Soluplus self-assembled hybrid nanoparticles for enhancing the oral drug bioavailability

Journal Pre-proofs Bile acid transporter mediated STC/Soluplus self-assembled hybrid nanoparticles for enhancing the oral drug bioavailability Shujuan Zhang, Dongmei Cui, Jiawei Xu, Jiandong Wang, Qi Wei, Subin Xiong PII: DOI: Reference:

S0378-5173(20)30104-6 https://doi.org/10.1016/j.ijpharm.2020.119120 IJP 119120

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

19 September 2019 2 February 2020 4 February 2020

Please cite this article as: S. Zhang, D. Cui, J. Xu, J. Wang, Q. Wei, S. Xiong, Bile acid transporter mediated STC/Soluplus self-assembled hybrid nanoparticles for enhancing the oral drug bioavailability, International Journal of Pharmaceutics (2020), doi: https://doi.org/10.1016/j.ijpharm.2020.119120

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Bile acid transporter mediated STC/Soluplus self-assembled hybrid nanoparticles for enhancing the oral drug bioavailability Shujuan Zhang a, Dongmei Cui a, Jiawei Xu a, Jiandong Wang a, Qi Wei a, Subin Xiong a,b* a. College of Pharmaceutical Sciences, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310032, PR China b. Shanghai Anbison Laboratory Co., Ltd., 889 Yishan Road, Shanghai, 200233, PR China. *Corresponding Author Subin Xiong, Ph.D 18 Chaowang Road Zhejiang University of Technology Hangzhou, 310032, P.R.China Email: [email protected]

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ABSTRACT The nano-particulate system for oral delivery faces a big challenge across the gastrointestinal bio-barriers. The aim was to explore the potential applications of bile acid transporter mediated the self-assembled hybrid nanoparticles (SHNPs) of taurocholic acid (STC) and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol (Soluplus) for augmenting the oral delivery of poorly water-soluble drugs. Felodipine (FLDP) was chosen as a model drug. The self-assembly of STC with Soluplus to load FLDP and the microstructure of the SHNPs were confirmed using molecular simulation, STC determination by HPLC and transmission electron microscope. Results showed that STC was integrated with Soluplus on the surface of nanoparticles by hydrophobic interactions. The permeability of FLDP loaded STC/Soluplus SHNPs was STC dependent in the ileum, which was inhibited by the higher concentrations of STC and the inhibitor of apical sodium-dependent bile acid transporter (ASBT). STC/Soluplus (1:9) SHNPs significantly improved the drug loading of FLDP, achieved the highest permeability of FLDP and realized 1.6-fold of the area under the curve (AUC) of Soluplus self-assembled nanoparticles (SNPs). A water-quenching fluorescent probe P4 was loaded into the STC/Soluplus SHNPs, which verified that the SHNPs were transferred intactly across the ileum. In conclusion, STC/Soluplus SHNPs via ASBT are a potential strategy for enhancing the oral bioavailability of poorly water-soluble drugs. Keyword: felodipine; taurocholic acid; Soluplus; self-assembled hybrid 2

nanoparticles; ASBT; P4.

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1. Introduction Both biopharmaceutics classification system (BCS) Ⅱ and Ⅳ drugs are poorly water-soluble. The low water solubility limited their oral bioavailability (Boyd et al. 2019). Nano-particulate systems such as liposomes, emulsions, polymeric nanoparticles, self-assembled nanoparticles and nano-crystals have been widely utilized to load and deliver poorly water-soluble drugs (Kawabata et al. 2011, Goke et al. 2018). Among them, self-assembled nanoparticles composed of amphiphilic copolymers are automatically formed in aqueous mediums with low input energy and high drug loading (Danquah et al. 2010, Dimchevska et al. 2017). Soluplus® is a novel graft copolymer consisting of a polyvinyl caprolactam-polyvinyl acetatepolyethylene glycol, which has been applied as self-assembled nanoparticle gels to deliver tacrolimus (Wu et al. 2017) or polymeric crystal inhibitors to stabilize the amorphous solid dispersions (Chen et al. 2018, Chen et al. 2018) in our previous studies. The hybrid nanoparticles or micelles of Soluplus with surfactants such as dalpha tocopherol acid polyethylene glycol 1000 succinate (TPGS) (Bernabeu et al. 2016), Solutol® HS (Hou et al. 2016), sodium dodecyl sulfate (SDS) (Xia et al. 2016) to increase drug loading or promote the drug dissolution from particles have been reported. ASBT mediated active transport is a specific and highly effective re-absorption pathway of bile acid in the intestine (Swaan et al. 1996, Park et al. 2017, Fan et al. 2018, Wu et al. 2019). Bile acid transport systems such as the conjugates of N(4)4

