filter papers to leave just enough water to bathe the fish body. Five μL suspension of ACNP at the concentration of 5 mg/mL was injected into the yolk sac of zebrafish in an IM 300 microinjection instrument (Narishige, Japan) (Figure S3). All ACNP suspensions were freshly dispersed by ultrasonication for 10 min before use. During the whole period of experiment, zebrafish that received microinjection of ACNP exhibited no significant differences in death rate, development, teratogenesis, and cardiovascular toxicity in comparison with control (Figure S4). Ligation of common bile duct and injection of ACNP into the mice The common bile duct was ligated with the method originally described by Cameron and Oakley 23 with modifications. Briefly, Kunming mice of 20-25 g were anaesthetized with pentobarbital (25 mg/kg) and fixed onto a wood surgical sheet. A midabdominal incision was made, and the abdominal tissues were separated carefully to clearly expose the CBD. Two sterile nylon medical surgical sutures (Unic Surgical Sutures, Mfg., Co., Ltd., Suzhou, China), 0.2 mm in diameter, were put through under the CBD, and two nodes were made at both ends of a segment of CBD (Figure S5), and the CBD was then cut off between the two ends. After closure of abdomen, ACNP were suspended in 0.9% NaCl to a final concentration of 5 mg/mL and injected through tail veins in a dose of 50 mg/kg (i.e., 10 mL/kg). All the suspensions of ACNP were freshly dispersed by sonication for 10 min before use. After the injection of ACNP, the animals' body weight, behavior, and number of blood cells were monitored (Figure S6). At the end of the experiments, these physical parameters showed no significant differences from those of control (Figure S6). Histopathological examination On the 4th day after injection of ACNP, the mice were anaesthetized and their internal organs were taken out and fixed in 40% formaldehyde. 5 μM thick sections were made on a Lecia RM2135 Rotary Microtome. The sections were stained in hematoxylin and eosin or alcian blue and observed under an Olympus BH2 phase contrast microscope (Japan). Quantitative evaluation of the efficiency of IGCSP for ACNP
Pigment inhibition in zebrafish Pigmentation genes were suppressed by adding 1-phenyl, 2-thiourea (PTU) (Sigma, USA) into fish water in a final concentration of 75 μM in the developmental stage of 28 somites. 22 This concentration produced enough pigment inhibition with no adverse effects on the hatching and survival.
The quantitative evaluation was carried out with rates occupied by ACNP-containing GCs in the total number of the GCs seen under a phase light microscope in 10 visions in 3 HEstained histopathological sections, which were calculated by a formula: Rates (%) = Number of ACNP containing GCs/total number of seen GCs×100%. 120 Mice without CBD ligation were randomly divided into 4 groups and 30, 60, 100 and
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Figure 1. ACNP characteristics. (A) The AFM images of the prepared ACNP of 30, 60, 100, and 200 nm. (B) The SEM image of 100 nm ACNP (left), which is spheric with many micropores in the sphere (right).
200 nm ACNP were intravenously injected respectively. The different organs of every 3 mice in each group were respectively taken out for quantitative histopathological examination on day 1-180 after ACNP injection. Results
Because HBS is a known excretion pathway of nanoparticles, we paid special attention to the distribution of ACNP in liver and gallbladder. Surprisingly, there were little ACNP found in liver and no ACNP in gallbladder (Figure 3, D), which does not support the hypothesis that HBS is the main pathway for in the excretion of ACNP.
Characteristics of ACNP
Excretion of ACNP through intestinal tract of zebrafish
Structurally, ACNP belong to a large family of carbonbased nanoparticles composed of graphene sheets 24–26. The prepared ACNP had diameters of 30, 60, 100, and 200 nm, respectively, as imaged with AFM (Figure 1, A) and SEM (Figure 1, B), which are spheres with mesopores (Figure 1, B). XRD revealed that the graphite microcrystals of ACNP have a thickness of 0.707 nm, a width of 2.007 nm and a layer distance of 0.372 nm. The ζ electropotentials are 0.65-6 mV in pH 7.4 saline. The measurements of some structures and properties of ACNP in different sizes are listed in Table S1. Withdrawing test demonstrated that the suspension was stable within 2 h.
