Influence of Particle Geometry on Gastrointestinal Transit and

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Influence of Particle Geometry on Gastrointestinal Transit and Absorption following Oral Administration Dong Li, Jie Zhuang, Haisheng He, Sifan Jiang, Amrita Banerjee, Yi Lu, Wei Wu, Samir Mitragotri, Li Gan, and Jianping Qi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11821 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 18, 2017

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ACS Applied Materials & Interfaces

Influence of Particle Geometry on Gastrointestinal Transit and Absorption following Oral Administration Dong Lia,b,1, Jie Zhuangc,1, Haisheng Hea, Sifan Jianga, Amrita Banerjeed, Yi Lua, Wei Wua, Samir Mitragotrid, Li Ganb,*, Jianping Qia,* a

School of Pharmacy, Fudan University, Key Laboratory of Smart Drug Delivery of MOE, Shanghai 201203, China b

Department of Pharmaceutical Engineering, School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China

c

School of Pharmacy, Institute of Nanotechnology and Health, Shanghai University of Medicine & Health Sciences, Shanghai 201318, China

d

Department of Chemical Engineering, University of California at Santa Barbara, Santa Barbara, CA 93106, USA

KEYWORDS: particles, shape, geometry, oral delivery, in vivo

ACS Paragon Plus Environment

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ABSTRACT Geometry has been considered as one of the important parameters in nanoparticle design since it affects cellular uptake, transport across the physiological barriers and in vivo distribution. However, only a few studies have been conducted to elucidate the influence of nanoparticle geometry in their in vivo fate after oral administration. This paper discloses the effect of nanoparticle shape on transport and absorption in gastrointestinal (GI) tract. Nanorods and nanospheres were prepared, and labeled using fluorescence resonance energy transfer (FRET) molecules to track the in vivo fate of intact nanoparticles accurately. Results demonstrated that nanorods had significantly longer retention time in GI tract compared to nanospheres. Furthermore, nanorods exhibited stronger ability of penetration into space of villi than nanospheres, which is the main reason of longer retention time. In addition, mesenteric lymph transported 1.75% nanorods within 10 h, which was more than nanospheres (0.98%). Fluorescent signals arising from nanoparticles were found in the kidney but not in the liver, lung, spleen or blood, which could be ascribed to low absorption of intact nanoparticles. In conclusion, nanoparticle geometry influences in vivo fate after oral delivery and nanorods should be further investigated for designing oral delivery systems for therapeutic drugs, vaccines or diagnostic materials.


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INTRODUCTION Nanoparticles are developing as a promising carrier for oral drug delivery by avoiding drug degradation, increasing retention in gastrointestinal (GI) tract and promoting permeability across the intestinal epithelium

1-3

. In order to optimize nanoparticles, the effect of

physiochemical characteristics on their in vivo fate must be understood. The effect of size on nanoparticle behavior has been well documented where smaller particles (500 nm) are taken up by M cell and transported into intestinal lymph

5-6

. Recently, there has been a

surge in interest in nanoparticle geometry in drug delivery 7-8. The phagocytosis, circulation, and distribution of nanoparticles have been demonstrated to be shape-dependent

9-12

. For

instance, polystyrene nanorods are not easily taken up by macrophages compared to their spherical counterparts 10. In addition, a plethora of inorganic nanoparticles have demonstrated shape-dependent interaction with cells and biodistribution 9, 13-15. Nanoparticle geometry can also be a significant parameter affecting oral delivery, however, this has not been extensively studied. Our previous study showed that rod-like polystyrene nanoparticles were transported 1.5-fold more than nanospheres across intestinal co-culture model representing M cell model in vitro

16

. Nevertheless, cellular uptake is only an aspect

contributing to oral delivery since nanoparticles are required to traverse through a complicated gastrointestinal environment before arriving at the intestinal epithelia. One report suggested that silica nanorods may penetrate into intestinal mucus more efficiently than spheres 17. However, most studies have been performed using single stable fluorescent probes to label nanoparticles for tracking in vivo fate, which makes the conclusions unclear since the fluorescence signals from the inside and the outside of the particles can’t be distinguished