ursodeoxycholic acid/cytarabine (Zhang et al. 2016) and deoxycholic acids/low molecular weight heparin (Al-hilal et al. 2014) have achieved the 2 and 5-fold AUCs of a single drug in rats, respectively. Felodipine (FLDP), a BCS II hydrophobic drug with poor water solubility of 0.58 µg/mL (Japanese pharmacopeia 17th edition), was chosen as a model drug. The objectives of this study were to explore the mechanisms of absorption and potential application of STC/Soluplus SHNPs for oral delivery of poorly water-soluble drugs. The particle size, zeta-potential, microstructure, and the structure integrity of the FLDP loaded STC/Soluplus SHNPs in the mimic gastrointestinal tract were investigated. P4 (Ma et al. 2017, Xie et al. 2018) was used as a water-quenching fluorescent probe to reveal whether the STC/Soluplus SHNPs could permeate the intestinal bio-barriers as nanoparticles. The in vivo pharmacokinetics and mechanisms of ASBT mediated absorption of FLDP loaded STC/Soluplus SHNPs following oral administration were also compared. 2. Materials and methods 2.1. Materials FLDP was purchased from Changzhou Rui Ming Pharmaceutical Co., Ltd (Changzhou, China). Soluplus® was kindly provided by BASF (Ludwigshafen, Germany). STC was purchased from Sigma (St. Louis, USA). Fluvastatin sodium (FVS) was purchased from Beijing Novartis Pharma Co., Ltd (Beijing, China). The lipophilic fluorescent probe coumarin derivative (CouD, λabs/λem=360 nm/494 nm) 5

was kindly provided by professor Jianhong Jia in Zhejiang University of Technology (Hangzhou, China). The water-quenching NIR fluorescent probe P4 (λabs/λem=651 nm/662 nm) was kindly provided by professor Wei Wu in Fudan University (Shanghai, China). HPLC grade methanol and acetonitrile were purchased from Tedia (Ohio, USA). All other reagents were of analytical grade. ICR mice (male, 18-22 g) and SD rats (male, 250-300 g) were purchased from Zhejiang Academy of Medical Sciences. All animals were fed and maintained under constant conditions at temperature of 20-25 ℃ and humidity 55 ± 5%, with 12 h light and 12 h dark cycles. Water and food were accessible to animals ad libitum. All studies were performed with the approval of the Institutional Animal Ethics Committee of Zhejiang University of Technology (Hangzhou, China). 2.2. Preparation of FLDP loaded STC/Soluplus SHNPs FLDP loaded STC/Soluplus SHNPs were prepared by a two-phase mixing method. STC and Soluplus were added in 9 mL purified water and stirred for 30 min to obtain a light blue opalescent dispersion. FLDP was dissolved in 1 mL ethanol, injected into the STC/Soluplus dispersion under magnetically stirring at room temperature, and stirred for another 30 min. The CouD or P4 loaded STC/Soluplus SHNPs were also prepared using the same procedure. 2.3. Encapsulation efficiency, drug loading and particle size 1 mL of samples was pipetted into the regenerated cellulose ultrafiltration tube 6

(Millipore, molecular exclusion size of 3 kDa), and centrifuged (ST16R Centrifuge, Thermo fisher, Germany) at 4,500 rpm for 1 h to remove the free drug. The collected FLDP loaded STC/Soluplus SHNPs were dissolved in ethanol. FLDP was analyzed by ultraviolet spectrophotometer (UV2450, Shimadzu, Japan) at 363 nm. Encapsulation efficiency (EE) and drug loading (DL) were calculated according to the equations as follows. All tests were conducted in triplicate. 𝑊𝑙𝑜𝑎𝑑𝑒𝑑 𝐹𝐿𝐷𝑃

(1)

EE(%) = 𝑊𝑎𝑑𝑑𝑒𝑑 𝐹𝐿𝐷𝑃 × 100 DL(%) =

W𝑙𝑜𝑎𝑑𝑒𝑑 𝐹𝐿𝐷𝑃 𝑊𝑎𝑑𝑑𝑒𝑑 𝐹𝐿𝐷𝑃, 𝑆𝑇𝐶 𝑎𝑛𝑑 𝑆𝑜𝑙𝑢𝑝𝑙𝑢𝑠

(2)

× 100

The particle size and zeta potential were determined by dynamic light scattering method (Delsa Nano C, Beckman, USA). 2.4. Ultrastructure of FLDP loaded STC/Soluplus SHNPs 2.4.1. Transmission electron microscopy Morphology of FLDP loaded Soluplus SNPs and STC/Soluplus SHNPs was observed by transmission electron microscopy (TEM, JEM-1200EX, JEOL, Tokyo, Japan) after negative staining with 2% uranyl acetate (w/v). 2.4.2. Molecular simulation Materials Studio 7.0 software (Accelrys, USA) with dissipative particle dynamics (DPD) simulation was performed to explore the self-assembling mechanisms of FLDP, STC and Soluplus in the ethanol-aqueous solution. Firstly, according to coarse-grained models, Soluplus was grouped into polyethylene glycol (PEG), polyvinyl acetate (PVA), and polyvinyl caprolactam (PVCL) as orange, green 7