Since there were little distribution of ACNP in liver and no ACNP found in gallbladder, there should be no ACNP in intestinal tract. Again to our surprise, a great amount of ACNP was observed in and excreted through intestinal tract with the peristalsis of the intestines as demonstrated in Figure 2. After microinjection, ACNP appeared as small aggregated particles (circles) in the yolk sac at the fourth hour, when the intestinal tract had not developed (Figure 2, A). On the third day, the intestinal tract (green arrow) had developed, and most yolk sac had been absorbed with a small part remaining (yellow arrow). No ACNP were in the intestinal tract; most ACNP still existed in the residual yolk sac, and some of them appeared in the tissues between intestinal wall (red arrow) and abdominal skin (Figure 2, B). On the fourth day, yolk sac was completely absorbed. ACNP began to appear in the intestinal wall (red arrows) and gut lumen (green arrow) (Figure 2, C). On the fifth day, more ACNP entered the intestinal tract (green arrow, Figure 2, D). On the sixth day, the quantity of ACNP in intestinal tract (green arrow) reached its peak (Figure 2, E). On the seventh day, ACNP had moved downward to the lower segment of the gut lumen with the intestinal peristalsis and were finally excreted through cloaca (yellow arrow, Figure 2, F). On the eighth day, while previously observed ACNP had been completely excreted out, ACNP continuously entered the intestinal tract from other tissues (Figure 2, G). These results suggested that
Tissue distribution of ACNP in zebrafish Under microscope, the black ACNP were well visible in the transparent embryonic zebrafish. The suspension of ACNP appeared in the yolk sac as cloud-like immediately after the injection (Figure S3, B). Within 3 h, most ACNP aggregated into larger recognizable particles (Figure S3, B). In 0-4 h after injection, ACNP mainly accumulated in the yolk sac (Figure 2, A). With the development of zebrafish, ACNP gradually distributed to intestinal wall (Figures 2 and 3, A), extracranial tissues (Figure 3, B), myocardium (Figure 3, C), kidney (Figure 3, D), and blood vessels (Figure 3, E).
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Figure 2. The dynamic changes of ACNP in gut lumen of zebrafish after ACNP microinjection in yolk sac. (A) At the fourth hour. (B) On the third day. (C) On the fourth day. (D) On the fifth day. (E) On the sixth day. (F) On the seventh day. (G) On the eighth day.
the yolk sac injected ACNP were effectively excreted through intestinal tract. Blood transportation of ACNP in zebrafish The above results gave rise to the question of how ACNP got to the intestinal tract. To answer this question, we first need to know the transportation of ACNP in animal body. The observation of the behavior of ACNP in blood vessels demonstrated that yolk sac-injected ACNP was transported through blood circulation. With the development of cardiovascular system in zebrafish, ACNP were first found to migrate from tissues to the wall of blood vessels. Figure 3, E demonstrated the case of ACNP in the wall of zebrafish tail veins. The yolk sac microinjected ACNP first migrated to
and then temporarily accumulated in the walls of blood vessels, where they were finally released into the blood stream. After injection, ACNP in the walls of blood vessels gradually decreased with their release into the blood stream. These results indicated that the ACNP were transported by the blood stream. Elimination of ACNP from various tissues To obtain the data supporting the excretion of ACNP from various tissues through intestinal tract, we investigated the relations between the elimination and intestinal excretion of ACNP. Results demonstrated that the intestinal excretion of ACNP was accompanied by their elimination from various tissues. ACNP in the tissues around the upper end of intestinal
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Figure 3. Dynamic changes of ACNP in the tissues outside of intestinal tract and the relation between ACNP and the mucous of GCs in the intestines of zebrafish. (A) ACNP in TAUEI (red circles). (B) ACNP (arrows) in the extracranial tissues. (C) ACNP (arrows) in the myocardium. (D) ACNP in the kidney, liver, and gallbladder. Blue circle: kidney; red circle: gallbladder; green circle: liver. (E) ACNP in the wall of tail veins. hpt: hours post treatment; dpt: days post treatment. (F) Alcian blue staining of the control intestines of zebrafish. (G) Alcian blue staining of the intestinal wall of zebrafish with yolk sac injection of ACNP.