18

. Fluorescence resonance energy transfer (FRET) probes were therefore


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employed in this study to indicate what these intact nanoparticles would undergo after oral administration. FRET is a reliable and effective approach to identify intact particles 19-20. It is a process of transferring radiation energy from an excited-state donor fluorophore to a ground-state acceptor molecule via dipole−dipole interaction. The energy transfer is highly dependent on the distance between the donor fluorophore and acceptor molecule, which occurs when the distance of two fluorophores is within the range of 10 nm. FRET fluorescence disappears when the distance is more than 10 nm or one fluorophore exist alone

21

. Therefore, in this

study, two FRET paired fluorescent molecules were utilized to label the nanoparticles for accurately tracking the transport of intact nanoparticles in GI tract. In order to clarify the shape-dependent oral delivery of nanoparticles, this article investigated the gastrointestinal retention, absorption, intestinal epithelial uptake and lymphatic transport influenced by nanoparticle shape utilizing polystyrene nanospheres and nanorods labelled with FRET molecules. The results of this study demonstrate that the geometry holds great significance in the design of orally deliverable nanoparticles.

EXPERIMENTAL SECTION Materials Polystyrene particles were provided by Polysciences Inc. (Warrington, PA, USA). Polyvinyl alcohol (PVA) was purchased from Sigma-Aldrich (St Louis, MO, USA). Lipophilic fluorescents dyes (DiO, DiI, DiD and DiR) were purchased from AAT Bioquest Inc. (Sunnyvale, CA, USA). 4’,6-diamidino-2-phenylindole (DAPI) was purchased from Yeasen Bio-tech Co., Ltd. (Shanghai, China). Simulated fluid mixed powders (FaSSIF, FeSSIF & FaSSGF) were purchased from Biorelevant.com (Croydon, UK). Glycerinum, dimethyl


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silicon oil, pepsin, trypsin, tetrahydrofuran (THF), isopropyl alcohol and absolute ether were purchased from Sinopharm Chemical Reagent Co., Ltd.(Shanghai, China). Preparation of nanoparticles labelled and the mixed solution with FRET molecules Polystyrene nanorods were engineered from polystyrene spherical particles using the filmstretching procedure reported in our earlier publications

16, 22

, and have not been mentioned

here. Nanoparticles were labelled with two pairs of FRET dyes (DiO and DiI, DiD and DiR) by swelling nanoparticles in organic solvents. Briefly, nanoparticles were spun down by centrifugation at 11,000 g for 15 min. FRET probes (DiO and DiI or DiD and DiR) were dissolved in tetrahydrofuran (THF) to obtain 1 mg/ml solution. 50 µl of FRET probe solution was added to nanoparticles and vortexed for 5 min, followed by addition of 500 µl of pure water and vortexing for 1 min. Thereafter, the nanoparticles were spun down and washed for five times to remove all excess fluorescent molecules. The nanoparticles labeled with DiO and DiI were used in observation by confocal laser scanning microscopy (CLSM) and those labeled with DiD and DiR were used in semi-quantification by IVIS Spectrum Live Imaging System (Perkin-Elmer, USA). The FRET effect of labeled nanoparticles were characterized by Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, CA, USA). The mixed solution used as control was prepared as follows. Briefly, FRET probes (DiO and DiI or DiD and DiR) were dissolved in tetrahydrofuran (THF) to obtain 1 mg/ml probes solution. 50 µl FRET probes solution were dried by nitrogen. The dried residues were dissolved by 50 µl dimethyl sulfoxide (DMSO) and followed by addition of 450 µl of pure water. And then the mixed solution was obtained. Characterization of nanoparticles