and blue beads, respectively. STC was grouped into steroid (S) and sulfonic acid group (T) as kelly and yellow beads, respectively. One FLDP, three ethanol and five water molecules were grained as three red, two grey and one sky blue beads, respectively. Then, the Flory-Huggins parameters (χij) and interaction parameters (aij) were calculated using the Amorphous Cell and Forcite analysis module with the Compass II force field at T = 298K. Finally, the formation of self-assembling nanoparticles was simulated at 20×20×20 rc3 (the cut-off radius rc of 7.5 Å) box with the spring constant C of 4, the friction coefficient of 4.5 and the noise amplitude of 3.0. 200,000 steps with an integration time step of 0.05 ns were run to obtain the thermodynamic equilibrium of the system. 2.4.3. STC determination To elucidate the binding of STC with Soluplus in the FLDP loaded STC/Soluplus (1:9, 9:1, w/w) SHNPs, free STC in the dispersions were removed by tangential flow diafiltration using a Millipore Pellicon XL cartridge with molecular weight cut off (MWCO) of 30 kDa. The bound STC was then dissolved in ethanol and assayed by high performance liquid chromatography (HPLC, Waters 2695 with UVvisible detector 2487, USA) with a C18 column (Yilite, 250 mm×4.6 mm, 5μm). The mobile phase consisted of acetonitrile and 0.4 % KH2PO4 (pH 3.0) (50:50, v/v), and the flow rate was 1.0 mL/min. The detection wavelength was 200 nm and column temperature was 30 °C. 2.5. The structural integrity and In vitro release 8

To explore the structural integrity of STC/Soluplus SHNPs upon dilution in the gastrointestinal fluids, FLDP loaded Soluplus SNPs and STC/Soluplus SHNPs with Soluplus 10 mg/mL were diluted with 0.1 M HCl (pH 1.2), acetate buffer (pH 4.5) and phosphate buffer solution (PBS, pH 6.8) to 0.2 mg/mL, respectively. The drug leakage and particle size of SHNPs were tested. The in vitro release of FLDP loaded STC/Soluplus SHNPs was also investigated using a dialysis method. 1 mL of FLDP loaded Soluplus SNPs and STC/Soluplus SHNPs (1 mg/mL FLDP) were placed in a dialysis bag (MWCO 3 kDa), immersed in 50 mL PBS (50 mM, pH 6.8) containing 0.05 % SDS, and shaken at 100 rpm in 37℃ water bath. The medium (2 mL) was withdrawn at the preset times and replaced with 2mL fresh medium. FLDP was measured with a UV method as described previously. Each test was repeated in triplicate. 2.6. In situ single pass perfusion In situ single pass perfusion was utilized to compare the permeability of FLDP loaded Soluplus SNPs and STC/Soluplus (1:9, 1:4 and 9:1, w/w) SHNPs in the intestinal tract. The rats were fasted for 12 h with free access to water before the experiment. They were anesthetized with 10% chloral hydrate solution (0.35 g/kg, ip), and 10 cm of duodenum, jejunum, ileum and colon were exposed, respectively. The segments were gently rinsed with 37 ℃ Kerbs Ringer’s buffer (7.8 g/L NaCl, 0.35 g/L KCl, 1.37 g/L NaHCO3, 0.32 g/L NaH2PO4, 0.32 g/L CaCl2, 0.02 g/L MgCl2, and 1.4 g/L 9

glucose, pH 7.4), and perfused with the corresponding formulations for 30 min at a constant flow rate of 0.20 mL/min to reach a steady state using constant syringe pump (LSP04-1A, Longer Pump, Baoding, China). Samples were collected at 15 min intervals for 120 min. The length and radius of the segments were measured at the end of perfusion. The inlet and outlet perfusates were weighed. The collected samples were dissolved in ethanol and analyzed by a UV method. The effective permeability (Peff) was calculated as follows. 𝐶𝑜𝑢𝑡𝑉𝑜𝑢𝑡 𝐶𝑖𝑛𝑉𝑖𝑛

𝑙𝑛

𝑃𝑒𝑓𝑓 = ―𝑄 ×

(3)

2𝜋𝑟𝐿

Where Q is the flow rate (0.2 mL/min)，Cin is the concentration of FLDP in the donor fluid, Cout is the measured FLDP in the outlet perfusate, Vin and Vout are the inlet and outlet volumes of the perfusate, L and r are the length and radius (in cm) of the intestinal segments. 100 μg/mL FVS was pre-perfused for 30 min to block ASBT transporter to explore the uptake mechanisms of FLDP loaded STC/Soluplus SHNPs. 2.7. In vivo pharmacokinetic study The fasted ICR mice were randomly divided into five groups with 5 in each group. They were FLDP loaded Soluplus SNPs, FLDP loaded STC/Soluplus (1:9, 9:1, w/w) SHNPs, oral solution composed of Cremophor EL-ethanol-water (1:1:4, v/v/v) and FLDP suspension (0.3 % carboxymethylcellulose sodium, w/v). A single dose of FLDP 75 mg/kg for nanoparticles and oral solution, and 150 mg/kg for suspension was given by oral garage. At 5 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 6 h and 10 h 10

after administration, animals were sacrificed. Blood was collected in tubes containing heparin and centrifuged at 3000 rpm for 6 min to obtain the plasma. 100 µL of plasma was pipetted into 1.5 mL polythene tubes. Methanol (30 µL) and acetonitrile (2.7 mL) were added, mixed for 2 min by vortex and then centrifugated at 10000 rpm for 10 min. A sample (15 µL) was injected into HPLC for analysis. FLDP was assayed by HPLC method with the mobile phase composed of acetonitrile and 10 mM ammonium acetate buffer (pH 5.0) (55:45, v/v) at a flow rate of 1.2 mL/min and the absorption wavelength of 240 nm. Pharmacokinetic parameters, such as the peak plasma concentration (Cmax), time to reach peak concentration (Tmax), AUC and elimination half-life (t1/2) for FLDP were calculated using the PK Solver 2.0 software by linear trapezoidal method. 2.8. Transportation mechanisms across the ileum CouD and P4 were used as fluorescent markers to explore the mechanisms of insoluble drug and SHNPs across the ileum using the in situ ligated intestinal loop method. The ICR mice were anesthetized with chloral hydrate (0.35 g/kg). 3 cm of ileum was segmented. 0.3 mL of CouD loaded Soluplus SNPs (40 µg/mL CouD), P4 loaded Soluplus SNPs (1.5 µg/mL P4) and P4 loaded STC/Soluplus (1:9, w/w) SHNPs (1.5 µg/mL P4) were added into the ileum and ligated at both ends, respectively. The control groups were CouD oral solution (Cremophor EL-ethanol-water 1:1:4, v/v/v) and water-quenched P4 suspension. 100 μg/mL FVS to block ASBT for 30 min was 11