tract (UEIT) had the fastest and most complete elimination, though there was a temporary increment on the fifth day (Figure 3, A). Vast majority of the ACNP in UEIT was eliminated on the sixth day. On the eighth day after injection, the ACNP in UEIT were completely eliminated. In extracranial tissues, ACNP gradually decreased, and there were significantly less ACNP on the eighth day (Figure 3, B). The yolk sacmicroinjected ACNP also distributed in myocardial muscles and were eliminated slowly too. On the eighth day, ACNP in the myocardium significantly decreased (Figure 3, C). In the kidney, there was little distribution of ACNP, which were eliminated slowly. On the eighth day after ACNP microinjection in yolk sac, there were still some ACNP in the kidney. The ACNP in the wall
of blood vessels had the slowest elimination. On the eighth day, there were still some ACNP in the vascular walls (Figure 3, E), which may be explained by the function of blood vessel walls as a relay station in the process of ACNP transportation from tissues to blood stream; therefore, a complete elimination from the walls of blood vessels is possible only after the complete elimination of ACNP from other tissues over the whole body. The relation between ACNP and the mucus in the intestinal membrane of zebrafish In the HBS pathway, nanoparticles cross the intestinal wall with the bile through CBD. To understand how ACNP got into intestinal tract through the intestinal wall without involving of
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Figure 4. ACNP in the intestinal contents of the mice with CBD ligation. (A) The schematic illustration of the relations of HBS with intestinal tract. (B) The ligation of common bile duct. (C) ACNP (arrows) in the intestinal contents of mice injected with ACNP through tail vein.
HBS, we observed the cellular distribution of ACNP in intestinal wall of zebrafish. In alcian blue-stained histopathological sections the mucus was stained into blue color (Figure 3, F) and ACNP appeared as dense black particles (Figure 3, G) in the mucous, suggesting that ACNP may be secreted by GC in the intestinal mucous membrane and enter into gut lumen with mucus.
transported only by the blood stream. In these experiments, ACNP were again observed in the intestinal contents of mice fed particle-free foods 21 (Figure 4, C) and injected with 30-200 nm ACNP while there were no particles seen in those of the control mice (Figure 4, C), which provided a conclusive evidence for non-HBS intestinal pathway of excreting ACNP.
Intestinal excretion of ACNP in mice with CBD ligation
Cell distribution of ACNP in intestinal wall in mice with CBD ligation
To definitely prove that the intestinal excretion of ACNP without involving HBS, we carried out experiments in mice with CBD ligation (Figures 4, A and B, S5), which absolutely blocked the connection between HBS and intestinal tract (Figure 4, A and B). The intravenous injection made sure that ACNP was
As shown in Figure 5, in HE stained sections, the main cell populations of intestinal wall included the absorptive columnar epithelial cells (CECs) and excretory GCs. Each GC contained a large vacuole of mucus contents, which made them easily be recognized (Figures 5, A1 and 3). There were no black particles
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Figure 5. The distribution of ACNP in intestinal GCs of mice with CBD ligation. (A) The histopathological microsection (HE staining) of the jejunum and colon tissues of the mice treated with ACNP injection through tail veins at the dose of 50 mg/kg, once a day for 10 days. The GCs were easily identified for containing a large intracellular vacuole because of the mucins. (A1) The jejunum of control mice treated with saline injection through tail vein; (A2) The jejunum tissue of the mice treated with intravenous ACNP injection; (A3) The colon tissue of the control mice with intravenous saline injection; (A4) The colon tissue of the mice treated with intravenous ACNP injection. (B) The secretion of ACNP by intestinal GCs of the mice treated with intravenous ACNP injection. (B1) Some ACNP ready to be secreted by a GC (arrow); (B2) About half of ACNP had been expelled out from GC (arrows); (B3) Majority of ACNP had been expelled out from a GC (arrow); (B4) All of the ACNP had been completely expelled out of a GC (arrow). (C) The TEM images of different phases of the secretion of ACNP from GCs. C1: the ACNP in GC (yellow arrow); (C2) Some ACNP were being secreted from GC (green arrow) while some were still inside the cell (yellow arrow); (C3) Some ACNP had been secreted out from GC (red arrow) while some were being secreted (green arrow) and some were still in GC (yellow arrow). (C4) Some ACNP had entered into the gut lumen (red arrow) and some had just been secreted out (green arrow); and some were still inside the GC (yellow arrows).