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The particle size and polydispersity index (PDI) of different shaped nanoparticles were measured utilizing a Zetasizer Nanos (Malvern Instruments, Malvern, UK) equipped with a 4 mW He-Ne laser (633 nm) at 25 ℃. The morphologies of nanoparticles were observed by transmission electron microscopy (TEM) (Tecnai G2 F20, FEI, Holland) at 200 kV. The nanoparticles were diluted to appropriate concentration and dropped on copper grids. The nanoparticles were stained with 2% (w/v) uranyl acetate for 5 min at room temperature. The nanoparticles on the grids were observed and photographed with TEM. Stability of nanoparticles in simulated physiological fluids To test the stability of labeled nanoparticles in gastrointestinal fluid, they were incubated in ultrapure water, simulated gastric fluid (SGF), fasted state simulated gastric fluid (FaSSGF), fasted state simulated intestinal fluid (FaSSIF), fed state simulated intestinal fluid (FeSSIF) respectively. FaSSGF, FaSSIF and FeSSIF were obtained by dissolving a patented powder (biorelevant.com) in water according to vendor instructions. SGF was prepared according to USP35/NF30. The labeled nanospheres and nanorods were added to the aforementioned simulated media and incubated at 37 °C for 24 h with constant shaking at 150 rpm. At specified time points, quantitative samples were withdrawn and detected for fluorescent intensity, size and PDI. Fluorescent stability of nanoparticles in physiological fluids Three rats weighing 250 ± 20 g for each, were anaesthetized by injection of 10% chloral hydrate solution intraperitoneally at a dose of 5 ml/kg. The blood was collected from the abdominal aorta with 1% heparin sodium solution. And the plasma was obtained by centrifuging blood at 3, 000 g for 5 min. Following this, the abdomen was opened and cut off the segment from duodenum to the end of ileum. The rat intestinal contents were flushed out


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from intestinal segment with 2 ml physiological saline solution. The intestinal medium was got by homogenizing the rat intestinal contents. The labeled nanospheres and nanorods were added to blood, plasma and rat intestinal medium, and then incubated at 37 °C for 12 h with constant shaking at 150 rpm. At specified time points, 200 µL samples were withdrawn and detected for fluorescent intensity by the IVIS spectrum live imaging system (PerkinElmer, USA).

Ex vivo imaging For the purpose of investigating the fate of different shaped nanoparticles in vivo, the residence time in gastrointestinal tract and the distribution in organs and tissues were observed. Nine male SD rats for three groups, weighing 250 ± 20 g for each, were raised in the Experimental Animal Centre of Fudan University and all ethical guidelines on experiments involving the use of animals were followed. Exactly 0.5 ml of different shaped nanoparticles suspensions were administered to the rats by oral gavage and 0.5 ml of mixed solution was administered as control. The animals were sacrificed and dissected at specific time points. The fluorescence intensity and distribution in whole gastrointestinal tract and organs was detected using the IVIS Spectrum Live Imaging System (Perkin-Elmer, USA). The total radiant efficiency after taking off background was used as a quantification index.

Intestinal epithelial uptake Nine male SD rats for three groups, weighing 250 ± 20 g for each, were anaesthetized by injection of 10% chloral hydrate solution intraperitoneally at a dose of 5 ml/kg and maintained at 37 ℃ on a surgical plate. Following this, the abdomen was opened to select a 4 cm segment of jejunum and ileum (containing peyer's patches). After washing with sterilized


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saline to remove the intestinal contents and ligating the segments using medical suture carefully, 1.5 ml of nanospheres or nanorods was injected into each segment slowly and the mixed solution (1.5 ml) was used as control. One hour later, all intestinal segments were cut off from the rats and washed with saline. Thereafter, the intestinal segments were immobilized with 4% paraformaldehyde for 12 hours and then dehydrated in 30% sucrose solution for 24 hours. After being embedded into the OCT compound, the frozen circular sections with depth of 10 µm were collected and stained with DAPI (5µg/ml). Then the frozen sections were observed and photographed by Zeiss LSM 510 confocal laser scanning microscopy (CLSM) (Carl Zeiss Inc., Germany). Lymphatic transport of nanoparticles Lymphatic transport of different shaped nanoparticles was investigated by a conscious lymph duct cannulation mode in rats 23. Nine male SD rats for three groups, weighing 330 ± 20 g for each, were orally given 2.5 ml peanut oil for filling of the lymphatic vessels and visualization of the lymph. Rats were anaesthetized by injection of 10% chloral hydrate solution intraperitoneally at a dose of 5 ml/kg and maintained at 37 ℃ on a surgical plate. The mesenteric duct was cannulated immediately. After cannulation, the mesenteric lymph cannulas were tunneled beneath the skin and fixed to a tube on the ventral side of the animal. When the rats woke up from anesthesia and drank water freely, 1.0 ml nanoparticles were administered to the rats by oral gastric gavage. Lymph samples were collected every 2 h and fluorescent intensity was determined using the IVIS spectrum live imaging system (PerkinElmer, USA). The transport amount of nanoparticles was calculated by standard curve of nanoparticles in lymph. In addition, the nanoparticles in lymph were concentrated for TEM observation. Briefly, the 1 ml lymph samples were diluted by 3 ml pure water and centrifuged at 3,000 g for 5 min. And then, the supernatant was taken out and centrifuged at 11,000 g for 15 min. The nanoparticles were spun down in the sediment and washed with


