Nanostructured lipid carriers with exceptional gastrointestinal stability and inhibition of P-gp efflux for improved oral delivery of tilmicosin

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Nanostructured lipid carriers with exceptional gastrointestinal stability and inhibition of P-gp efflux for improved oral delivery of tilmicosin Qian Zhanga, Haifeng Yanga,b, Benazir Sahitoa, Xinyu Lia, Lin Penga, Xiuge Gaoa, Hui Jia, Liping Wanga, Shanxiang Jianga, Dawei Guoa,* a b

Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China Department of Animal Pharmacy, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Tilmicosin Nanostructured lipid carriers Caco-2 cells Solubility Permeability Oral bioavailability Piglets

Tilmicosin (TMS) is widely applied to treat porcine bacterial respiratory diseases in veterinary medicine. However, oral administration of TMS is greatly limited due to its physicochemical properties, such as poor water solubility, gastric acid sensitivity and bitterness. Therefore, nanostructured lipid carriers (NLCs) were developed as an oral delivery system for TMS by the high shear method combined with ultrasonic techniques in this study. The results showed that TMS-NLCs were approximately spherical with a hydrodynamic diameter of 283.03 nm and a zeta potential of -30.04 mV. TMS was almost entirely encapsulated in the NLCs by interacting with the lipid matrix, as characterized by differential scanning calorimetry and fourier transform infrared spectroscopy. Thus, TMS-NLCs had an excellent encapsulation efficiency and loading capacity with values of 93.46% and 9.23%, respectively. TMS-NLCs maintained good stability not only during storage at 4 ℃, 25 ℃ and 40 ℃ for 90 days but also in stimulated gastrointestinal (GI) fluids at 37 ℃ for 7 days. Therefore, TMS-NLCs displayed low and sustained release in vitro without an initial burst release in stimulated GI fluids. Furthermore, TMS-NLCs showed higher oral bioavailability in piglets compared to the API suspension. Subsequently, Caco-2 cell monolayers were utilized to analyze the mechanism of NLC-enhanced oral adsorption of TMS. The data revealed that NLCs not only increased cellular uptake of TMS but also inhibited the efflux of P-gp in Caco-2 cells. Additionally, TMSNLCs mainly entered Caco-2 cells via the caveolae/lipid raft-mediated endocytosis pathway. Moreover, nanoparticles were transported across Caco-2 cell monolayers in the intact form to the basolateral side, as identified by transmission electron microscopy, indicating that TMS-NLCs escape lysosome degradation. Taken together, these results indicate that NLCs are a potential delivery carrier for improving the solubility, permeability and oral bioavailability of TMS.

1. Introduction Tilmicosin (TMS) is a semisynthetic macrolide antibiotic that inhibits the protein synthesis of bacteria, and it is widely applied in veterinary medicine [1]. Currently, TMS has been approved for treating bacterial respiratory diseases in pigs infected with Pasteurella multocida, Streptococcus suis, Actinobacillus pleuropneumoniae and Mannheimia (Pasteurella) hemolytica [1–3]. Oral administration is the most commonly used form of delivery of TMS in pigs due to its high lethal risk of injection [4–6]. However, TMS has a bitter taste, which influences TMS uptake in pigs [5,6]. In addition, the insolubility and gastric sensitivity of TMS also decrease its oral bioavailability [7]. Therefore, it is urgent to develop a novel drug delivery system to improve the gastrointestinal adsorption and reduce the amount of TMS.

⁎

Lipid-based drug delivery systems are expected to be promising oral carriers with the potential to increase solubility and improve oral bioavailability of poor water soluble and/or lipophilic drugs. It has been reported that solid lipid nanoparticles (SLNs) with hydrogenated castor oil (HCO) is a promising formulation for subcutaneous administration of TMS without inflammation at the injection site and systemic toxicity [4,8,9]. Importantly, SLNs significantly enhance the therapeutic efficacy of TMS against Staphylococcus aureus [10] and Streptococcus agalactiae [11], as determined by lower CFU counts and a decreased degree of inflammation. In addition, TMS and florfenicol encapsulated by SLNs with HCO have synergistic or additive antibacterial activity against Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae, exhibiting its potential for bovine mastitis therapy [12]. Moreover, SLNs significantly improve the oral

Corresponding author at: Laboratory of Veterinary Pharmacology and Toxicology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, PR China. E-mail address: [email protected] (D. Guo).