in all intestinal tissue cells in control mice (Figures 5, A1 and 3), while many ACNP were distributed in GCs in mice intravenously injected with ACNP (Figures 5, A2 and 4). There were no ACNP found in CECs. With the increase of GCs, the ACNP in the intestinal wall increased (Figure 5, A3 and 4). These results were in coincidence with those obtained by the observation of alcian blue-stained sections of the intestines of zebrafish, suggesting that ACNP entered into gut lumen through GCs. Secretion phase of ACNP from GCs Histopathological examination further revealed the roles of GC in excretion of ACNP. In HE-stained histopathological sections, ACNP were seen being excreted by GCs. As shown in
Figure 5, B, ACNP were spotted both in the fused intracellular secretory vacuoles and in the vesicles undergoing exocytosis. Figure 5, B1 shows some ACNP located at the margin of a GC ready to secrete. Figure 5, B2 shows some ACNP passing through the membrane of a GC. Figure 5, B3 displays some ACNP that had been secreted out by a GC and some were still in it. Figure 5, B4 shows that some ACNP had been completely expelled out from a GC by exocytosis. TEM also revealed ACNP in various secretory phases in GCs (Figure 5, C). Figure 5, C1 shows the ACNP inside a secretory vesicle of GC, Figure 5, C2 demonstrates the scene of ACNP being secreted by a GC. In Figure 5, C3, some ACNP had already been expelled out of a GC, and Figure 5, C4 indicates that ACNP had been secreted into the gut lumen from GCs.
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Figure 6. The time course of IGCSP for ACNP excretion and its relations with sizes. (A) Histopathological observation of the changes in quantity of ACNP in some important internal organs of mice within 180 days. (B) The time course of intestinal ACNP excretion and its relations with sizes.
Time course and the effect-size relation of IGCSP The excretion of ACNP through IGCSP had a rather long time course. As shown in Figure 6, A, the intravenously-injected ACNP of 200 nm were widely distributed in various organs or tissues including liver, which was different from the case in zebrafish because of the difference in the way of injection. The ACNP in various tissues decreased obviously on day 30. The half elimination time was about 2 months. The complete excretion seemed to need 180 days (Figure 6). Taking the rates occupied by ACNP-containing GCs in total number of the observed GCs (RAC) as a parameter, the time course of the excretion of ACNP from mouse bodies may be studied quantitatively, which was shown in Figure 6, B. 30-200 nm ACNP appeared in the GCs of intestinal mucous membrane within 30 min, reached its highest value on day 10, began to decrease on day 30 and completely eliminated within 180 days. These results indicated that IGCSP was a highly efficient pathway for the excretion of nanoparticles, though it was very slow in speed. It can be observed that the height of RAC–time curves increased with the increment in sizes, indicating a positive correlation of the ability of IGCSP with sizes, which was identical with the results of Manabe et al. 3 RAC– time curves of 30-200 nm ACNP were quite similar, which indicate that IGCSP is the common mechanism for the excretion of ACNP with different sizes. Aggregation modes of ACNP in tissues and cells As shown in Figures 2-6, the ACNP existed in tissues and cells in clusters formed by aggregation. There were two modes
for ACNP aggregation. In tissues of zebrafish and the cells of liver, spleen, kidney and lung of mice, the aggregation of ACNP took Eden mode. 27 The clusters appeared as spots with a high density. In GCs, the aggregation of ACNP took DLA mode. 28 The clusters were dendritic with a loose configuration. These results indicated that NPs take different aggregation modes in different tissues or cells. Discussion To study the excretion of nanoparticles, we selected biologically inert ACNP as a model, because many nanoparticles used as diagnostic agents and drug carriers contain metal or nonmetabolizable cores such as gold, 29 iron, 30 silica, 31 and carbonaceous nanoparticles, 25,26 though their organic out-shells can be degraded by various enzymes. 32 ACNP, as carbonaceous mesoporous nanoparticles, have been used as drug carriers. 33,34 Because of their biological inertness, ACNP represent pure “particulate materials.” Although the excretion of nanoparticles is influenced by their chemical activities, their particulate properties are also an important affecting factor. The results of the present work are significant for the evaluation of the particulate effects of the non-metabolizable nanoparticles and the non-metabolizable cores of nanoparticles. From the inertness point of view, the results obtained from ACNP can be extrapolated to other non-metabolizable nanoparticles and the non-metabolizable cores of nanoparticles on their excretion. Of course, further investigations are required, since the present work is only a start point for the relative studies.
