An Intestinal âTransformersâ-like Nanocarrier System for Enhancing

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An Intestinal “Transformers”-Like Nanocarrier System for Enhancing the Oral Bioavailability of Poorly Water-Soluble Drugs Er-Yuan Chuang, Kun-Ju Lin, Tring-Yo Huang, Hsin-Lung Chen, Yang-Bao Miao, Po-Yen Lin, Chiung-Tong Chen, Jyuhn-Huarng Juang, and Hsing-Wen Sung ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b00470 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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An Intestinal “Transformers”-Like Nanocarrier System for Enhancing the Oral Bioavailability of Poorly Water-Soluble Drugs Er-Yuan Chuang, 1,‡ Kun-Ju Lin,2,‡ Tring-Yo Huang,3 Hsin-Lung Chen,3 Yang-Bao Miao,3 Po-Yen Lin,3 Chiung-Tong Chen,4 Jyuhn-Huarng Juang, 5,* and Hsing-Wen Sung,3,* 1

Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University,

Taipei, Taiwan (ROC) 2

Department of Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan (ROC)

3

Department of Chemical Engineering and Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, Taiwan (ROC)

4

Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Miaoli, Taiwan (ROC)

5

Division of Endocrinology and Metabolism, Chang Gung University and Memorial Hospital, Taoyuan, Taiwan (ROC)

‡

E. Y. Chuang and K. J. Lin contributed equally to this work.

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ABSTRACT

Increasing the intestinal dissolution of orally administered poorly water-soluble drugs that have poor oral bioavailability to a therapeutically effective level has long been an elusive goal. In this work, an approach that can greatly enhance the oral bioavailability of a poorly water-soluble drug such as curcumin (CUR) is developed, using a “Transformers”-like nanocarrier system (TLNS) that can self-emulsify the drug molecules in the intestinal lumen to form nanoemulsions. Owing to its known anti-inflammation activity, the use of CUR in treating pancreatitis is evaluated herein. Structural changes of the TLNS in the intestinal environment to form the CURladen nanoemulsions are confirmed in vitro. The therapeutic efficacy of this TLNS is evaluated in rats with experimentally induced acute pancreatitis (AP). Notably, the CUR-laden nanoemulsions that are obtained using the proposed TLNS can passively target intestinal M cells, in which they are transcytosed and then transported into the pancreatic tissues via the intestinal lymphatic system. The pancreases in rats that are treated with the TLNS yield approximately 12 times stronger CUR signals than their counterparts receive free CUR, potentially improving the recovery of AP. These findings demonstrate that the proposed TLNS can markedly increase the intestinal drug dissolution, making oral delivery a favorable noninvasive means of administering poorly water-soluble drugs.

KEYWORDS: oral drug delivery, poorly water-soluble drug, M cells, intestinal lymphatic transport; pancreatitis


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For patients, the oral route is the most convenient and comfortable route for administering drugs. The dissolution step of an orally administered drug in the intestinal fluid is the most critical ratelimiting step in achieving the desired pharmacological concentration.1 Unfortunately, many small-molecule drugs that are currently used clinically are poorly water-soluble, as they become insoluble aggregates upon exposure to bodily fluids, reducing their oral bioavailability. Increasing the oral absorption of drugs that have poor oral bioavailability to a therapeutically useful level has long been an elusive goal for formulation scientists. To address this issue, this work proposes a “Transformers”-like nanocarrier system (TLNS) that allows a poorly watersoluble drug such as curcumin (CUR) to self-emulsify in the intestinal lumen to form nanoemulsions and then to be absorbed effectively, improving its oral bioavailability. CUR is a natural phytochemical compound with numerous pharmacological activities but has low water solubility and oral bioavailability2 as well as fast hepatic metabolism.3 Owing to its known antiinflammation activity, the potential of CUR in treating pancreatitis has been evaluated in animal models and in humans.4,5 The proposed TLNS is derived from a bubble-carrier system that self-assembles in the intestinal tract, following oral administration of an enteric-coated gelatin capsule that contains a powdered mixture of an acid initiator [diethylene triamine pentaacetic acid (DTPA) dianhydride], a foaming agent (sodium bicarbonate; SBC), a surfactant (sodium dodecyl sulfate; SDS), and a poorly water-soluble drug (CUR). The hydrolysis of DTPA dianhydride in water yields an acid solution, while the reaction of SBC with an acid generates a bubbly CO2 gas.6 The SDS is an anionic surfactant molecule that consists of a hydrophilic head and a lipophilic alkyl tail; the use of surfactant-based formulations can improve the oral bioavailability of poorly water-soluble drugs by increasing the fluidity of the intestinal cell membrane.7,8 The preparation of the