https://doi.org/10.1016/j.colsurfb.2019.110649 Received 30 May 2019; Received in revised form 15 November 2019; Accepted 16 November 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Qian Zhang, et al., Colloids and Surfaces B: Biointerfaces, https://doi.org/10.1016/j.colsurfb.2019.110649

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bioavailability of TMS or florfenicol and enhance their therapeutic effect against Actinobacillus pleuropneumoniae, Streptococcus suis and Hemophilus parasuis in pigs, suggesting that SLNs may be a useful formulation for oral administration [13]. Nanostructured lipid carriers (NLCs) are a new generation of lipid nanoparticles prepared with a blend of both solid and liquid lipids, which are generally recognized as safe (GRAS), to form a controlled nanostructure [14,15]. Imperfections of binary lipids provide spaces to accommodate drugs in the matrix, resulting in the maximum drug loading capacity compared to SLNs [16,17]. Recently, it has been found that NLCs are promising carriers for TMS with a better drug encapsulation capacity, release behavior and antibacterial activity than SLNs [2]. In addition, NLCs have a bright future in enhancing the oral bioavailability and other pharmacokinetic properties of drugs to be administered orally [15,18]. Currently, there are various formulation techniques for the production of NLCs, among which high pressure homogenization has the highest potential for scaling up to industrial production scale [17]. Therefore, NLCs are promising oral delivery carriers to promote clinical applications of TMS in veterinary medicine. In this study, nanostructured lipid carriers of tilmicosin (TMS-NLCs) were prepared by a high shear-ultrasonic method and were then characterized in terms of their physicochemical properties, drug loading capacity, encapsulation efficiency and in vitro release behaviors. The pharmacokinetics of TMS-NLCs were further evaluated to confirm their enhanced oral bioavailability in piglets. Finally, the mechanism of NLCs improving intestinal adsorption of TMS was elucidated using Caco-2 cell monolayers.

2.2.2. Hydrodynamic diameters, polydispersity index and zeta potentials of TMS-NLCs The mean hydrodynamic diameter (HD), polydispersity index (PDI) and zeta potential (ZP) of TMS-NLCs were measured by dynamic light scattering (DLS) using a Malvern Zetasizer (Nano ZS90, Malvern Instruments, UK) at an angle of 90° at 25 ℃. 2.2.3. Transmission electron microscopy of TMS-NLCs The morphology of TMS-NLCs was examined by transmission electron microscopy (TEM; Tecnai 12, Philips, Netherlands). A drop of TMSNLCs was mounted on 300 mesh copper grids, followed by negative staining with a 2% (w/v) phosphotungstic acid solution for 2 min. The negative staining samples were then dried overnight at room temperature before TEM analysis. 2.2.4. Differential scanning calorimetry of TMS-NLCs TMS-NLCs were lyophilized with 10% sucrose prior to differential scanning calorimetry (DSC) and fourier transform infrared (FT-IR) spectroscopy. Thermal analysis of the drug (TMS), solid lipids (SAs), the lipid/drug physical mixture, the cryoprotectant (sucrose) and freezedried TMS-NLCs were performed using a Differential Scanning Calorimeter (DSC 8500, PerkinElmer, Francisco, USA). 2.2.5. Fourier transform infrared (FT-IR) spectroscopy FT-IR spectra were recorded for the drug (TMS), solid lipids (SAs), the lipid/drug physical mixture, sucrose and lyophilized TMS-NLCs using a FT-IR Spectrometer (IS5&N380, Nicolet, USA). Prior to conducting FT-IR, samples were mixed with potassium bromide (KBr) at a ratio of 1:100 and pressed into a pellet using a high pressure hydraulic machine. The infrared scanning wave numbers were from 4000 to 500 cm−1.

2. Materials and methods 2.1. Materials

2.3. Stability evaluation of TMS-NLCs

Tilmicosin (TMS, 91.4% w/w) was supplied by Ningxia Teirui Pharmaceutical Corporation (Yinchuan, China). Stearic acid (SA) was purchased from China Pharmaceutical Group Chemical Reagents Corporation (Shanghai, China). Tween 80 (T80) was obtained from Nanjing Chemical Reagent Corporation (Nanjing, China). Poloxamer188 (P188) was supplied by Shanghai Yuanhong Chemical Preparations and Accessories Technology Corporation (Shanghai, China). Oleic acid (OA), propranolol hydrochloride, verapamil hydrochloride and chlorpromazine hydrochloride (CHI) were purchased from Aladdin (Shanghai, China). Lucifer Yellow was provided by Sigma Aldrich (Shanghai, China). 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) was supplied by ApexBio (Houston, USA). Methyl-β-cyclodextrin (MβCD) was obtained from Energy Chemical (Shanghai, China). Dynasore was purchased from Abcam (Cambridge, UK). Pure water was prepared by a Milli-Q system with 18.2 MΩ/cm (Millipore, Boston, USA).