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pure water for three times. The nanoparticles were stained with 2% (w/v) uranyl acetate for 5 min at room temperature and observed with TEM. Data analysis All data are represented as mean ± standard deviation (SD). The Graphpad of Prism 6.0, (GraphPad Software, LaJolla, CA) was used to plot the graphs and conduct statistical analysis with Student's t-test.

RESULTS Characterization of nanoparticles The nanoparticles were characterized based on their size, charge and morphology. The nanospheres were obtained by the same process as nanorods without stretching in order to compare as control. The particle size of nanospheres was similar to their respective manufacturer labels (200 nm) and quite uniform as evident from the extremely low PDI values of 0.006 (Table 1). The nanorods prepared by stretching showed larger particle sizes than nanospheres according to dynamic light scattering (DLS), but the particle size distribution remained uniform at a PDI of about 0.015. The zeta potential did not change significantly after the nanospheres were stretched into nanorods.

Table 1. Particle size, PDI and zeta potential of the nanoparticles (n=3). Particles

Size (nm)

PDI

Zeta potential (mV)

Nanospheres

184.2±12.1

0.006±0.001

-11.3±0.7

Nanorods

298.1±3.8

0.015±0.006

-12.8±1.2

The TEM images of both nanospheres and nanorods revealed particle size and PDI in accordance with the size obtained from DLS (Figure 1). The nanospheres were in the size


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range of 150-220 nm, while nanorods had a major axis length of approximately 450-500 nm and a minor axis size of approximately 100-150 nm, which indicated the aspect ratio was controlled by stretching.

Figure1. TEM images of nanospheres (A) and nanorods (B), and particle size distribution of nanospheres (C) and nanorods (D). FRET effect of nanoparticles Figure 2 showed the FRET effect of nanoparticles loaded DiD and DiR by fluorescence spectrophotometer. When the excitation wavelength of DiD was used, no fluorescent signal was detected in the emission wavelength of DiR from a THF solution of DiD and DiR. However, strong fluorescence was detected in the emission wavelength of DiR when DiD and DiR were loaded into the nanoparticles simultaneously, which confirmed that FRET effect occurred as some energy of DiD was transferred to excite DiR. Meanwhile, FRET effect was also observed by IVIS spectrum live imaging system which was utilized to quantify the nanoparticles. The fluorescent intensity of nanospheres and nanorods were 1.47 x 109 and


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1.27 x 109 [p/s] / [µW/cm2] by IVIS spectrum respectively. Similarly, other fluorophores DiO and DiI also showed FRET effect when both were entrapped in nanoparticles, which were used to observe confocal laser scattering microscope (CLSM) based images due to their relatively short wavelength of excitation and emission.

Figure 2. Fluorescent emission spectrum of DiD and DiR in THF solution or after loading in nanoparticles upon excitation at the excitation wavelength of DiD at 650 nm. Stability of nanoparticles in simulated physiological fluids In order to accurately evaluate the behavior of nanoparticles in vivo, stability of nanoparticles, including fluorescent intensity, particle size and PDI, was investigated in various simulated physiological fluids (Figure 3). The FRET fluorescence intensity of nanospheres and nanorods did not change significantly upon 24 h incubation except for a slight decline in FaSSGF, which indicated fluorescent molecules would remain encapsulated in nanospheres and nanorods for at least 24 h. The particle size and PDI of both nanoparticles showed slight increase in various media, especially in FeSSIF and FaSSIF, which could be as a result of aggregation induced by the presence of high amount of electrolytes in these media. However, PDI of nanospheres and nanorods remained stable for 24 h after introducing them into various media.


