In Vivo Fate of Biomimetic Mixed Micelles as Nanocarriers for

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In Vivo Fate of Biomimetic Mixed Micelles as Nanocarriers for Bioavailability Enhancement of Lipid-Drug Conjugates Yuhua Ma, Haisheng He, Wufa Fan, Yingxia Li, Wei Zhang, Weili Zhao, Jianping Qi, Yi Lu, Xiaochun Dong, and Wei Wu ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 2, 2017

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ACS Biomaterials Science & Engineering

In Vivo Fate of Biomimetic Mixed Micelles as Nanocarriers for Bioavailability Enhancement of Lipid-Drug Conjugates

Yuhua Ma†,‡, Haisheng He‡, Wufa Fan‡, Yingxia Li‡, Wei Zhang‡, Weili Zhao‡, Jianping Qi‡, Yi Lu*,‡ , Xiaochun Dong*,‡, and Wei Wu*,‡

†

Key Laboratory for Tibet Plateau Phytochemistry of Qinghai Province, School of

Pharmacy, Qinghai Nationalities University, Xining 810007, China ‡

Key Laboratory of Smart Drug Delivery of MOE and PLA, School of Pharmacy,

Fudan University, Shanghai 201203, China

*Correspondence: Wei Wu & Yi Lu, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China. Tel. & fax: +862151980084. E-mail: [email protected]; [email protected]; [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT: The transformation of lipid-based nanovehicles into mixed micelles (MMs) upon lipolysis plays an indispensable role in enhancement of the oral bioavailability of poorly water-soluble drugs. Therefore, this study employs biomimetic MMs as functional vehicles to enhance the oral bioavailability of the lipid conjugates of a model drug silybin. The main objective is to explore the in vivo fate and underlying mechanisms of facilitated absorption by MMs. Pharmacokinetics in rats indicate bioavailability enhancement by 7~9 folds as compared to a fast-release silybin solid dispersion formulation. Confocal laser scanning microscopy reveals evidence of cellular uptake of integral MMs into the cytoplasm of both Caco-2 and Caco-2/HT29-MTX co-culture cells lines. The recovery of a definite amount of prototype silybin but negligible or traces of lipid-silybin conjugates from the cells, as well as the limited trans-monolayer transport, confirms fast disruption of MMs and fast degradation of the conjugates as well. The MMs survive the gastrointestinal environment with relatively high integrity for about 4 h, and are found locating to intestinal villi surfaces in higher density but in lower density to the basolateral tissues. By scanning the organs, a small amount of integral MMs are observed to distribute mainly to the livers with peak time around 4-8 h. The total amount of lymphatic absorption monitored by cannulation is negligible. It is concluded that biomimetic MMs might be taken up by enterocytes and be digested there to release the prototype drug, which is further transported to the circulation, and only a limited amount of integral MMs could be absorbed into the circulation. KEYWORDS: Lipid-drug conjugate; silybin; mixed micelles; drug delivery; in vivo fate; oral; fluorescent probes

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INTRODUCTION

Lipid-based nanoparticles (LBNs), including solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), lipid-drug conjugates (LDCs), nanoemulsions, microemulsions or self-microemulsifying drug delivery systems (SMEDDS) and so on, mimics the digestion of food lipids in the gastrointestinal tract (GIT) and hold great promise for the enhancement of oral bioavailability of poorly water-soluble and poorly permeable drugs.1-6 Despite the popularity of LBNs, the underlying mechanisms have not been fully understood yet. It is now clear that LBNs will be broken down by lipases in the GIT, and form secondary structures such as liquid crystalline phases and vesicular vehicles, which are further broken down to form mixed micelles (MMs).7-10 It is for sure that MMs play an indispensable role in lipid metabolism, and in our opinion MMs might perform as the terminal shuttles that convey the drugs to their destination of absorption in GIT. However, the general perception of the virtual role that MMs play in oral absorption is still ambiguous. The difficulties partly lie in the inability to collect or recover MMs from gastrointestinal fluid for a full-scale analysis. What we have learnt to date is largely based on speculations. In our previous study,11 the mechanisms of enhanced oral absorption of silybin, a poorly water-soluble and poorly permeable model drug isolated from the seed extract of Silybum marianum plant,12-13 by LDCs as encapsulated in SLNs were explored by tracking either the drug or LDCs or SLNs. Results indicated that SLNs were primarily digested in the GIT and the products of lipolysis, i.e. the drug silybin and the conjugates, were absorbed by the enteric epithelia.11 Since the inherent solubility of the drug or the conjugates in intestinal fluid is extraordinarily low, it is natural to speculate that MMs might work as shuttle vehicles to solubilize and transport the drug 3