also compared. After 15 min of incubation, the mice were sacrificed by cervical dislocation. The ileums were thoroughly washed with normal saline, fixed with 4 % paraformaldehyde for 12 h, dehydrated for 12 h in 15 % sucrose and then stored in 30 % sucrose. After that, the tissues were quickly frozen in cryo-embedding media, and sliced into 10 μm pieces (Cryotome FSE, Thermo, USA). The confocal laser scanning microscope (CLSM, FV1000, Olympus, Japan) was used to observe the fluorescence distribution in the slices and the pictures were captured. 2.9. Statistical analysis Data were reported as the mean ± standard deviation. Statistical significance was measured by student t-tests and significant difference was defined at p < 0.05. 3. Results and discussion 3.1. Preparation and characterization of STC/Soluplus SHNPs FLDP loaded Soluplus SNPs and STC/Soluplus SHNPs dispersions showed a typically colloidal appearance. However, the color changed from blue to brown when STC/Soluplus mass ratio was 9:1 (Fig. 1A). As shown in Table 1, all the average volume particle sizes were not more than 110 nm. In comparison with the Soluplus SNPs, STC significantly decreased the particle sizes of Soluplus dominated SHNPs (STC/Soluplus 1:9 to 5:5, p<0.05), while increased the particle sizes of STC dominated SHNPs (STC/Soluplus 7:3 and 9:1). FLDP at their highest drug loadings did not significantly enlarge the particle sizes compared to the corresponding blank 12

nanoparticles (p0.05). The zeta potential of FLDP loaded Soluplus SNPs was -2.0 mV, which meant that the nanoparticles were neutral. With the increasing of STC in the FLDP loaded nanoparticles, the zeta potential was significantly raised from -11.6 mV to -23.1 mV for Soluplus dominated SHNPs (STC/Soluplus 1:9 to 5:5, w/w, p<0.05), and reached the platform when the ratios of STC was more than Soluplus. The results suggested that molecular STC could integrate with Soluplus at the surface to form the selfassembled hybrid nanoparticles. The surface charges could condense the particles and result in smaller hydrodynamic diameter and higher zeta potential at the mass ratio of STC/Soluplus till 5:5. When STC was more than Soluplus, the surplus STC could insert into the internal parts of nanoparticles, loose the nanoparticles and result in larger particle size and steady zeta potential. When the concentration of Soluplus in the formulations was kept consistent, STC significantly improved FLDP content in the SHNPs formulations in comparison with FLDP loaded Soluplus SNPs (p<0.05). Interestingly, FLDP content was similar in the Soluplus dominated SHNPs (STC/Soluplus 1:9 to 5:5), and sharply elevated in the STC dominated SHNPs (STC/Soluplus 7:3 and 9:1). When the STC/Soluplus was more than 14:1, FLDP in the dispersions precipitated after an overnight storage. For those nanoparticles with stability for more than 30 days, the DL and EE were further compared. The EE of all FLDP loaded nanoparticles were above 90 %, while the highest DL was found in SHNPs with STC/Soluplus of 1:9 (w/w). To understand the 13

loading behaviors, the content of FLDP in the ethanol-water (10:90, v/v) solution with 10 mg/mL STC was determined and it was only 0.27 mg/mL. The results indicated that the solubilization of STC to FLDP was limited. The STC/Soluplus hybrid nanoparticulate delivery system with STC (not more than 30 %) contributed the higher loading of FLDP. 3.2. Ultrastructure of FLDP loaded STC/Soluplus SHNPs 3.2.1. Transmission electron microscopy The morphology of the FLDP loaded nanoparticles was investigated. As shown in Fig.1B, when STC/Soluplus was not more than 3:7, FLDP loaded nanoparticles were uniform and spherical. Once STC/Soluplus was more than 5:5, the nanoparticles were multi-dispersions under TEM, full of small particles and some spherical aggregates. These results suggested that Soluplus dominated STC/Soluplus SHNPs can maintain the rigid structures of nano-particulates, while STC dominated STC/Soluplus SHNPs became soft and looked like the mixed micelles. Both the hydrodynamic diameter and microstructure under TEM were the critical quality attributes of SHNPs. 3.2.2. Molecular simulation To help understanding the self-assembling mechanisms of STC/Soluplus SHNPs, DPD was employed. Based on the above experimental results, all the grain beads had an average volume of 144 Å3 (Fig. 2A) and the formulations of FLDP composed of FLDP and STC/Soluplus (0:10, 1:9, 9:1, w/w) were simulated. Their corresponding 14