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described in situ self-assembled TLNS is quite straightforward, involving simply filling a gelatin capsule with a mixture of the specified compounds. This delivery platform can be readily scaledup for industrial purposes. Figure 1 schematically depicts the mechanisms of generation and transformation of the bubble carriers containing CUR which are self-assembled in the intestinal tract from the orally administered capsule; it also depicts the delivery of CUR across the epithelial barrier, ultimately accumulating in the pancreas following intestinal lymphatic transport. To evaluate the therapeutic efficacy of the proposed TLNS, rats with experimentally induced acute pancreatitis (AP) are used as an animal model. AP is a sudden inflammation of the pancreas, which can cause severe pain to human patients with a mortality rate of approximately 10%.9


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Figure 1. Mechanism of formation of nanoemulsions of CUR-laden SDS micelles that are derived from proposed TLNS that self-assembles in intestinal tract, following oral administration of enteric-coated capsule that contains powdered mixture of acid initiator (DTPA dianhydride), foaming agent (SBC), surfactant (SDS), and poorly water-soluble drug (CUR). Mechanism of delivery of CUR across epithelial barrier via intestinal lymphatic transport, and its specific accumulation in pancreas for treatment of AP is also shown.

Following oral administration of the enteric-coated capsule and its dissolution in the small intestine, its contained DTPA dianhydride is exposed to intestinal fluid, generating an acidic environment, in which SBC decomposes to form CO2 bubbles that are stabilized by a monolayer of surfactant molecules (SDS). The lipophilic alkyl tails of these surfactant molecules cause them to self-assemble into a nanofilm that incorporates the poorly water-soluble CUR. As the CO2 bubbles travel to the water/air interfaces in the intestinal lumen, this self-assembled monolayer


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carrier system transforms into double-layer nanoassemblies. After the bubbles burst at the water/air interfaces, the surfactant molecules (SDS) readily transform the double-layer nanoassemblies into micellar nanoemulsions that contain CUR (CUR-laden SDS micelles). The negatively charged SDS micelles are then internalized by the microfold cells (M cells) of which most are located in Peyer’s patches with some scattered on the lateral surfaces of villi, ultimately accumulating in the pancreatic tissues through the mesenteric lymphatic system, enabling them to treat AP. Oral drug delivery typically involves two types of intestinal cell, which are enterocytes and M cells; transcytosis of the negatively charged particles occurs mostly across M cells.10 A drug that is delivered through the lymphatic system can bypass the hepatic first-pass metabolism, making oral delivery a favorable noninvasive means of administering poorly watersoluble drugs.

RESULTS AND DISCUSSION Once exposure to water, DTPA dianhydride decomposes, releasing protons, yielding an acidic solution; SBC swiftly reacts with the acid to produce CO2 bubbles, which are then stabilized by a monolayer of the SDS surfactant molecules. The alkyl tails of the SDS monolayer, which are lipophilic and approximately 1 nm in length,11 may function as carriers to transport poorly water-soluble drugs, such as CUR. DTPA is biocompatible12 and SBC is a buffering agent that is commonly used in standard cell culture media,13 while SDS is approved as an excipient for oral formulations.14 The formulation of empty bubble carriers that consist of DTPA/SBC/SDS (empty vehicles) was firstly optimized, based on the amount of CO2 bubbles that could be generated in the presence of water in a test tube. According to Figure 2a, as soon as water was added to the test


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tube that contained a powdered mixture of DTPA/SBC/SDS, CO2 bubbles were immediately formed. Notably, the formulation with a DTPA/SBC/SDS weight ratio of 8/5/7 produced the most CO2 bubbles (empty vehicles). Consequently, this formulation was used to prepare bubble carriers throughout the rest of this study.