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and the LDCs to the site of absorption, simultaneously. Herein, biomimetic MMs composed of phospholipids and bile salts are employed to enhance the oral bioavailability of silybin, and mechanistic investigations are carried out to explore the role that biomimetic MMs might play in oral absorption. Owing to its poor compatibility with MMs, silybin is first conjugated to form LDCs, and subsequently encapsulated into MMs to facilitate loading. Then, the in vitro and in vivo performance of silybin LDCs-loaded MMs are investigated by tracking either silybin or the conjugates or the MMs. Both silybin and the conjugates are analyzed by high-performance liquid chromatography. Tracking of integral MMs is achieved using a technology of monitoring integral nanoparticles developed in our lab.14-16 The core science of this technology lies in the water-quenching fluorescent probes used to label nanoparticles so as to discriminate the vehicles from free probes. It is believed that released or free probes form aggregates immediately and quench completely to give no fluorescence at all upon contact with water. Therefore, the fluorescence observed represents integral MMs. By analyzing the evidence of mass transportation, as well as by comparing with previous results from SLNs,11 the role of MMs in oral absorption is carefully expounded.

EXPERIMENTAL SECTION Materials. Near-infrared aza-BODIPY fluorescent probes, P2 and P4, were

synthesized in our lab according to previous procedures.17,18 Silybin (SB) was purchased from Acetar Bio-Tech Inc. (Xi’an, China). Fatty acid-silybin conjugates (FA-SBs) were synthesized by a one-step reaction of silybin with acyl (6C, 12C and 18C) chlorides in pyridine environment according to our previous procedures.11 Lecithin (Lipoid E100, containing 100% of egg phosphatidylcholine) was from Lipoid GmbH Company (Ludwigshafen, Germany). Sodium taurocholate (STC) was 4


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purchased from TCI (Shanghai) Development Co., Ltd. (Shanghai, China). Silica gel for flash chromatography was provided by Santai Technologies Inc. (Suzhou, China). Polyvinylpyrrolidone (PVP, Plasdone K29/32®) were kindly donated from China Division, ISP Chemicals Co. (Shanghai, China). Non-pareil pellets (sugar spheres, 710-850 µm) were obtained from Gocheng Biotech & Health Co., Ltd. (Hangzhou, China). β-Glucuronidase Helix (Type Hp-2, aqueous solution, ≥ 100,000 U/mL) were purchased from Sigma-Aldrich (Shanghai, China). Isoflurane was supplied by RWD Life Science Co., Ltd. (Shandong Keyuan Pharmaceutical Co., Ltd., China). Ultrapure deionized water was prepared by a Milli-Q purification system (Millipore, Molsheim, France). HPLC grade acetonitrile and methanol were provided by Tedia Company Inc. (USA). Caco-2 and HT29-MTX cells were from Cell Bank of Chinese Academy of Sciences (Shanghai, China) and China Center for Type Culture Collection (Wuhan, China), respectively. All cell culture media were purchased from Gibco (USA). Male Sprague-Dawley (SD) rats were obtained from Shanghai Laboratory Animal Center (Shanghai, China). The animal research protocols were approved by the Institutional Animal Care and Use Committee at School of Pharmacy, Fudan University, and were in full compliance with international guidelines on animal welfare. Preparation of FA-SB MMs. FA-SB MMs was prepared by a modified thin-film dispersion method.19 First, lecithin, STC and FA-SBs were dissolved in tetrahydrofuran/methanol (2:1, v/v). The organic solution was evaporated by a rotary evaporator to form a thin film on the bottom wall of a round-bottom flask. Then, the residue was hydrated with 7 mL phosphate buffer (pH 7.4) at 37°C under stirring for 10 min to form drug-loaded MMs. For fluorescence labeling, the water-quenching fluorescent

probes,

P2

(35

µg)

or

P4

(4

µg),

were

dissolved

in

tetrahydrofuran/methanol during the preparative process of FA-SB MMs. P2, with 5



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maximum absorption/emission wavelengths (λabs/λem) of 708/732 nm, was suitable for live imaging, while P4, with λabs/λem of 651/662 nm, was used for confocal laser scanning microscopy (CLSM). Characterization of FA-SB MMs. The morphology of FA-SB MMs was characterized