volume fractions were same as their weight ratios, and the molar ratios of FLDP/STC/Soluplus were 5.1:0:3.1, 5.6:0.9:1.7 and 3.0:13.0:0.3, respectively. The mole ratio of water to ethanol in the mediums was fixed at 13.8. As shown in Fig. 2B-2D, the conjugation morphologies of FLDP, Soluplus and STC in the ethanol-water systems changed with the simulation time. At the beginning of the simulation (0 step), all the grains were randomly dispersed in the box. At 500 steps, Soluplus and FLDP molecules with or without STC self-aggregated to form the small clusters under the attractive forces of FLDP/Soluplus/STC and the repulsive forces of water and ethanol. Continuously simulating to 200,000 steps, the thermodynamic equilibrium was achieved, the spherical nanoparticles were obtained, and the particle sizes of nanoparticles were not changed with simulation time. From the cross-sections of the simulated blank and FLDP loaded nanoparticles, it can be found that single Soluplus formed the spherical nanoparticles with the core of hydrophobic PVA/PVCL and the shell of hydrophilic PEG, condensing FLDP in the PVA/PVCL core (Fig. 2B). In the Soluplus dominated systems of STC/Soluplus (1:9, w/w), the molar ratio of Soluplus was 1.9-fold of STC. The sulfonic acid of STC and the PEG of Soluplus corporately coated the hydrophobic core of steroid and PVA/PVCL groups. Because of the similar repulsion parameters of steroid with PVA and PVCL (Table 2), the strong hydrophobic attractions of STC and Soluplus and the polymer chains entanglement provided the powerful force and space to conjugate much more FLDP in the core (Fig. 2C). These gave the insight into the highest drug 15

loading of FLDP loaded STC/Soluplus(1:9) SHNPs. For the STC dominated systems of STC/Soluplus (9:1, w/w), the actual molar ratio of small molecular STC was 43.3fold of Soluplus polymers. As shown in Fig. 2D, although FLDP was still covered in the core of nanoparticles, the polymeric chains of Soluplus were completely surrounded by STC, and the molecular interactions of STC donated the attractive forces to form the loose nanoparticles. Therefore, the particle sizes of blank and FLDP loaded SHNPs (STC/Soluplus, 9:1, w/w) were significantly larger, while the drug loading was significantly lower than the corresponding Soluplus SNPs. 3.2.3. STC determination The above results have interpreted the self-assembly processes of FLDP/STC/Soluplus in the ethanol-aqueous systems and their nanoparticle properties based on the molecular thermodynamics and kinetics. To further investigate the binding amount of STC on the FLDP loaded SHNPs, ultrafiltration tubes have been applied to separate the free STC and bound STC for HPLC determination. The results showed that the binding ratios of STC on the FLDP loaded STC/Soluplus 1:9 (w/w) and 9:1(w/w) SHNPs were 50.0±1.2% and 12.4±8.2%, respectively. It suggested that STC did conjugate with Soluplus to shape the hybrid nanoparticles and the binding amount was dependent on the Soluplus ratio. 3.3. The structural integrity and In vitro release The structural integrity of nano-particulate delivery systems is the premise to explore the carrier mediated trans-membrane behaviors. The biorelevant pH-dilution 16

method using an orbital shaker at 37°C and100 rpm has been applied to assess the performance of the formulations in the dynamic gastrointestinal transit environment. The dilution factors and shaking times were 1:0.5 in pH 4.0 HCl solution for 0.25 h, 1:0.9 in duodenum fasted state simulated intestinal fluid (FaSSIF) for 0.2 h, 1:4.8 in the jejunum/ileam FaSSIF for 2h, and 1:1.9 in the cecum/colon FaSSIF for 4.5 h and 7.7 h. Accordingly, the total dilution fold was 48 (Yi Gao et al. 2010). As shown in Table 3, when FLDP loaded Soluplus SNPs and STC/Soluplus (1:9) SHNPs containing Soluplus 10 mg/mL were diluted to 0.2 mg/mL, the particle sizes were not significantly changed and the FLDP leakages were less 2% (data were not shown). However, in the same dilution regimen, the aggregation of FLDP loaded STC/Soluplus (9:1) SHNPs in the 0.1 M HCl were found. Their sizes after dilution in the acetate buffer and PBS were significantly decreased, but the FLDP leakages were still not more than 2%. Although the critical micelle concentration of Soluplus was 7.6 μg/mL (23℃, data on file, BASF, Pharma Ingredients & Services, Germany; Technical Information 2009), the particle sizes of nanoparticles with Soluplus less than 0.2 mg/mL could not be accurately tested by DLS. The results suggested that Soluplus dominated STC/Soluplus SHNPs maintained integrity in the gastrointestinal tract after dilution for 50-fold, while STC dominated STC/Soluplus SHNPs with may face the risks of aggregation in the stomach. These findings were in accordance with the above molecular simulation and TEM results. In our previous study, we found pH 6.8 PBS with 0.05 % SDS was a 17