Figure 2. (a) Foaming ability of powders of DTPA/SBC/SDS (empty vehicle) in test tube after addition of water. Fluorescence images showing loading capacity of CUR in vehicle at predetermined dose of CUR: (b) photomicrographs taken by fluorescence microscope and (c) quantitative results obtained by microplate spectrophotometer.

A carrier system must have a high loading capacity for efficient drug delivery. To determine the loading capacity of the as-prepared empty vehicles herein, a pre-determined dose of CUR was mixed with the DTPA/SBC/SDS powders (at a weight ratio of 8/5/7) and subsequently treated with water. Since CUR is a fluorescence agent owing to its phenolic groups,15,16 the CUR


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loading capacity of the bubble carriers was determined by detecting its intrinsic fluorescence. According to Figure 2b, the formation of CUR-laden bubble carriers was clearly detected; the fluorescence intensity of the bubble carriers, which indicates the amount of CUR loaded, increased with the amount of CUR used until a DTPA/SBC/SDS/CUR weight ratio of 8/5/7/4 was reached. Further increasing the amount of CUR used to a DTPA/SBC/SDS/CUR weight ratio of 8/5/7/8 dramatically reduced the fluorescence intensity of the bubble carriers, probably because the dose of CUR that was used in this case was too high so these poorly water-soluble molecules interacted freely with each other, resulting in drug precipitation, before they were loaded into the bubble carriers. The encapsulation efficiency of the bubble carriers, which is the percentage of the drug that is successfully encapsulated, was estimated by subtracting the fluorescence intensity of free CUR from the total fluorescence intensity.17 According to Figure 2c, most of the drug molecules were effectively encapsulated by the bubble carriers at all tested doses of CUR (80–90%) except at a DTPA/SBC/SDS/CUR weight ratio of 8/5/7/8 (20%). Therefore, bubble carriers were formulated with a DTPA/SBC/SDS/CUR weight ratio of 8/5/7/4 for use in the following experiments. CUR is nontoxic and can be orally ingested in large quantities.18 However, upon exposure to the intestinal fluid, poorly water-soluble drugs undergo drug-drug interactions that are stronger than the drug-water interactions, causing them to become insoluble aggregates, reducing their oral bioavailability. As presented in Figure 3a, upon exposure to water, free CUR molecules tended to aggregate and precipitate.


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Figure 3. (a) Photographs of aggregation and precipitation of free CUR in water taken by camera and fluorescence microscope. Results and schematic depictions of structural changes of TLNS and subsequent formation of nanoemulsions of CUR-laden SDS micelles in aqueous environment obtained by (b) X-ray solution scattering (SAXS) and (c) fluorescence microscopy. (d and e) Characteristics of nanoemulsions of CUR-laden SDS micelles analyzed by DLS and TEM.

In this work, a method for substantially enhancing the oral bioavailability of a poorly watersoluble drug is developed, using the proposed TLNS that delivers the drug in the form of nanoemulsions. Structural changes of this TLNS were investigated by small angle X-ray scattering (SAXS) and fluorescence microscopy. When pure SDS in powdered form came into contact with water, its SAXS profile exhibited two sharp peaks with a position ratio of 1/2 (Figure 3b), revealing that the surfactant molecules self-assembled into a multilamellar structure with alternating lipophilic layers (composed of two sublayers of the alkyl tails) and hydrophilic