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Figure 3. Stability of nanospheres (A, fluorescence intensity; B, particle size and C, PDI) and nanorods (D, fluorescence intensity; E, particle size and F, PDI) in different simulated physiological fluids (n=3). Fluorescent stability of nanoparticles in physiological fluids Figure 4 demonstrated the fluorescent stability of nanospheres and nanorods in blood, plasma and rat intestinal contents. Basically, there were no significant loss of the fluorescent intensity within 8 h in various physiological fluids except for rat intestinal contents. Until 12


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h incubation, there were only around 6%, 4% and 7% loss of fluorescent intensity in blood, plasma and rat intestinal contents respectively.

Figure 4. Fluorescent stability of nanospheres (black circle) and nanorods (red square) in blood (A), plasma (B) and rat intestinal contents (C). * represents significant difference compared to initial fluorescent intensity. The in vivo transport of nanoparticles determined by ex vivo imaging The transport of nanoparticles in GI tract after oral administration is typically shown in Figure 5A and all images for three rats are placed in Figure S1. There were more nanorods in the stomach at 2 h after oral administration and a large number stayed in the small intestine compared to nanospheres (Figure 5B-D). At the end of 8 h, approximately 30% nanorods were still present in the small intestine compared to less than 10% nanospheres which were mostly excreted and present in cecum and colon (Figure 5E). Nanorods exhibited much longer retention time in jejunum and ileum than nanospheres, which offered more opportunities for intestinal absorption.


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Figure 5. Typical pictures of gastrointestinal segments from SD rats after ex vivo imaging (A) and quantification of nanoparticles in stomach (B), duodenum and jejunum (C), ileum (D), cecum and colon (E) based on total fluorescence after oral administration (n=3). * represents significant difference compared to nanospheres (p < 0.05). Figure 6 shows the typical distribution of nanoparticles in various reticuloendothelial organs after absorption and all images for three rats are shown in Figure S2. No fluorescent signal was observed in the organs except kidney, which was low for both nanospheres and nanorods. However, interestingly, the fluorescent signals from nanospheres were observed in kidney much earlier than that from nanorods.


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Figure 6. Typical ex vivo imaging of reticuloendothelial organs isolated from rats following oral administration of nanoparticles. The uptake of nanoparticles by intestinal epithelial The intestinal uptake of nanoparticles was investigated by observing the frozen section of small intestine after bowel loops treatment by CLSM. FRET fluorescence was observed to elucidate the uptake of nanoparticles by intestinal epithelia. There was no any FRET signal in intestine after administration of the mixed solution. In jejunum (Figure 7), there were few


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nanospheres at the lumenal side near the epithelial cells and only a small amount attached on the surface of mucus. In contrast, the nanorods largely distributed not only the surface of the jejunal villus but also penetrated between villus to increase the retention and absorption opportunity. The similar results were observed in ileum (Figure S3).

Figure 7. Confocal laser scanning microscopy (CLSM) images of frozen sections of jejunum after bowel loops treatment with the mixed solution, nanospheres and nanorods. (Magnification time of 100 for large images and 400 for small images in merge column) Lymphatic transport


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The FRET signals were used to represent the intact nanoparticles in lymph because the FRET fluorescent intensity was pretty stable in lymph within 24 h and the free FRET molecules could not aggregate to exhibit the FRET effect (Figure S4). The lymphatic transport of both nanoparticles began within the first 2 h of oral administration that peaked at around 4 h, slowed down after 6 h and extended to as long as 10 h as shown in Figure 8. A significantly higher amount of nanorods were transported into lymph between 2-4 h and 4-6 h than nanospheres. The cumulative transport efficiency of nanorods within 10 h was 1.75%, while that of nanospheres amounted to only 0.98%, which were calculated by standard curve of nanoparticles in lymph samples (Figure S5). However, there were relatively large individual difference between rats, and the lymph flow rate influenced the final results. Despite these differences, the work suggests that more nanorods were transported by lymph. At the same time, the intact nanospheres and nanorods were observed clearly in lymph samples, which indicated the nanoparticles were indeed transported into lymph (Figure 9).