discriminative medium for the FLDP/Soluplus immediate releasing solid dispersions (Chen et al. 2018). The in vitro release profiles of FLDP from nanoparticles in this medium are shown in Fig. 3. The cumulative released FLDP from Soluplus SNPs, STC/Soluplus (1:9 and 9:1) SHNPs were only 32.3 %, 28.1 % and 38.3 % after 8 h， respectively. It also suggested that these nanoparticles could deliver FLDP across the gastrointestinal tract. 3.4. In situ single pass perfusion In situ single pass perfusion was carried out to investigate the permeability of FLDP loaded Soluplus SNPs and STC/Soluplus SHNPs. As summarized in Table 4, the permeability of FLDP loaded Soluplus SNPs did not show significant differences in the duodenum, jejunum, ileum and colon (p>0.05). In comparison with the Soluplus SNPs, the permeation of FLDP in the ileum was augmented 1.6-fold via STC/Soluplus (1:9) SHNPs with STC concentration of 44 μg/mL (p<0.05), while no significant changes were found in the duodenum, jejunum and colon. However, as the increasing of STC from 0.1mg/mL to 3.6 mg/mL in the nanoparticle perfusates, the permeability of FLDP in the ileum was significantly decreased (p<0.05). The previous publications have verified the expression of ASBT on the apical membrane of the ileum (Shneider et al. 1995, Donkers et al. 2019). Higher concentration of STC decreased the permeation of taurocholic acid-linked heparin-docetaxel conjugates across the Caco-2 cells (Khatun et al. 2014). And the re-absorption of taurocholic acid was inhibited by the STC(Stahl et al. 1993). Therefore, we can conclude that STC 18

mediated ASBT enhanced the permeability of FLDP loaded STC/Soluplus SHNPs, but higher concentration of STC in the formulations may inhibit the ASBT transporter. FVS is a strong inhibiter of ASBT. It has reported that 100 μg/mL FVS could completely block the active uptake of taurocholic acid in the ileum (Li et al. 2018). In our study, after perfusion of 100 μg/mL FVS for 30 min, the permeability of FLDP loaded STC/Soluplus (1:9) SHNPs in the ileum was significantly inhibited, showing the reduction of 58.9 % (p<0.05). These findings confirmed that ASBT did mediate the active transportation of FLDP loaded STC/Soluplus SHNPs. To explore the reasons that FVS did not completely block the absorption of STC/Soluplus (1:9) SHNPs in the ileum, the in vitro single pass perfusion of FLDP loaded nanoparticles through dead intestines were compared. The dead duodenum, jejunum, ileum and colon were obtained by putting the freshly isolated intestines in 0.9 % saline at 4 ℃ for 12 h. As shown in Table 5, the permeability of three FLDP loaded nanoparticles across the dead intestines was decreased in comparison with the in situ perfusion. STC/Soluplus SHNPs induced the higher permeation of FLDP across the dead intestines than Soluplus SNPs. It is well known that the dead intestines are lack of active transporters, and the cell membranes are semipermeable to allow passive diffusion of small molecular drugs. The above in vitro release data also indicated that partial release of FLDP in the medium of pH 6.8 PBS with 0.05 % SDS. The results suggested that the released molecular FLDP transported the 19

intestines via passive diffusion, which was dependent on the STC concentrations to a degree. 3.5. In vivo pharmacokinetic study In situ single pass perfusion has demonstrated that STC/Soluplus(1:9) SHNPs enhanced the permeability of FLDP. The in vivo animal study in rats was carried out and the pharmacokinetic parameters of five FLDP formulations were summarized in Table 6. The Cmax and AUC0-6h were 0.4 µg/mL and 1.1 μg/mL·h after oral administration of 150 mg/kg FLDP suspensions. Due to the enhanced absorption, the dosage of the FLDP solution and nanoparticles for pharmacokinetics was adjusted to 75 mg/kg. The highest oral bioavailability of FLDP was achieved at FLDP loaded STC/Soluplus (1:9) SHNPs, with Cmax of 1.3-fold and AUC of 1.6-fold in comparison with the FLDP loaded Soluplus SNPs (p<0.05). A significantly decreased Cmax and AUC was found in the FLDP loaded STC/Soluplus (9:1) SHNPs (p<0.05), which was in agreement with the in situ perfusion results. We think it may come from the aggregation of FLDP loaded STC/Soluplus (9:1) SHNPs in the stomach, and the blockage of ASBT mediated active transportation by excess STC in the formulation. Fig. 4 shows the curves of FLDP plasma concentration versus time. Two peaks of Cmax were observed after administration of FLDP loaded STC/Soluplus (1:9) SHNPs. Based on the in vitro release and the in vitro perfusion through the dead intestines results, it can be understood that the fast absorption of the released FLDP via passive transport provided the first peak, and the ABST mediated active transport 20

produced the second peak. The double peaks phenomena in the pharmacokinetics of tetrameric deoxycholic acid/camptothecin conjugates (Xiao et al. 2019) and STC/pluronic P123 micelles (Zhang et al. 2016) have also been reported. 3.6. Transportation mechanisms across the ileum To further investigate the hydrophobic free drug and the STC/Soluplus hybrid nanoparticles how to penetrate the bio-barriers of gastrointestinal tract, CouD and P4 were chosen as fluorescence agents. As shown in Fig. 5, the green fluorescence of CouD around the intestine villi were observed, and fluorescence strength of CouD solution was stronger than that of Soluplus SNPs. Because the fluorescence strength of CouD was not influenced by its physical states, the higher concentration of free CouD, the more amount trans-membrane and the stronger fluorescence. P4 is a water-quenching fluorescent probe, which has been used to monitor the in vivo fate of many nano-carriers (Wu et al. 2017, Xie et al. 2018). Once P4 was quenched in suspensions, whose fluorescence was negligible (Fig. 6A and 6B). The weak purple fluorescence at the surface of villi was observed in the P4 loaded Soluplus SNPs treated ileum, which indicated that very small amount of Soluplus SNPs transported (Fig. 6C). The strong purple fluorescence in the villi and the basolateral side was found in the ileum treated by P4 loaded STC/Soluplus (1:9) SHNPs (Fig. 6D), and the intensity was significantly reduced after blockage of ASBT by FVS (Fig. 6E). CouD and P4 worked as probes to provide the strong evidences that the enhanced absorption of FLDP loaded STC/Soluplus (1:9) SHNPs were realized 21