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layers (composed of the headgroups and water molecules), as schematically depicted in the right panel of Figure 3b. The interlamellar distance that was calculated from the primary peak position was ca. 4.0 nm. Upon formation of the bubble carriers in the absence of CUR (the empty vehicle with DTPA/SBC/SDS), the sharp primary peak was converted into a broad peak and the second-order peak vanished, revealing loss of most of the stacking order of the surfactant layers. The film that surrounds the bubble carriers comprised only a few layers of SDS; that is, the original multilamellar structure that formed in the pure SDS powder became an oligolamellar structure upon foaming. The reduction of the number of stacked surfactant layers allowed their lipophilic alkyl tails to come into contact with CO2 within the bubble carriers, which was thermodynamically favorable since CO2 is basically hydrophobic. In the bubble carriers that were laden with CUR (DTPA/SBC/SDS/CUR), the broad scattering peak almost vanished (indicated by the black arrow) when foaming, suggesting that the surfactant layers that covered the bubble carriers became unilamellar. This finding was verified by our fluorescence images; as shown in Figure 3c, the shells of the bubble carriers were monolayers of fluorescing CUR. A significant proportion of the bubbles with monolayer fluorescence that developed in water eventually arrived at the water/air interfaces. When these bubbles were observed under a microscope, nanoassemblies that exhibited double-layer fluorescence were observed. When the bubble carriers burst, the double-layer fluorescence nanoassemblies were immediately transformed into CUR-laden SDS micelles, in whose hydrophobic cores CUR was incorporated. The size distribution and surface charge of the CUR-laden SDS micelles were


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evaluated by dynamic light scattering (DLS). According to Figure 3d, the size distribution of the SDS micelles was in the range of 300–900 nm and they had a zeta potential of about –60 mV. The lack of significant aggregation of the transformed SDS micelles within 24 h was as a result of the electrostatic repulsion between the negatively charged particles. The TEM micrograph of the CUR-laden micelles showed that they had slightly smaller sizes (200–800 nm, Figure 3e) than indicated by the DLS results, as a result of the dehydration process that was used in the preparation of the TEM samples. The above in vitro results indicate that the TLNS that was derived from the CO2 bubble carriers effectively dispersed the poorly water-soluble drug in an aqueous environment, by forming nanoemulsions of CUR-laden SDS micelles, which markedly increased the dissolution of the drug, a prerequisite for its effective intestinal absorption.14 In

the

animal

study,

an

enteric-coated

capsule

was

filled

with

powdered

DTPA/SBC/SDS/CUR that could produce the CO2 bubble carriers (Figure 2) and then be transformed into CUR-laden SDS micelles (Figure 3) in a “Transformers”-like fashion (“Transformers”-like CUR). A capsule that contained only free CUR served as a control (free CUR). To investigate the biodistributions of CUR, test capsules were orally administered to a rat model using a dosing syringe. At predetermined times, the test rats were sacrificed, and their gastrointestinal (GI) tracts and organs were harvested and then examined using an in vivo imaging system (IVIS). Moreover, the accumulations of CUR in organ tissues were quantified using high-performance liquid chromatography (HPLC) with a fluorescence detector. Figure 4a demonstrates that the CUR molecules that were released from the capsule that contained free CUR aggregated in particular spots along the small intestine (indicated by white arrows). Conversely, those released from the capsule with “Transformers”-like CUR were dispersed throughout the intestinal tract, likely resulting from the intestinal formation of CUR-


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laden nanoassemblies, causing the dispersion of the poorly water-soluble CUR molecules, mitigating the difficulty of drug absorption. According to Figure 4b, only the pancreas yielded a detectable CUR signal in both cases. Notably, the pancreas in the rats that were treated with “Transformers”-like CUR yielded significantly (around 12 times) stronger CUR signals than their counterparts received free CUR (Figure 4c).

Figure 4. (a) Fluorescence images of biodistributions of CUR in (a) GI tract and (b) major organs, following oral administration of test capsules that contained free CUR or “Transformers”-like CUR. (c) Amounts of CUR accumulated in organs determined by HPLC. n.s.: not significant; *: statistically significant (P < 0.05).