using

transmission

electron

microscopy

(JEM-1230

Electron

microscope, JEOL, Japan). The average particles size, polydispersity index (PDI) and zeta potential were analyzed by Zetasizer Nano® (Malvern Instruments, Malvern, UK) equipped with a 4-mW He–Ne laser (633 nm) at 25°C. The amount of SB and FA-SBs was measured by an Agilent 1260 HPLC system.11 C18 column (Agilent ZORBAX SB-C18, 5 µm, 4.6 mm × 250 mm, USA), kept at 40°C, was used to separate SB or FA-SBs from impurities. The mobile phase is composed of methanol and 2% acetic acid in a ratio of 45:55 (v/v) for SB, or acetonitrile/water in a ratio of 62:38, 86:14 and 98:2 for 6C-SB, 12C-SB and 18C-SB, respectively. The flow rate was set to 1.0 mL/min, and the detection wavelength was set to 288 nm. The MMs suspension was dissolved using methanol and analyzed by HPLC for the total drug amount (WT). An ultrafiltration method was used to separate free drugs from encapsulated drugs.20 Briefly, 500 µL of the FA-SB MMs dispersion was diluted by five times with purified water, placed in Amicon® Ultra-0.5 (30 kD, Merck Millipore Ltd., USA), and centrifuged (Shanghai Anting Scientific Instrument Factory, China) under 8000 r/min for 15 min. The drug content in the filtrate was measured (WF), representing the amount of free drug. Drug loading (DL %) and entrapment efficiency (EE %) was calculated as follows: % =

% =

−

× 100%

−

× 100% + − 6


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where WL was defined as the amount of phospholipid and bile salt as a whole. Pharmacokinetic study. The FA-SB MMs formulations were administered to male SD rats (250 ± 20 g) by gavage (equivalent to 9 mg/kg of SB). The reference is a fast-release SB solid dispersion formulation in the form of small pellets, prepared by a fluid-bed coating technique using PVPk30 as a carrier (SB/PVP = 1/4, w/w) following previous procedures.21 Plasma samples were collected by sampling 500 µL blood from the eye socket (n = 6) at 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 and 12 h post administration, followed by centrifugation at 7000 rpm/min for 5 min. The supernatant was collected and stored at −20°C until analysis. The SB concentration in the plasma samples was then determined by HPLC.11 In brief, 100 µL of plasma was mixed with 150 µL acetate buffer (pH 5.0, 0.2 M) and 10 µL of β-glucuronidases, then incubated at 37°C for 3 h to release SB. Afterwards, 40 µL of internal standard (1-naphthol, 10 µg/mL) was added. SB was extracted using 3 mL ice-cold acetone (30 min, −20°C), and the solvent was evaporated by blowing nitrogen at 45°C. The residues were dissolved using the mobile phase followed by centrifugation at 12,000 rpm for 5 min. The supernatant was injected into HPLC for analysis. The same chromatographic conditions as for in vitro SB analysis was employed. The limit of detection was 0.062 µg/mL, and the linearity of this method ranged from 0.094 to 23.97 µg/mL (r = 0.9998). Accuracy of this HPLC method was 99.08 ± 0.01%, and extraction recovery of SB in plasma was 85.56 ± 0.03%. The pharmacokinetic parameters were calculated by software Kinetica 4.4 (Thermo Fisher). Cell culture. Caco-2 and HT29-MTX cells were separately cultured in DMEM with 10% FBA, 1% nonessential amino acids, 1% penicillin and streptomycin and incubated under standard sterile conditions for cell cultures (5% CO2, 37°C).14,16,22,23 Raji cell were cultured under the same conditions except that DMEM was replaced 7



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with RPMI 1640. Cellular uptake. Caco-2 and Caco-2/HT29-MTX (7:3) cells were seeded into 96-well (for fluorescent detection) or 24-well plates (for content determination) at a density of 15 × 104 cells per well. The culture medium was changed every other day. The cell model was ready for cellular uptake study when a homogeneous cell monolayer was formed. The culture media were discarded and washed with HBSS. FA-SB MMs suspensions were diluted with HBSS by three times, Then, 200 µL (for fluorescent detection) or 1 mL MMs suspension (for content determination) were added into each well. After incubation for 2 h, the MMs solution was discarded and washed with HBSS for three times. The cellular fluorescence intensity was measured using IVIS live imaging systems (Perkin Elmer, USA). The cell nuclei were stained with DAPI and images were taken by Zeiss LSM510 confocal laser scanning microscope (Carl Zeiss Inc., Germany). For quantitative analysis in cells, the cells were broken down by alternate freezing and thawing cycles, and lysed by probe ultrasonication (Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China). The concentration of SB and corresponding FA-SB in cell lysates was measured by HPLC and normalized to the total cellular protein content (BCAAssay Kit). Transport

across

cell

monolayers.