through passive diffusion of free FLDP and ASBT mediated active transport of nanoparticles. 4. Conclusion A novel STC/Soluplus SHNPs system via ASBT to enhance oral absorption of poorly water-soluble FLDP was developed. The strong hydrophobic interactions of STC and Soluplus induced the self-assembly of hydrophobic nanoparticles. The composition, integrity, microstructure, drug loading, in vitro release, in situ permeability, in vivo pharmacokinetics and transportation mechanisms have been comprehensively explored. The results demonstrated that STC/Soluplus (1:9) SHNPs significantly improved the drug loading of FLDP, and enhanced the permeability and oral bioavailability FLDP via both passive delivery of free drug and ASBT mediated active transport of nanoparticles. Overall, STC/Soluplus SHNP is a promising strategy for augmenting the oral bioavailability of poorly waster-soluble drugs. Disclosure and acknowledgments There is no conflict of interest in this work. We appreciate Prof. Jianhong Jia in Zhejiang University of Technology and Prof. Wei Wu in Fudan University for providing fluorescence probes. The present research was supported by grants from Zhejiang Natural Science Foundation Committee (LGF20H300009). References Al-hilal T. A., Park J., Alam F., Chung S. W., Park J. W., Kim K., Kwon I. C., Kim I. S., Kim S. Y.,Byun Y., 2014. Oligomeric bile acid-mediated oral delivery of low 22

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Figure legends: Fig.1 (A) Formulation appearance of FLDP loaded nanoparticles at Soluplus concentration of 10 mg/mL. (B) TEM images of FLDP loaded nanoparticles. Soluplus SNPs (1); STC/Soluplus 1:9 SHNPs (2); STC/Soluplus 3:7 SHNPs (3); STC/Soluplus 5:5 SHNPs (4); STC/Soluplus 7:3 SHNPs (5); STC/Soluplus 9:1 SHNPs (6).

Fig.2 DPD simulation of FLDP/STC/Soluplus in the ethanol-water solution. (A) Molecular structures and coarse-grained models of Soluplus, STC, FLDP, ethanol and water. (B) Configurations of FLDP/Soluplus (1:4, w/w) particles at different simulated steps, cross-section of FLDP loaded Souplus SNPs and blank Souplus SNPs. (C) Configurations of FLDP/STC/Soluplus (4.5:1:9, w/w/w) particles at different simulated steps, cross-section of FLDP loaded STC/Souplus (1:9) SHNPs and blank STC/Souplus (1:9) SHNPs. (D) Configurations of FLDP/STC/Soluplus (1.5:9:1, w/w/w) particles at different simulated steps, cross-section of FLDP loaded STC/Souplus (9:1) SHNPs and blank STC/Souplus (9:1) SHNPs.

Fig.3 In vitro release profiles of FLDP loaded nanoparticles (n=3).

Fig.4 FLDP plasma concentration versus time after a single oral dose of FLDP loaded Soluplus SNPs, STC/Soluplus SHNPs, oral solutions and suspensions (n=5, 75 mg/kg).

Fig.5 Fluorescent images of cryo-sections of ileum after administration of CouD loaded nanoparticles to rats. (A) Blank; (B) CouD loaded Soluplus SNPs; (C) CouD solutions.

28

Fig.6 Fluorescent images of cryo-sections of ileum after administration of P4 loaded nanoparticles to rats. (A) Blank; (B) Quenched P4 suspension; (C) P4 loaded Soluplus SNPs; (D) P4 loaded STC/Soluplus (1:9) SHNPs; (E) P4 loaded STC/Soluplus (1:9) SHNPs blocked by FVS.

29

Table 1 Particle size, zeta potential, FLDP content, drug loading (DL), and entrapment efficiency (EE) of FLDP loaded STC/Soluplus SHNPs (10 mg/mL Soluplus) STC/Solupl

FLDP/Solup

Blank

FLDP loaded nanoparticles

us

lus

particle

Particle

Zeta

FLDP

DL

EE

(w/w)

(w/w)

size

size

potentia

content

(%)

(%)

(nm)

(nm)

l

(mg/mL)

19.1±0.

100±0.0

2.0±0.8

02

1

30.3±0.

(mV) 54.4±4. 0:10

52.9±5.0

2.5±0.04 4

1:9

-

1:4

5:10

30.4±3.

40.9±5.4

-

6*

#

11.6±1.

5.1±0.1#

3#

9# 38.7±6. 3:7

5:10

44.5±0.6

6*

1

12.0±1.

26.8±0. 5.2±0.1#

4#

2# 40.5±0.