Much evidence in rodent models validates the potential anti- inflammatory, cardioprotective, and antidiabetic effects, as well as the anticoagulant activity of CUR that is orally administered


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at doses of 50−300 mg/kg.19 This study evaluates the therapeutic efficacy of the capsules that contained “Transformers”-like CUR at a dose of 100 mg/kg that were orally administered to a rat model with L-arginine-induced AP; the oral ingestion of test capsules with free CUR at an equivalent dose or of empty vehicles (DTPA/SBC/SDS) was also investigated. The normal rats (normal control) and untreated AP rats (untreated control) served as the positive and negative controls, respectively. Blood samples were collected from the tail veins of test rats at predetermined intervals before and after dosing, and the plasma levels of interleukin-6 (IL-6) and amylase were analyzed to monitor the severity of AP. Pancreatitis is associated with an increase in IL-6 level, which is a pro-inflammatory cytokine; additionally, when the pancreas is inflamed, amylase is released into the blood.20 After the rats were sacrificed, the pancreatic tissues of each test group were harvested and processed for histological examination using a light microscope to visualize their morphological changes. As shown in Figure 5a, the administration of L-arginine in test rats induced a marked increase in the serum levels of IL-6 and amylase, revealing the successful induction of AP (untreated control). No significant reduction in IL-6 or amylase levels was detected in the group that had been treated with the capsules containing empty vehicles (P > 0.05), while the group that received the capsules with free CUR exhibited slight improvements. Treatment with the capsules that contained “Transformers”-like CUR significantly suppressed the serum levels of IL-6 and amylase (P < 0.05). These findings demonstrate that the therapeutic effects of the “Transformers”-like CUR were more pronounced than those of free CUR, since substantially more CUR molecules accumulated in the pancreas in the former case than in the latter case, as revealed by our biodistribution study (Figures 4b and 4c).


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Figure 5. Results obtained in AP rats that were treated with multiple daily doses of various CUR formulations: (a) levels of plasma IL-6 and amylase vs. time and (b) histological photomicrographs of pancreatic tissues that are stained with H&E. *P < 0.05 vs. untreated control.

Histological analyses of pancreatic tissues were also carried out. AP is known to be associated with a reduction in the number of acinar cells, which is accompanied by the appearance of cytosolic vacuoles.21 Unlike the normal control group, the untreated AP group exhibited histopathologic signs of AP, but treatment with free CUR slightly improved the integrity of the acinar cells (Figure 5b). The group that was orally treated with the “Transformers”-like CUR exhibited significantly reduced acinar cell vacuolization, revealing the recovery of the pancreas from histological injury.


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Owing to the accelerated metabolism of inflammatory cells, AP may cause a large glucose uptake or considerably increase the accumulation of

18

F-2-fluoro-2-deoxy-D-glucose (18F-

FDG),22 which is a radiopharmaceutical analog of glucose and can be visualized by positron emission tomography/computed tomography (PET/CT). Given its high spatial–temporal resolution, PET/CT is a very sensitive molecular imaging technique that is used clinically.23 To verify the therapeutic efficacy of various treatments for AP, the accumulation of 18F-FDG in the pancreas, indicative of the severity of tissue inflammation, was evaluated by PET/CT in the transverse and coronal tomographic views, and the intensity of

18

F-FDG was measured by

analyzing their corresponding standardized uptake values (SUV).24 18

F-FDG clearly significantly accumulated in the pancreas (as indicated by the white arrow)

in the untreated control (Figures 6a and 6b) relative to the normal control, reflecting the association of severe inflammation with AP. Slight but insignificant improvements in

18

F-FDG

accumulation were identified in the group that received free CUR (P > 0.05) possibly because the amount of CUR that was orally delivered to the inflamed pancreatic tissues was under the therapeutic threshold (Figures 4b and 4c). Minimal accumulation of

18

F-FDG was observed in

the group that was orally treated with “Transformers”-like CUR (P < 0.05), indicative of the amelioration of AP, likely because of a significant increase in CUR that was delivered to the inflamed tissues. The above observations can be readily confirmed from the time-lapse 3D video that is provided in the Supplementary Information, which reveals the spatial distribution of 18FFDG metabolic activity in the body.