Caco-2,

Caco-2/HT29-MTX,

and

Caco-2/HT29-MTX/Raji cells were employed to evaluate the trans-monolayer transport of different FA-SB MMs. The transport of SB and FA-SBs across Caco-2 and Caco-2/HT29-MTX were monitored by HPLC, whereas the transport of integral MMs across Caco-2, Caco-2/HT29-MTX and Caco-2/HT29-MTX/Raji was assayed by monitoring fluorescence using the IVIS live imaging system. Caco-2 and Caco-2/HT29-MTX (7:3) cells were seeded onto the apical (AP) side of PCF membrane inserts (Millipore, Carrigtwohill, Co. Cork, Ireland) and cultured for 14 d. 8


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Raji cells were then added to the basolateral (BL) side and co-cultured for 4-5 d until the trans-epithelial electrical resistance (TEER) value declined obviously, indicating successful infiltration of Raji cells. After washing with HBSS, the cell models were ready, and 400 µL of the diluted MMs and 600 µL of HBSS were added to the AP and BL side of the membrane, respectively. The AP→BL transport of the compounds or MMs was monitored at various time intervals for up to 4 h. In vivo live imaging. Male SD rats (160–180 g) were fasted overnight but allowed free access to water before experiment. P2-labled FA-SB MMs (0.8 mL) were given to each rat by gavage (n = 3). P2 signals were captured at various time intervals post administration using IVIS Spectrum Live Imaging System. Throughout the imaging process, delivery of isoflurane to each rat was maintained via a nosecone. Biodistribution. Biodistribution studies were carried out using male SD rats weighing 160–180 g. P2-labled FA-SB MMs (0.8 mL) were administered by gavage to the animals. The rats were sacrificed and dissected at specific time intervals for 24 h post administration to collect major organs like liver, lung, spleen, kidney and the whole GIT. The organs were washed with cold saline, sopped up by filter paper, and imaged with IVIS. To determine the concentration of MMs in blood, 200 µL of blood samples were collected from the eye socket at various time points after administration, and the fluorescent intensity of blood samples was measured by IVIS. Histological examination of the uptake of MMs in small intestine. To observe the bioadhesion behaviors and membrane permeability of MMs in the GIT. The intestinal segment was dissected 1 h after administration, fixated, dehydrated and frozen in

Optimal

Cutting Temperature

compound

(OCT, Surgipath®, Leica,

USA). The frozen tissues were cut into 10-µm slices using a Leica Microm CM3050S cryostat (Leica Inc., Germany) and stained with DAPI to reveal the nuclei. Tissue 9



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sections were observed by CLSM. Transport via mesenteric lymphatics. To identify the contribution of lymphatic transport, mesenteric lymph duct cannulation was performed following reported procedures with minor modifications.24,25 In brief, 1 h before administration, the SD rats (about 350 g) were given 2 mL of peanut oil by oral gavage to induce the production of lymph. One hour later, the rats were anaesthetized with 10% chloral hydrate. A midline abdominal incision was made to the xiphoid cartilage, and the superior mesenteric lymph duct was cannulated with a tubing. The cannula was fixed and externalized through a stab wound in the right flank. The revived animals were allowed to recover overnight with free drinking of 5% glucose solution. At time intervals after oral administration, lymph samples were collected continuously and the cumulative fluorescence was measured using IVIS.

RESULTS Preparation and characterization of MMs. FA-SB MMs, irrespective of the

chain lengths of the lipids used, are spherical as observed under TEM (Figure 1). The particle size distribution of all FA-SB MMs ranges from 20 to 100 nm, with an average size of 56.99 ± 2.58 nm, 57.10 ±1.09 nm and 50.60± 2.24 nm for 6C-, 12Cand 18C-SB MMs, respectively (Figure 1). The PDIs are all less than 0.15, indicating uniform dispersity (Table 1). Due to high lipophilicity, all FA-SBs can be encapsulated into MMs completely with EE % of 100% and DL around 9%. Pharmacokinetics. After administration of FA-SB MMs, none of the conjugates can be detected in rat plasma. Therefore, only the pharmacokinetics of SB are evaluated. The primary pharmacokinetic parameters of FA-SB MMs and SB solid dispersion pellets are presented in Table 2, while the mean plasma SB concentration vs. time curves are shown in Figure 2. The control group (SB solid dispersion pellets) 10


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shows much lower levels of SB exposure in circulation than any MM formulation. FA-SBs with shorter chain lengths show smaller Tmax (0.40, 0.50 and 1.17 h for 6C-, 12C- and 18C-SB, respectively), indicating faster absorption. However, 12C-SB with a median-length FA shows the highest Cmax. Compared to the solid dispersion pellets, the relative bioavailability of MMs has been increased to 7.13, 8.17 and 7.85 folds for 6C-, 12C- and 18C-SB, respectively. Cellular Uptake. No fluorescence signals are observed in the cells incubated with quenched P4 solution, which is a criterion that precludes the interference from fluorescence rekindling (Figure 3A). There are no significant difference in fluorescence