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Aggregation is a common phenomenon for nanoparticles. To fulfill the needs of injection and blood transportation, aggregation must be prevented. Ultrasonication just before injection can make ACNP disperse in suspension. After they were injected into yolk sac or veins, the concentration of ACNP was greatly decreased by the dilutive effects of yolk sac fluid and blood, which prevent ACNP from aggregation. At the same time, the movement of animals and the flow of blood are also helpful for prevention of the aggregation. However, the case in tissues and cells was completely different from that in blood. The concentration of ACNP in tissues would be much higher than that in blood because of EPR effects, and the flow of tissue fluid was much slower than that of blood. The case in cells that can phagocytose ACNP was the same as that in tissues: high ACNP concentration and low flow rate, which explain the aggregation of ACNP in tissues and cells. There were two aggregation modes for ACNP in tissues and cells: the Eden mode 27 and the DLA mode, 28 which can be more clearly seen in hitopathological sections of mice, because the magnification of the direct observation of ACNP clusters in living zebrafish was too low to recognize the details. In tissue space and cells of liver, spleen, lungs and kidney of mice, the aggregation took Eden mode, in which the clusters were spots with high density. 27 In intestinal GCs, the aggregation took DLA mode, in which the clusters were dendritic with a loose configuration. 28 These results suggested that the aggregation of nanoparticles is a rather complex process and the growth of clusters can take different modes in different microenvironments. GCs had unique mucin contents, and, therefore, ACNP aggregation in them took unique DLA mode. Whether biological systems can remove or excrete clusters of nanoparticles is another question requiring answers for practical use of nanoparticles. The results of this study indicate that GCs have the capability to secrete clusters of nanoparticles. As for the clusters in tissues and cells, the removing process may be more complex. They must disaggregate into dispersed nanoparticles or clusters small enough for blood transportation, which is theoretically possible. The concentration of ACNP in the blood decreased gradually with time after the injection. When blood concentration was low enough, the ACNP in tissues began to enter into blood, and hence the concentration of ACNP in tissues began to decrease. When tissue concentration was low enough, some of ACNP at the edge of cluster tended to separate; leading to the decrease in size and finally disaggregation of the cluster. In spite of the difficulty, this process must happen, sooner or later, faster or slower. As discussed above, the aggregation only influences the speed but cannot prevent the excretion of nanoparticles. To understand how the ACNP cross the intestinal wall and are released into the gut lumen, HBS was firstly considered because it is a well-known excretion system. There have been many literatures that believe nanoparticles can be excreted through HBS based on the results that nanoparticles can be simultaneously seen in liver and intestinal tract. In the late 1990s, Renaud et al found that the low-density lipoprotein20 nm colloidal gold (CG) could be excreted through HBS but albumin- or polyvinylpyrrolidone-CG could not. 35 This selective excretion was rationally attributed to the function of
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the lipoprotein receptor of liver cells. In other words, the nanoparticles without low-density lipoproteins could not be excreted through HBS. Later studies found that negatively charged gold nanoparticles 9 and polystyrene carboxylated nanoparticles 36 could also be excreted through HBS, although it was limited to no larger than 20 nm and the excretion quantity was very low. Souris et al 11 reported that 80 nm nanoparticles can be excreted through intestinal tract and the concentration in feces was very high while the concentration in liver was very low. In comparison with liver, the nanoparticle concentration was much higher in intestinal wall. It seems easier to explain the intestinal excretion of nanoparticles by intestinal secretion than by HBS pathway. More recently, in one study by Manabe et al, it was found that that nanoparticles as large as 500 nm, far beyond the ability of HBS, can be eliminated from fish body and the elimination of 500 nm particles was faster and more efficient than 50 nm particles no matter whether modified or not, 10 which cannot be explained by HBS pathway. Obviously, there were paradoxes between the results of the experiments on HBS for nanoparticle excretion. These paradoxes are unexplainable based on the belief that HBS is the only pathway for excretion of nanoparticles through intestinal tract. Especially, the results of Manabe et al strongly suggest the existence of another pathway for intestinal excretion of nanoparticles without involving HBS, and the results of Souris et al suggest that intestinal secretion is a possible pathway for nanoparticle excretion, since the concentration of nanoparticles was more identical with that in feces than that in liver. 