45.4±1.3

100±0.0

99.6±0. 04

-

19.4±0. 4.7±0.02

5:5

5:10

8*

23.1±0.

5

99.5±0.

#

4# 63.9±2. 7:3

8.5:10

60.1±1.8

8

-

05 7.6±0.1#

21.7±1.

19.5±1. 0

3#

9:1

15:10

1

84.7±8.

108.8±1.

-

13.0±0.1

12.0±0.

3*

8#

23.2±0.

#

7#

3#

30

98.6±0.

90.7±0. 9

--14:1

---

---

19.6±0.0

---

---

---

----

20:10 1# ---

20:1

---

---

7.5±0.01

8:10 #

---: Non-determined for FLDP precipitation after an overnight storage * p < 0.05, in comparison with the blank Soluplus SNPs #

p <0.05, in comparison with the FLDP loaded Soluplus SNPs

31

Table 2 Interaction parameters (aij) between various beads in the DPD simulation. aij

F

PEG

PVA

PVCL

F

25.0

PEG

47.1

25.0

PVA

25.0

47.4

25.0

PVCL

25.0

45.3

25.1

25.0

S

25.0

46.8

25.0

25.0

25.0

T

33.2

27.8

30.9

28.8

30.1

25.0

E

27.3

35.1

27.4

26.7

27.2

31.5

25.0

W

107.9

44.4

108.7

104.0

107.4

31.0

25.0

32

S

T

E

W

25.0

Table 3 Integrity of FLDP loaded nanoparticles before and after dilution in various mediums (n = 3) FLDP loaded

Before dilution

After dilution (Soluplus 0.2mg/mL)

nanoparticles

(Soluplus 10 mg/mL)

0.1M HCl

Acetate buffer

PBS

(pH 1.2)

(pH 4.5)

(pH 6.8)

Soluplus

52.9±5.0

51.5±3.3

51.1±1.2

54.6±4.8

STC/Soluplus 1:9

40.8±2.7

31.2±5.4

37.9±2.3

39.6±2.3

STC/Soluplus 9:1

105.2±7.4

aggregation

75.5±0.1*

75.2±3.5*

*

p < 0.05, in comparison with the particle size before dilution

33

Table 4 In situ single-pass intestine perfusion of FLDP loaded nanoparticles in rats. (100 μg/mL FLDP, n = 3) FLDP loaded nanoparticles

Peff×10-3 (cm/min) Duodenum

Jejunum

Ileum

Colon

Soluplus

7.1±1.0

7.3±1.0

9.5±1.4

9.9±1.0

STC/Soluplus 1:9

10.3±2.6

10.0±2.1 15.1±1.6* 9.8±2.1

STC/Soluplus 1:4

8.1±1.6

11.0±2.3 9.7±2.3#

8.1±0.5

STC/Soluplus 9:1

5.3±0.8#

6.3±2.1

7.2±1.6#

7.5±1.3

FVS+STC/Soluplus 1:9

6.6±1.8

7.8±0.6

6.9±1.3#

6.5±1.7

* p＜0.05, #

in comparison with FLDP loaded Soluplus SNPs

p＜0.05, in comparison with FLDP loaded STC/Soluplus (1:9) SHNPs

34

Table 5 In the dead intestine perfusion of FLDP loaded nanoparticles. (100 μg/mL FLDP, n = 3) FLDP loaded nanoparticles

Peff×10-3 (cm/min) Duodenum

Jejunum

Ileum

Colon

Soluplus

4.3±0.7

4.2±0.6

3.9±0.9

6.7±0.3

STC/Soluplus 1:9

5.7±0.8

6.1±0.2*

6.1±0.4*

6.8±2.1

STC/Soluplus 9:1

6.8±0.4*

6.4±0.5*

6.3±1.3*

6.5±1.4

*

p＜0.05, in comparison with FLDP loaded Soluplus SNPs

35

Table 6 Pharmacokinetic parameters of FLDP formulations after oral administration in rats. (n=5, 75 mg/kg) Formulations

Cmax

AUC 0-6h

(μg/mL)

(μg/mL·h)

Oral solutions

4.8±1.2

11.1±1.1

0.6±0.3

1.8±0.3

Soluplus SNPs

5.5±1.1

10.0±0.4

0.5±0.3

2.2±0.8

STC/Soluplus 1:9 SHNPs

7.1±0.7*

15.9±4.8*

0.3±0.04

2.3±0.7

STC/Soluplus 9:1 SHNPs

1.4±0.4*

3.4±0.9*

2.2±2.6

6.9±4.5

Suspensions#

0.4±0.1*

1.1±0.03*

2.0±0.02* 2.4±0.2

*

Tmax

t1/2

(h)

(h)

p＜0.05, in comparison with FLDP loaded Soluplus SNPs

#: 150 mg/kg

36

Graphical abstract

37

Credit Author Statement

Shujuan Zhang: Conceptualization, Data Curation, Methodology, Investigation, Writing - review & editing； Dongmei Cui：Supervision, Writing - review & editing； Jiawei Xu: Formal analysis, Validation, Investigation; Jiandong Wang: Formal analysis, Data Curation; Qi Wei: Visualization, Investigation; Subin Xiong: Resources, Supervision, Validation, Writing - review & editing.

38

Conflict of Interest ☑The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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