intensity

among

three

MM

groups

in

either

Caco-2

or

Caco-2/HT29-MTX cell models (P > 0.05) (Figure 3A and 3B). To investigate the cellular uptake of MMs further, the Z-stack model of CLSM were used to locate the position of P4-labeled MMs. As shown in Figure 3C, the red fluorescence signals are found on both AP and BL sides in each cell model, implying that MMs can be internalized no matter whether there is a layer of mucusa or not. However, the fluorescence signals locate in higher density to the AP side in the Caco-2/HT29-MTX cell model than in the Caco-2 cell model, highlighting that mucosa might function to trap MMs via bioadhesion. Figure 3D shows the quantitative results of SB ingested by Caco-2 and Caco-2/HT29 cells after incubation with MMs. Remarkably, the amount of SB in the 6C-SB MMs (12.61 µg/mg) group was 4.87- and 26.83-fold higher than that of 12C-SB MMs (2.59 µg/mg) and 18C-SB MMs (0.47 µg/mg), respectively. However, no conjugates are detected for 6C- and 12C-SB MM groups in either Caco-2 or Caco-2/HT29-MTX cells after incubation for 2 h, whereas 18C-SB was detected in both cell models (Figure 3E). The uptake of 18C-SB in Caco-2/HT29-MTX cells was 11



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2.43-fold higher than that in Caco-2 cells. Trans-membrane transport of FA-SB MMs. The transport of SB and FA-SB across the Caco-2 and Caco-2/HT29-MTX cell monolayers is shown in Figure 4A-D. 6C-SB MMs showed 7- and 15-time higher cumulative AP→BL transport of SB than the 12C- and 18C-SB counterparts did in both cell models, respectively. Only negligible amount of SB can be transported across both cell models for 18C-SB MMs. However, the amounts of FA-SB transportation across both cell models are far less than SB, with a maximum around 0.5%. In addition, much more amount of transportation of FA-SB is found for 18C-SB, whereas there is none for 6C-SB. Fluorescence signals of P4 are monitored to track the transportation of integral MMs. No fluorescence signals are detected for all three MM groups in the reception cell of either Caco-2 or Caco-2/HT29-MTX cell models. However, fluorescence signals can be detected in the reception cell of Caco-2/HT29-MTX/Raji model for all three MM groups (as shown in Figure 3E), indicating penetration across the cell monolayers. The AP→BL transportation curves for either 6C- or 12C- or 18C-SB MMs are similar, with cumulative amount of 3.02%, 3.23%, and 3.40%, respectively. In vivo fate of FA-SB MMs in GIT. All MMs, irrespective of the cargos, show similar in vivo fate (Figure 5). As shown in Figures 5A, the fluorescence is confined to the abdominal region for up to 12 h. The transition of MMs down through the GIT is also observed through isolated gastrointestinal segments at different intervals post administration (Figure 5B). FA-SB MMs reach the distal end of the small intestine at 0.5 h post administration and scatter throughout the whole GIT at around 2 h. However, at 8 h only weak fluorescence can be observed in GIT. To investigate the interaction of MMs with intestinal epithelia, the segment of small intestine were collected and analyzed by CLSM. As shown in Figure 6, there are red 12


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signals near the epithelial cells in all three MM groups. The MMs distribute uniformly along the surfaces of the intestinal villi with strong intensity, which thus confirms the penetration and absorption of the MMs by intestinal epithelia. Biodistribution and lymphatic transport of FA-SB MMs. After oral administration of P2-labled FA-SB MMs, all major organs, including the liver, lung, spleen, and kidney, were then harvested and imaged ex vivo. Figure 7A indicates that MMs accumulate gradually in liver, beginning at 2 h (for 12C- and 18C-SB) or 4 h (for 6C-SB) after administration and lasting for about 12 h, and fluorescence intensity reaches the maximum at around 8 h (4 h for 6C-SB), after which the signals attenuate gradually (Figure 7B). However, no fluorescent signals are observed in lung, spleen, and kidney tissue, indicating that the liver was the major metabolic organ of FA-SB MMs. Only a limited amount of MMs are found in blood with a peak time at around 12 h after administration of MMs (Figure 7C), whereas lymph-borne fluorescence signals are negligible with approximately 0.25%, 0.28% and 0.29% recovery of 6C-, 12C- and 18C-SB MMs from mesentery lymph (Figure 7E) and a peak time at around 8 h (Figure 7D).