Figure 8. Lymphatic transport of nanoparticles every two hours for 10 h (A) and cumulative transport (B) after oral administration (n=3). * represents significant difference compared to nanospheres (p< 0.05).


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Figure 9. The nanospheres (A) and nanorods (B) in lymph samples observed by TEM.

DISCUSSION Oral route is a preferred way to deliver drugs due to high patient compliance. However, many challenges, such as poor water solubility, low stability in GI tract, poor permeability across the intestinal epithelia and short retention time induced by gastrointestinal movements remarkably reduce oral bioavailability of many therapeutic drugs 24. Currently, nanoparticles are considered to have high potential in enhancing oral bioavailability of poorly water soluble drugs, proteins or peptides

25-26

. However, in vivo fate of nanoparticles could be affected by

various physicochemical characteristics including particle size, charge, surface modification or geometry. Our previous work had revealed that nanorods were more efficiently transported by M cell model in vitro compared to nanospheres and nanodiscs

16

. However, in many

instances in vitro data may not correlate well with in vivo findings, which makes it necessary to assess in vivo fate of nanoparticles of different shapes after oral administration to validate in vitro results. Nanorods were earlier found to demonstrate higher transport across M cells in vitro compared to spheres of comparable volumes. Therefore, nanorods and nanospheres were selected to be tested in this work. In order to understand the in vivo behavior accurately, near


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infrared FRET molecules were used to label the nanoparticles. FRET effect occurs only when the distance of two molecules are very close. In this work, FRET fluorescence was observed when DiD and DiR were loaded into nanoparticles simultaneously and it disappeared while either DiD or DiR leached out of nanoparticles, which helped track the intact nanoparticles in vivo. Polystyrene nanoparticles swell to produce numerous pores in organic solvents like tetrahydrofuran and shrink in water after removal of organic solvents 27, which was utilized to load the DiD and DiR. The nanoparticles loaded with DiD and DiR (1:1, mole ratio) showed strong fluorescence peak at 780 nm which was the emission wavelength of DiR when the excitation wavelength of DiD was utilized, which confirmed occurrence of FRET phenomenon in the nanoparticles. The DiD and DiR could penetrate into nanoparticles by swelling pores simultaneously because they have similar properties and structure. And then they would keep closer distance due to affinity of similar structure when the nanoparticles shrank in aqueous environment. In addition, the stability of nanoparticles in physiological fluids is very important to study their in vivo fate. The particle size and PDI of nanospheres and nanorods increased in the fluids with high electrolyte and surfactant contents such as FaSSGF, FaSSIF and FeSSIF due to change in surface charge, that would lead to nanoparticle aggregation

28

. However, fluorescent intensity of both nanoparticles did not decrease

significantly in various simulated physiological fluids, which indicated that DiD and DiR did not leach out of the polystyrene nanoparticles. Furthermore, there were a little loss of the fluorescent intensity after 12 h incubation of nanoparticles in blood, plasma and rat intestinal contents, but the fluorescent intensity maintains stable within 8 h and the fluorescent intensity was not significant difference between nanospheres and nanorods at any time points in various media. Therefore, the FRET fluorescent signals can represent the intact nanoparticles in vivo and intensity is used to determine the relative amount of nanoparticles.


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In order to evaluate the whole process of nanoparticles in GI tract after oral administration, the two aspects were considered respectively, including the GI transit and in vivo absorption. In the first place, there was evidence that the GI transit of rods differs to that of spheres with greater residence time of the former. Nanoparticles dispersed in large area in GI tract after oral administration. After 0.5 h, a large number of nanoparticles were found in jejunum and stomach. And, nanorods exhibited much longer retention time than nanospheres in intestine, which indicated the nanoparticles shape could affect the movement route or rate (Figure 5). But the lymphatic transport of nanorods was more than that of nanospheres, which looks like contrary to the results of intestinal retention because the fluorescent intensity was used to characterize the relative amount of nanoparticles. Although nanoparticles could be absorbed into systemic circulation, the absolute amount is very low (

Influence of Particle Geometry on Gastrointestinal Transit and

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