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Figure 6. (a) PET/CT images of 18F-FDG accumulations in rats that were treated with multiple daily doses of various CUR formulations and (b) their semi-quantitative radioactivity concentrations (SUV) that were accumulated in pancreatic tissues, indicative of severity of inflammation.

To better understand the route of drug delivery, rats were treated with the capsules that contained “Transformers”-like CUR using a loop method, which has been widely used for studying the intestinal absorption mechanisms of drugs.25 The route of CUR delivery was then investigated by immunofluorescence staining, IVIS fluorescence examination, and transmission electron microscopy (TEM).26 Analyses of the immunofluorescence staining results demonstrate that the signals of CUR-laden SDS micelles (negatively charged) were highly co-localized with the M cells, most of which are located in Peyer’s patches with some scattering on the lateral surfaces of the villi.27 Following uptake by the M cells, the CUR-laden particles were detected in Peyer's patches (as indicated by the white arrows in Figure 7a). M cells easily undergo efficient


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endocytosis and transcytosis of nanoparticles because their apical surfaces have broad membrane microdomains from which endocytosis occurs.28,29 Thus, the detection of CUR fluorescence signals in Peyer's patches is hypothesized to result from transcytosis of CUR-laden particles across M cells. One possible mechanism by which M cells to capture the negatively-charged particles is known to be partially related to their lower electrostatic repulsion than that of enterocytes.10 The microvilli at the apical surface of enterocytes contain numerous negativelycharged molecular species,10 so the particles (CUR-laden SDS micelles) were drawn toward the more neutral M cells that lack microvilli. Furthermore, the cellular uptake of nanoparticles depends on the size of the particles: small particles (< 50–100 nm) can be internalized by enterocytes, and larger particles are more likely taken up by M cells.30

Figure 7. Route of CUR delivery following administration of test capsules that contained “Transformers”-like CUR using a loop method: (a) CLSM images showing co-localization of CUR-laden SDS micelles with intestinal M cells, followed by their transcytosis into Peyer's


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patches. (b) Schematic depiction, photograph, IVIS image, and TEM micrograph of harvested GI tract together with intestinal lymphatic system and pancreas. I: intestine; M: mesenteric lymphatic system; LN: lymph node; and P: pancreas.

Ex vivo IVIS images demonstrate that CUR was delivered to the pancreas via intestinal lymphatic transport. TEM reveals the electron density of the CUR-laden SDS micelles that were stained by osmium tetroxide (OsO4), a preferential staining agent for the double bonds of unsaturated compounds (such as SDS or CUR),31 in lymph nodes (as indicated by the red arrows in Figure 7b). However, whether the delivery of CUR-laden SDS micelles could enhance the absorption of endotoxins and other toxic compounds/particles that are present in the small intestine remains to be explored. CONCLUSIONS In summary, the above experimental results indicate that the nanoemulsions of CUR-laden SDS micelles that are derived from the proposed TLNS can passively target intestinal M cells, in which they are transcytosed and then accumulated in the pancreatic tissues by the mesenteric lymphatic system, successfully ameliorating the severity of AP. This TLNS may allow a broad spectrum of poorly water-soluble drugs to be effectively emulsified in the intestinal tract, increasing their solubility and thereby substantially enhancing their oral bioavailability.