11 Based on previous reports, we also believed that HBS is the most possible pathway for nanoparticle intestinal excretion. However, surprisingly, there was only a little distribution of ACNP in the liver and no ACNP in gallbladder during the whole period of experiments in zebrafish. These observations do not support HBS pathway because gallbladder is the only organ to store bile and CBD is the only way for excretion of ACNP from liver to intestinal tract. Thus, we believe that there must be an alternative mechanism for ACNP to enter the intestinal tract, which does not involve HBS. To confirm the existence of this pathway, we carried out the experiments in mice with ligation of CBD in mice, which completely cut off the way that ACNP must pass through to get to gut lumen from liver. As it was anticipated, ACNP were still observed in the feces of these mice with CBD ligation. The histopathological and TEM examination revealed that the mechanism of such non-HBS pathway for nanoparticle excretion was through the secretion of intestinal GCs. The distribution of ACNP only in GCs suggested IGCSP, which was then proved by the secretion phase observed in histopathological sections and TEM ultrasections. IGCSP had a long time course, which is quite in favor of that nanoparticles are used as drug carriers for long lasting treatments of chronic diseases. 37–39 As demonstrated in zebrafish and mice, most intravenously-injected ACNP can be excreted through IGCSP, which greatly ameliorates our concern about the eternal existence of biologically inert nanoparticles in animal body. GC is a main cell type of the intestinal mucosal epithelium, which undergoes continuous cycles of renewal. 40
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The major function of intestinal GCs and their main secretory product, mucin, is the formation of mucus overlying layers on intestinal membranes, which promotes the elimination of gut contents and provides the first line of defense against physical and chemical injury caused by ingested food, microbes and the microbial products. 13,41 There have been no reports on the roles of GC in the intestinal excretion of nanoparticles, let alone the mechanism for ACNP to enter into GC. In our experiments with zebrafish, the yolk sac-injected ACNP were distributed in various tissues with the formation of organs and then was transported by blood flow to intestines and excreted through intestinal tract, which suggested that the primary tissue distribution was related to the development of tissues and ACNP may enter GC during the differentiation. Because GC continuously undergo cycles of renewal, it is considered that the mechanism for ACNP getting into GC may be related to the differentiation of GC. The ACNP in GC combined or mixed with mucus and then were excreted into gut lumen by GC along with the secretion of mucus. There are also studies on the interaction between intra-tracheal nanoparticles and GCs in the airway 42,43; however, we didn't examine the distribution of ACNP in airway GC because the airway is not a main excretion organ for solid substance. So, IGCSP was the main pathway for the excretion of nanoparticles. In the present work, zebrafish and mice were used as animal models. Zebrafish has been used for drug screening. 19,44,45 Mice have gene sequences 80% identical with human and are frequently used as animal models in basic life science of human and preclinical studies on therapeutic agents and methods. 46 Both of them are vertebrate animals with similar intestinal tissues composed of mainly columnar and goblet cells. Therefore, the results of the present study are of great significance for evaluating the biosafety of nanoparticles in human. To date, IGCSP for nanoparticles has been found not only in marine animals but also in mammals. This pathway is especially important for those larger nanoparticles without modification, because the kidney 7 and HBS are only able to excrete nanoparticles modified with various chemical structures in small sizes. 