DISCUSSION

In this study, FA-SB incorporated mixed micelles were successfully prepared with the drug loading up to 9%. In fact, the uplimit of drug loading might be even higher. However, in order to make meaningful comparison between different FA-SBs, the drug loading was fixed to a moderate level of around 9%. We initially tried to encapsulate SB directly into mixed micelles but failed to obtain desirable formulations. SB readily leaks from MMs and results in very low drug loading. Although a drug loading of 1% has been achieved for SB, the size distribution of MMs so prepared was very wide. This is mainly ascribed to the poor lipophilicity of 13



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SB, which renders SB poorly soluble in the hydrophobic core of phospholipid/bile salt MMs. Modification with various lipid moieties of different chain lengths helps to increase the lipophilicity of SB and enhance its overall loading efficiency. Referring to our previous study with SLNs,11 the same effect of enhancement in drug loading is observed, except that the drug loading of SLNs is as high as 32% due to their relatively bigger size than MMs. Similar enhancement in entrapment efficiency has also been found for a variety of drug entities by lipid conjugation.26-28 In order to highlight the efficacy of the lipid assemblies, a fast-release SB formulation, solid dispersion pellets, is used as a reference to deduct the contribution of solulization. Neither of 6C-SB, 12C-SB and 18C-SB could be detected in plasma, which was in accordance with our previous results of FA-SB SLNs.11 There is a good chance that FA-SB, as well as the vehicles, has degraded completely in GIT or along the trans-epithelial absorption path. This is further proved by cellular uptake and trans-monolayer transport results. The fact that a certain amount of SB but no FA-SBs except a minute amount of 18C-SB is found in both cell lines implies that FA-SBs could be rapidly degraded in cell lines to produce SB. The same is true for the transport results. However, it should be noted that there is only a single monolayer in both the cellular uptake and transport test. If taking into account of the long passages of oral absorption, there is no way to assume transportation of integral FA-SBs across the epithelial membrane. Although we did not carry out studies on cellular uptake pathways, results by other researchers indicated that MMs compised phospholipids and a bile salt, deoxycholic acid, might be taken up via endocytosis and deliver their payloads directly into the cytosol.29 It is paradoxical to find that the cellular uptake of 18C-SB by Caco-2/HT29-MTX cell models is 2.43-fold higher thatn by Caco-2 cell models (Figure 3E) because the 14


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mucus layers coating the former are believed to present as barriers to penetration of particles. To provide an explanation, we need to make clear first that the measured value encompasses both internalized and adsorbed fractions. Our previous studies16 proved that either SLNs or MMs are prone to adhere to the surface mucus layers of Caco-2/HT29-MTX cell models, which serve as a “trap” to retain the nanoparticles. None the less, the Caco-2 cell models do not have a coating mucus layer, and thus “take up” lesss particles. On the other hand, it should be noted as well that LDCs are readily degraded by enzymes upon disruption of the cells during sample processing. That is why we only measured 18C-SB, but not 12C-SB or 6C-SB, that has a relativley slow degradation rate. Compared with our previous results of FA-SB SLNs,11 FA-SB MMs exhibited higher oral bioavailability. The significant difference in oral bioavailability as well as Tmax between 18C-SB MMs and 18C-SB SLNs highlights the importance of transformation of SLNs into MMs upon digestion. In this study, no significant difference in oral bioavailability between three FA-SB MMs is observed, although 12C-SB MMs seemed to have slightly higher bioavailability than the other two MMs. However, the delayed Tmax of 18C-SB MMs is possibly due to the relatively slower degradation of 18C-SB and release of SB into the blood circulation. In vivo live imaging indicates that all MMs groups can be emptied from the stomach quickly and reside in small intestine for prolonged time duration. After 4-8 h, the amount of intestine-borne MMs gradually decreases, possibly due to either degradation or absorption. It is very interesting to find that for all MM groups the intestine-borne MMs maintain at a relatively steady level for at least 2 h. Since the overall input of MMs from stomach into intestine before 2 h is very limited, it is reasonable to deduce that MMs maintain relatively high stability in the small intestine. 15



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In our previous study,11 free FA-SBs were found to be readily degraded in the presence of lipases or alkaline. MMs seem to have protective effect against intestinal digestion, which nevertheless could be explained by the enzyme-inhibiting effect of taurocholate that composes the MMs.30 Our previous results indicated that both free FA-SBs and SLNs loading FA-SBs could be digested by lipases in simulated intestinal fluids. The outcome of digestion is that a fraction of SB or FA-SB will be transferred into MMs directly, but a small fraction of free SB might be released prematurely in the intestinal lumen. This fraction of free SB might follow the same fate of the free SB formulation, such as the solid dispersion pellets in this study, whose oral bioavailability is extremely limited. Based on this analysis, MMs seem to be superior to SLNs because there is no need of a digestion/transformation process that might compromise the overall "absorbable" fractions.16,19,31-33 The presence of fluorescence in various organs means that there is absorption of a certain amount of integral MMs and subsequent transportation into various organs, especially liver, via systemic circulation. Mainly owing to the lack of available analytical tools, we are still unable to make out the exact percentage of MM accumulation in the organs. However, a rough estimation of the overall amount of absorption of integral MMs could be made based on our experience and through comparison with SLNs.16 In vitro evaluation in cell lines indicated that MMs could hardly be transported across cell monolayers and either MMs or FA-SBs could be degraded very quickly within cells. The same conclusion was obtained in the previous study for SLNs: disruption of the vehicles or degradation of the conjugates plays the leading role in enhancement of SB absorption.11 The contribution of absorption of integral nanovehicles is trivial. By comparing the hepatic and blood kinetics of MMs with SLNs, it is easy to conclude that the amount of absorption of integral MMs is 16