MATERIALS AND METHODS


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Materials. All chemicals and reagents used were of analytical grade and were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA) unless otherwise stated. Cell culture reagents were acquired from Life Technologies (Carlsbad, CA, USA). Optimization of Formulation of Bubble Carriers. To optimize the formulation in each test capsule for use in subsequent studies, the loading capacity of the generated bubble carriers was estimated using a powdered mixture of DTPA dianhydride (8 mg), SBC (5 mg), SDS (7 mg), and a pre-determined dose of CUR (0, 0.5, 1.0, 2.0, 4.0, or 8.0 mg). The contents of each test capsule with various amounts of CUR were initially exposed to deionized (DI) water. The formation of the bubble carriers and changes in their CUR loading capacity in DI water were then monitored using a camera and a fluorescence microscope (Axio Observer; Carl Zeiss, Jena, Germany). The fluorescence intensity of CUR was measured using a microplate spectrophotometer (SpectraMax M5, Molecular Devices, CA, USA), and the encapsulation efficiency of the bubble carriers was calculated by subtracting the fluorescence intensity of free CUR from the total fluorescence intensity.17 Structural Analysis of Bubble Carriers. The formation of bubble carriers and their structural changes as the gas bubbles transformed into CUR-laden SDS micelles were studied by SAXS, fluorescence microscopy, DLS (Zetasizer Nano-ZS, Malvern, Worcestershire, UK), and TEM (JEOL 2010F, Tokyo, Japan). The structure of the SDS films that surrounded the bubble carriers was characterized by SAXS at Endstation BL23A1 at the National Synchrotron Radiation Research Center (NSRRC), Hsinchu, Taiwan. The procedure and conditions of the SAXS experiments have been described in our earlier investigation.6 The SAXS experiment yielded a scattering intensity profile as a plot of the scattering intensity I(q) as a function of the magnitude of the scattering vector, q = (4π/λ)sin(θ/2) where λ and θ are the X-ray wavelength 19 Environment ACS Paragon Plus

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and scattering angle, respectively. To make TEM observations of the developed CUR-laden SDS micelles, a drop of sample solution was placed on the copper grid immediately after the reaction, and then blot-dried using filter paper; thereafter, 1% OsO4 solution was dropped onto the grid and blot-dried. Animal study. The animal studies involved Wistar rats with masses of approximately 250 g, and were performed in compliance with the “Guide for the Care and Use of Laboratory Animals”, which was prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press in 1996. The Institutional Animal Care and Use Committee of National Tsing Hua University approved all studies. To prepare the enteric-coated capsules for use in the animal studies, hard gelatin capsules (size 9; Torpac Inc., Fairfield, NJ, USA) were manually filled with a powdered mixture of DTPA dianhydride (8 mg), SBC (5 mg), SDS (7 mg), and CUR (4 mg), as per the manufacturer's instructions. The as-prepared capsules were dipped into a methanol solution that contained Eudragit® L100-55 (15% w/v, Evonik Industries, Parsippany, NJ, USA) and subsequently dried using an air-blower; this process was conducted three times. CUR Biodistribution. Test capsules that contained free CUR or “Transformers”-like CUR were individually administered orally to overnight-fasted rats (n = 3) via oral gavage using a dosing syringe. The untreated normal rats served as a control. Fasted animals are used in many experiments, mainly to reduce the variability of the parameters of interest and to facilitate comparisons of data.32,33 It is worth noting that food effects on pharmacokinetics must be carefully examined in non-fasted animals. Three hours after treatment, rats were sacrificed, and their GI tracts were harvested and visualized using an IVIS imaging system (Xenogen, Alameda, CA, USA).