8,11 Considering that many nanoparticles used as diagnostic and therapeutic agents have metal cores and are relatively large, 47–49 in the sizes that are beyond the excretory capability of kidney and HBS, this novel IGCSP provided greater promise for future application of nanoparticles in biomedical fields by eliminating the concerns about the persistent effects of nanoparticles. Obviously, the present results are only a starting point for the studies on IGCSP. The details, such as how ACNP cross the cell membrane and get into the cells and how they are secreted by the intestinal GCs, warrant further investigations. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2013.10.004.
References 1. Dreaden EC, Austin LA, Mackey MA, El-Sayed MA. Size matters: gold nanoparticles in targeted cancer drug delivery. Ther Deliv 2012;3(4):457-78. 2. Wahajuddin, Arora S. Superparamagnetic iron oxide nanoparticles: magnetic nanoplatforms as drug carriers. Int J Nanomedicine 2012;7:3445-71. 3. Zarogoulidis P, Chatzaki E, Porpodis K, Domvri K, Hohenforst-Schmidt W, Goldberg EP, Karamanos N, Zarogoulidis K. Inhaled chemotherapy in lung cancer: future concept of nanomedicine. Int J Nanomedicine 2012;7:1551-72. 4. Elder A, Gelein R, Finkelstein JN, Driscoll KE, Harkema J, Oberdorster G. Effects of subchronically inhaled carbon black in three species. I. Retention kinetics, lung inflammation, and histopathology. Toxicol Sci 2005;88:614-29. 5. Feng Y, Zong Y, Ke T, Jeong EK, Parker DL, Lu ZR. Pharmacokinetics, biodistribution and contrast enhanced MR blood pool imaging of GdDTPA cystine copolymers and Gd-DTPA cystine diethyl ester copolymers in a rat model. Pharm Res Aug. 2006;23:1736-42. 6. Hagens WI, Oomen AG, de Jong WH, Cassee FR, Sips AJ. What do we (need to) know about the kinetic properties of nanoparticles in the body? Regul Toxicol Pharmacol 2007;49:217-29. 7. Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, Bawendi MG, Frangioni JV. Renal clearance of quantum dots. Nat Biotecnol 2007;25:1165-70. 8. 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. Proc Natl Acad Sci U S A 2006;103(9):3357-62. 9. Hirn S, Semmler-Behnke M, Schleh C, Wenk A, Lipka J, Schäffler M, Takenaka S, Möller W, Schmid G, Simon U, Kreyling WG. Particle sizedependent and surface charge dependent biodistribution of gold nanoparticles after intravenous administration. Eur J Pharm and Biopharm 2011;77:407-16. 10. Manabe M, Tatarazako N, Kinoshita M. Uptake, excretion and toxicity of nano-sized latex particles on medaka (Oryzias latipes) embryos and larvae. Aquat Toxicol 2011:105576-81. 11. Souris JS, Lee CH, Cheng SH, Chen CT, Yang CS, Ho JA, Mou CY, Lo LW. Surface charge-mediated rapid hepatobiliary excretion of mesoporous silica nanoparticles. Biomaterials 2010;31:5564-74. 12. Kim YS, Ho SB. Intestinal goblet cells and mucins in health and disease: recent insights and progress. Curr Gastroenterol Rep 2010;12:319-30. 13. Behrens I, Pena AIV, Alonso MJ, Kissel T. Comparative uptake studies of bioadhesive and non-bioadhesive nanoparticles in human intestinal cell lines and rats: the effect of mucus on particle adsorption and transport. Pharm Res 2002;19:1185-93. 14. Jin Y, Song YP, Zhu X, Zhou D, Chen CH, Zhang ZR, Huang Y. Goblet cell-targeting nanoparticles for oral insulin delivery and the influence of mucus on insulin transport. Biomaterials 2012;33:1573-82. 15. des Rieux A, Fievez V, Garinot M, Schneider YJ, Préat V. Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach. J Control Releas 2006;116:1-27. 16. Dong YC, Feng SS. Poly(d, l-lactide-co-glycolide)/montmorillonite nanoparticles for oral delivery of anticancer drugs. Biomaterials 2005;26:6068-76. 17. Wang YY, Lai SK, Suk JS, Pace A, Cone R, Hanes J. Addressing the PEG mucoadhesivity paradox to engineer nanoparticles that “slip” through the human mucus barrier. Angew Chem 2008;47:9575-781. 18. Sun L, Yang LZ, Zhang YG. Study on the excretion pathway of nanoparticles. Chin Pharm J 2006;41:366-7. 19. Wang K, Ma JB, He M, Gao G, Xu H, Sang J, Wang YX, Zhao BQ, Cui DX. Toxicity assessments of near-infrared upconversion luminescent LaF3:Yb, Er in early development of zebrafish embryos. Theranostics 2013;3:258-66. 20. Westerfield M. The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio). Eugene, OR: University of Oregon Press; 1993.