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less than SLNs by about one third. As our previous findings showed that orally administered SLNs could hardly be absorbed integrally, it is reasonable to conclude that the overall absorption of integral MMs is negligible as well despite the observation of distribution of MMs to livers and several organs. Histological examination of cross sections of intestinal villi also confirms that integral MMs locate to villi surfaces in great amount and only a limited amount can permeate deep into the basolateral tissues. It is paradoxical to find that no FA-SBs are detected in blood although absorption of integral MMs implies that FA-SBs might be absorbed as well simultaneously. The main reasons might be ascribed to the single-pass clearance of the nanovehicles by liver and subsequent degradation of FA-SBs there. On the other hand, the disruption of the lipid conjugates during sample processing might contribute to it as well. To explore possible pathways for the entrance of MMs into systemic circulation, the lymphatic transport of MMs was monitored and quantified. It is obvious that the lymphatic absorption of MMs is negligible with a total transport percentage of less than 0.3% in contrast with about 6% for SLNs.16 The relatively larger size of SLNs might favor more transportation via lymph. On the other hand, the relatively longer lifetime of SLNs in tissues contributes to more translocation of integral SLNs than MMs.

CONCLUSIONS Conjugation of SB with lipids significantly improves its encapsulation efficiency in biomimetic MMs. MMs perform better than SLNs in enhancement of the oral bioavailability of SB as lipid conjugates. Taking together both in vitro and in vivo results obtained by tracking either SB or the conjugates or the vehicles, it is concluded that: (1) MMs can be taken up by intestinal epithelia but only a trivial amount can be 17



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absorbed as integral vehicles and transported into various organs via systemic circulation; (2) both MMs and the conjugates might be broken down in epithelial cells very quickly to release SB, which makes up of a majority of bioavailable SB; (3) the contribution of lymphatic absorption to overall bioavailability is negligible. All in one, MMs play an indispensable role in enhancement of oral absorption of SB, and might be an important intermediate vehicles that bridging the lipid-based vehicles and enhanced oral absorption.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]; [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This study was financially supported by National Natural Science Foundation of China (81573363, 81690263) and National Key Basic Research Program (2015CB931800).

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Table and Figure legends Table 1. Physicochemical properties of MMs containing different conjugates Table 2. Pharmacokinetic parameters of silybin after oral administration of 6C-SB MMs, 12C-SB MMs, 18C-SB MMs and SB solid dispersion pellets in SD rats (n = 6). Figure 1. Transmission electron microscope images and particle size distribution profiles of FA-SB MMs. Be aware that the small particles belong to the staining reagent. Figure 2. Plasma silybin concentration vs. time profiles after oral administration of a single dose (9 mg/kg SB equivalent) of 6C-, 12C- and 18C-SB MMs with SB solid dispersion pellets as a reference in rats Figure 3. Images of cellular uptake of MMs by IVIS live imaging systems (A); and corresponding quantification results of fluorescence (B). CLSM images of cellular uptake of SLNs by cell monolayers (C), the arrows point to fluorescent dots (red) of P4 signals that represent integral MMs at the AP side. Quantification of total silybin (D) and FA-SB conjugates (E) in cells. Figure 4. Cumulative transport of various FA-SB MMs across different cell monolayers for 4 h by measuring the content of SB (A-B), corresponding conjugates (C-D) and fluorescence intensity of P4 (E) Figure 5. In vivo live imaging of the digestion of MMs in rats (A); live imaging of ex vivo gastrointestinal segments (B); quantification of fluorescence of various gastrointestinal samples including stomach, small intestine and cecocolon of 6C- (C), 12C- (D), 18C (E)-SB MMs and the whole GIT (F), respectively. Figure 6. CLSM images of frozen section of the small intestine. Blue: nuclei of the 24


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intestinal epithelia. Red: P4-labled MMs. Figure 7. Ex vivo imaging of biodistribution in organs and tissues (A); quantification of liver-borne fluorescence (B); fluorescence intensity vs. time profiles in blood (C) after gavage administration of P2-labeled FA-SB MMs under a fasted state. Lymphatic transport percentage at each time points (D) and cumulative transport percentage (E) through lymphatics.