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Other tested rats from the same groups were sacrificed at 5 h following oral administration. Their major organs, including the heart, lung, liver, spleen, pancreas, and kidneys, were retrieved, and ex vivo fluorescent images of the retrieved organs were obtained and analyzed using the IVIS system that was described above. An aqueous solution that contained tissue lysis buffer (2 mL) was then added to each test tissue. The mixture was homogenized and centrifuged at 14,000 rpm for 30 min; the supernatant was lyophilized and resuspended in 2 mL methanol (with 1% acetic acid and 100 ng/mL 17β-estradiol). Finally, the fluorescence intensity of the solution was determined using HPLC.34 Anti-Pancreatitis Efficacy. To create an AP rat model, each test animal twice received Larginine (250 mg/100 g body weight) intraperitoneally with an interval of one hour.35 After the second injection of L-arginine, the rats were orally administered test capsules with free CUR, capsules with empty vehicles (DTPA/SBC/SDS), or capsules that contained “Transformers”-like CUR; the normal rats and untreated AP rats (six in each group) were the positive and negative controls, respectively. Food and water were removed for a period of 2 h before oral administration and subsequently returned, allowing ad libitum feeding. The rats were orally administered the test capsules three times (two capsules each time) at 5 h intervals. Blood samples were taken from the tail veins before dosing and at various time points after dosing (0, 6, 12, 16, 24, 36, and 48 h). These were centrifuged at 10,000 rpm for 10 min at 4 °C to collect plasma. Levels of amylase and IL-6 of the collected plasma samples were measured using a biochemical analyzer to detect amylase activity (DRI-CHEM 3500s, FUJI) and an IL-6 ELISA kit (Rat IL-6 Immunoassay, Quantikine® ELISA, R&D Systems, Abingdon, UK), respectively.36 For histological examination, specimens of pancreatic tissues were fixed in phosphate-buffered formalin, embedded in paraffin, cross-sectioned, and stained with hematoxylin and eosin (H&E).


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Inflammation Scan. Pancreatitis increases glucose update.37 This effect was assessed herein by PET/CT based on the methods that were described in our earlier study.37 The SUV was calculated as the ratio of the regional radioactivity concentration to the injected amount of radioactivity, normalized to body weight.38 Drug Delivery Route. The route of CUR delivery was studied in vivo by a loop method.25 Briefly, overnight-fasted rats were anesthetized by an intramuscular injection of Zoletil® (50 mg/kg, Virbac Laboratories, Carros, France). The abdominal cavity of the animals was opened by a midline incision, and the small intestine was exposed. The proximal end of the duodenum was tied up, and the test capsules that contained “Transformers”-like CUR were then introduced using a syringe. Three hours following loop administration, the GI tract and intestinal lymphatic system and pancreas were removed from euthanized rats and observed by IVIS to visualize the distribution of fluorescent CUR ex vivo. The retrieved loop segments were cryosectioned and then immunofluorescently stained by Cytokeratin 8 (anti-Cytokeratin 8 antibody [M20]–abcam, Alexa Fluor® 647 dye, Thermo Fisher Scientific)39 to identify the M cells in Peyer’s patches and on villi. The co-location of CUR with M cells was examined by inverted confocal laser scanning microscopy (CLSM). The dissected loops were fixed in 4% paraformaldehyde, and the fixed samples were further incubated with OsO4. After they had been rinsed in s-Collidine and phosphate buffered saline, tissue samples were processed for TEM examination, as detailed previously.40 Statistical Analysis. All results were presented as mean ± standard deviation. To compare the means of pairs of groups, Student's t test was used. One-way ANOVA was followed by the


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Bonferroni post hoc test to compare three or more groups. Differences were considered to be significant at P < 0.05.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Supplementary Video: Real-time PET/CT images of

18

F-FDG accumulations in organs in AP

rat that was treated with multiple daily doses of “Transformers”-like CUR; untreated rat was used as a control. Lateral tomographic views are presented, in terms of the standardized uptake value (SUV). H: heart; P: pancreas; D: diaphragm; I: intestine; and B: bladder. The authors declare no competing financial interest.

AUTHOR INFORMATION Corresponding Author *To whom correspondence should be addressed: [email protected] (J.H. Juang) and [email protected] (H.W. Sung).

ACKNOWLEDGMENT The authors would like to thank the Ministry of Science and Technology of Taiwan (ROC) for financially supporting this research (Contract No. MOST 106-2119-M-007-007 and 105-2314-B038-089-MY2). The PET imaging studies were carried out with the help of the Center of Advanced Molecular Imaging and Translation, Chang Gung Memorial Hospital, Linkou,


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Taiwan. The NSRRC is gratefully acknowledged for providing technical support for the synchrotron X-ray scattering experiment.


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An Intestinal âTransformersâ-like Nanocarrier System for Enhancing

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