B. Zhao et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 839–849 21. Yang Z, Zhang Y, Yang Y, Sun L, Han D, Li H, Wang C. Pharmacological and toxicological target organelles and safe use of single-walled carbon nanotubes as drug carriers in treating Alzheimer disease. Nanomedicine:NBM 2010;6:427-41. 22. Karlsson J, von Hofsten J, Olsson P-E. Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development. Mar Biotechnol 2001;3:522-7. 23. Cameron GR, Oakley CL. Ligation of the common bile duct. J Pathol Bacteriol 1932;35:769-98. 24. Geim AK, Novoselov KS. The rise of graphene. Nat Mat 2007;6:183-91. 25. Buseck PR, Tsipursky SJ, Hettich R. Fullerenes from the geological environment. Science 1992;257:215-7. 26. Allen MJ, Tung VC, Kaner RB. Honeycomb carbon: a review of graphene. Chem Rev 2010;110:132-45. 27. Herrmann HJ. Geometrical cluster growth models and kinetic gelation. Phys Rep 1986;136:517-30. 28. Meakin P, Tolman S. Diffusion-limited aggregation. Proc R Soc Lond A 1989;423:620-5. 29. Sadauskas E, Danscher G, Stoltenberg M, Vogel U, Larsen A, Hide HW. Protracted elimination of gold nanoparticles from mouse liver. Nanomedicine: NBM 2009;5:162-9. 30. Kumar A, Jena PK, Behera S, Lockey RF, Mohapatra, Hide SM. Multifunctional magnetic nanoparticles for targeted delivery. Nanomedicine: NBM 2010;6:64-9. 31. Liu S, Chen G, Ohulchanskyy TY, Swihart MT, Prasad PN. Facile synthesis and potential bioimaging applications of hybrid upconverting and plasmonic NaGdF4: Yb(3 +), Er(3 +)/silica/gold nanoparticles. Theranostics 2013;3:275-81. 32. Stanishevsky AV, Styres C, Yockell-Lelievre H, Yusuf N. Nanostructured carbon beads—properties and biomedical applications. J Nanosci Nanotechnol 2011;11:8705-11. 33. Yang Z, Ma S, Zhang Y. Using activated carbon nanoparticles to decrease the genotoxicity and teratogenicity of anticancer therapeutic agents. J Nanosci Nanotechnol 2010;10:8603-9. 34. Hu B, Zhao Y, Zhu HZ, Yu SH. Selective chromogenic detection of thiol-containing biomolecules using carbonaceous nanospheres loaded with silver nanoparticles as carrier. ACS Nano 2011;5:3166-71. 35. Renaud G, Hamilton RL, Havel RJ. Hepatic metabolism of colloidal gold-low density lipoprotein complexes in the rat: evidence for bulk excretion of lysosomal contents into bile. Hepatology 1989;9: 380-92. 36. Johnston HJ, Semmler-Behnke M, Brown DM, Kreyling W, Tran L, Stone V. Evaluating the uptake and intracellular fate of polystyrene
37.
38.
39.
40.
41.
42.
43.
44.
45.
46. 47.
48. 49.
849
nanoparticles by primary and hepatocyte cell lines in vitro. Toxicol Appl Pharmacol 2010;242:66-78. Dong J, Zhou G, Tang D, Chen Y, Cui B, Dai X, Zhang J, Lan Q, Huang Q. Local delivery of slow-releasing temozolomide microspheres inhibits intracranial xenograft glioma growth. J Cancer Res Clin Oncol 2012;138:2079-84. Ratanajiajaroen P, Ohshima M. Synthesis, release ability and bioactivity evaluation of chitin beads incorporated with curcumin for drug delivery applications. J Microencapsul 2012;29:549-58. Lee MJ, Jin SE, Kim CK, Choung HK, Kim HJ, Hwang JM. Effect of slow-releasing all-trans-retinoic acid in bioabsorbable polymer on delayed adjustable strabismus surgery in a rabbit model. Am J Ophthalmol 2009;148:566-72. Lievin-Le Moal V, Servin AL. The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota. Clin Microbiol Rev 2006;19:315-37. Dharmani P, Srivastava V, Kissoon-Singh V, et al. Role of intestinal mucins in innate host defense mechanisms against pathogens. J Innate Immun 2009;1:123-35. Tada Y, Yano N, Takahashi H, Yuzawa K, Ando H, Kubo Y, Nagasawa A, Ogata A, Nakae D. Acute phase pulmonary responses to a single intratracheal spray instillation of magnetite (fe(3)o(4)) nanoparticles in Fischer 344 rats. J Toxicol Pathol 2012;25:233-9. Jang S, Park JW, Cha HR, Jung SY, Lee JE, Jung SS, Kim JO, Kim SY, Lee CS, Park HS. Silver nanoparticles modify VEGF signaling pathway and mucus hypersecretion in allergic airway inflammation. Int J Nanomedicine 2012;7:1329-43. Vlasits AL, Simon JA, Raible DW, Rubel EW, Owens KN. Screen of FDA-approved drug library reveals compounds that protect hair cells from aminoglycosides and cisplatin. Hear Res 2012;294:153-65. Rovira M, Huang W, Yusuff S, Shim JS, Ferrante AA, Liu JO, Parsons MJ. Chemical screen identifies FDA-approved drugs and target pathways that induce precocious pancreatic endocrine differentiation. PNAS 2011;108:19264-9. Pennisi E. Sequence tells mouse, human genome secrets. Science 2002;298:1863-5. Alexis F, Pridgen E, Molnar LK, Farokhzad OC. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm 2008;5:505-15. Almeida JPM, Chen AL, Foster A, Drezek R. In vivo biodistribution of nanoparticles. Nanomedicine:NBM 2011;6:815-35. Cho K, Wang X, Nie S, Chen Z, Shin DM. Therapeutic nanoparticles for drug delivery in cancer. Clin Cancer Res 2008;14:1310-6.