25



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Abbreviations: AP: apical BL: basolateral BODIPY: 4,4′-difluoro-4-bora-3a,4a-diaza-s-indacene CLSM: confocal laser scanning microscopy DAPI: 4',6-diamidino-2-phenylindole DL: drug loading DMEM: dulbecco's minimum essential medium EE: entrapment efficiency FA-SB: fatty acid-silybin FBA: fetal bovine albumin GIT: gastrointestinal tract HBSS: Hank's Balanced Salt Solution HPLC: high-performance liquid chromatography IVIS: in vivo imaging system LBNs: lipid-based nanoparticles LDC: lipid-drug conjugate MMs: mixed micelles NLCs: nanostructured lipid carriers OCT: optimal cutting temperature compound PDI: polydispersity index PVP: polyvinylpyrrolidone SB: silybin SD: Sprague-Dawley SLNs: solid lipid nanoparticles STC: sodium taurocholate TEER: trans-epithelial electrical resistance 6C-SB: SB LDC with a 6C lipid chain length 12C-SB: SB LDC with a 12C lipid chain length 18C-SB: SB LDC with an 18C lipid chain length

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Table 1. Physicochemical properties of MMs containing different conjugates Characteristics

6C-SB MMs

12C-SB MMs

18C-SB MMs

Size (nm)

56.99 ± 2.58

57.10± 1.09

50.60± 2.24

Polydispersity index

0.15± 0.02

0.10± 0.01

0.14 ± 0.01

Zeta potential (mV)

-41.77± 1.53

-38.63± 1.80

-39.90 ± 2.79

Encapsulation efficiency (%)

100± 0

100± 0

100± 0

Drug loading of FA-SB (%)

8.78 ± 0.22

9.06± 0.15

8.91± 0.34

Table 2. Pharmacokinetic parameters of silybin after oral administration of 6C-SB MMs, 12C-SB MMs, 18C-SB MMs and SB solid dispersion pellets in SD rats (n = 6). Formulation

SB-pellets

6C-SB MMs

12C-SB MMs

18C-SB MMs

Cmax (µg/mL)

1.41±0.43

13.29±2.93**** 15.88±1.62****

12.65±1.51****

Tmax (h)

2.00±0.84

0.40±0.34***

1.17±0.52

AUC0-12 (µg⋅h/mL)

4.74±1.12

33.81±4.89**** 41.30±6.10****# 37.19±3.82****

AUC0-∞ (µg⋅h/mL)

4.81±1.09

34.52±4.46**** 41.74±6.12****# 37.89±4.29****

RBA (%)

100

713.29****

0.50 ±0***

871.16****##

RBA: relative bioavailability ***

P < 0.001, ****P < 0.0001: 6C-, 12C-, 18C-SB SLNs vs. Pellets

#

P < 0.05, ##P < 0.01: 12C-, 18C-SB SLNs vs. 6C-SB SLNs

27


784.51****


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Figure 1. Transmission electron microscope images and particle size distribution profiles of FA-SB MMs. Be aware that the small particles belong to the staining reagent.

28


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Figure 2. Plasma silybin concentration vs. time profiles after oral administration of a single dose (9 mg/kg SB equivalent) of 6C-, 12C- and 18C-SB MMs with SB solid dispersion pellets as a reference in rats

29



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Figure 3. Images of cellular uptake of MMs by IVIS live imaging systems (A); and corresponding quantification results of fluorescence (B). CLSM images of cellular uptake of SLNs by cell monolayers (C), the arrows point to fluorescent dots (red) of P4 signals that represent integral MMs at the AP side. Quantification of total silybin (D) and FA-SB conjugates (E) in cells. **P < 0.01, ****P < 0.0001.

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Figure 4. Cumulative transport of various FA-SB MMs across different cell monolayers for 4 h by measuring the content of SB (A-B), corresponding conjugates (C-D) and fluorescence intensity of P4 (E)

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Figure 5. In vivo live imaging of the digestion of MMs in rats (A); live imaging of ex vivo gastrointestinal segments (B); quantification of fluorescence of various gastrointestinal samples including stomach, small intestine and cecocolon of 6C- (C), 12C- (D), 18C (E)-SB MMs and the whole GIT (F), respectively.

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Figure 6. CLSM images of frozen section of the small intestine. Blue: nuclei of the intestinal epithelia. Red: P4-labled MMs.

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Figure 7. Ex vivo imaging of biodistribution in organs and tissues (A); quantification of liver-borne fluorescence (B); fluorescence intensity vs. time profiles in blood (C) after gavage administration of P2-labeled FA-SB MMs under a fasted state. Lymphatic transport percentage at each time points (D) and cumulative transport percentage (E) through lymphatics.

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for TABLE OF CONTENTS GRAPHIC 266x127mm (150 x 150 DPI)


In Vivo Fate of Biomimetic Mixed Micelles as Nanocarriers for

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