2.3.1. Storage stability TMS-NLCs were stored at 4 ℃, 25 ℃ and 40 ℃ in Drug Stability Cabinets (WD-A, Tianjin, China) for 90 days. Samples were removed to monitor the changes in HD, PDI and ZP of TMS-NLCs at different time points. 2.3.2. Stability of TMS-NLCs in simulated gastrointestinal (GI) fluids Simulated gastric fluids with pepsin (pH 1.2) and simulated intestinal fluids with trypsin (pH 6.8) were prepared to test the stability of TMS-NLCs in a GI environment. Briefly, TMS-NLCs were diluted 10-fold with simulated GI fluids and incubated at 37 ℃ for 168 h. The HD, PDI and ZP of TMS-NLCs were measured every 1 h for the first 4 h and for 168 h until the end of the study. 2.4. High performance liquid chromatography (HPLC) analysis of TMS

2.2. Preparation and characterization of TMS-NLCs

The concentrations of TMS were analyzed using a High Performance Liquid Chromatography (HPLC) System (1200, Agilent Technologies, USA) with a ZORBAX Eclipse XDB-C18 Reverse-phase Column (5 μm, 4.6 mm inner diameter ×250 mm length, Agilent, USA). A mixture of acetonitrile, tetrahydrofuran, water, and a n-dibutylamine phosphate buffer salt solution (12:5.5:80:2.5; v/v) was used as the mobile phase with a flow rate of 1 mL/min. The column temperature was kept constant at 30 ℃, and the UV detection wavelength was 291 nm. The injection volume was 20 μL. The samples were filtered through a 0.22 μm filter membrane before automatic injection into the HPLC system.

2.2.1. Preparation of TMS-NLCs NLCs were prepared by the high shear method together with ultrasonic techniques. Briefly, accurate quantities of SA, OA and T80 were well mixed at 80 ℃ with 500 rpm of magnetic stirring to form a uniform and clear lipid phase. The aqueous phase containing P188 was preheated at the same temperature. The hot lipid phase was then poured into the aqueous phase at 80 ℃ with magnetic stirring. The pre-emulsion was obtained under high shear at 11,000 rpm for 5 min with a High Shear Dispersion Emulsifying Machine (FM200, IKA, Germany) followed by ultrasonication at 300 W for 20 min with an Ultrasonic Cell Disruptor (JY96-Ⅱ, Scientz, Ningbo, China). Subsequently, the nanoparticle suspension was immediately dispersed in cold distilled water with high shear at 11,000 rpm for 1 min. For drug-loaded NLCs, 0.3 g TMS was added to the lipid phase.

2.5. Entrapment efficiency and drug loading An ultrafiltration centrifugation method was utilized to determine the entrapment efficiency (EE) and loading capacity (DL) of TMS-NLCs. The concentration of unincorporated TMS was determined by HPLC 2

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2.8. In vitro cell culture

after separation from NLCs in an ultrafiltration centrifugal tube with a molecular weight cut off (MWCO) of 100 kDa (Ultra-4, Millipore, USA). Briefly, TMS-NLCs were diluted 10-fold with distilled water, and 0.5 mL of the diluent was placed into the upper chamber of the tube and centrifuged at 2352 g for 20 min at 4 ℃ in a Sigma Centrifuge (3K15, Sigma, Germany). The amount of free drug (Wfree) was collected in the outer chamber and brought to 1 mL with methanol for analysis by HPLC. Additional diluent samples (0.5 mL) were directly extracted with 0.5 mL of methanol, vortexed for 2 min, demulsified for 10 min with ultrasound and centrifuged at 2352 g for 10 min. The supernatant was analyzed by HPLC, representing the total amount of TMS (Wtotal). The EE and DL were respectively calculated by the following equations:

2.8.1. Culture of Caco-2 cells and monolayers Caco-2 cells were maintained at 37 ℃ under a 5% CO2 atmosphere in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin mixture. Cells were seeded at a density of 2 × 105 cells per well on Transwell® polycarbonate inserts (12-well and 0.4 μm pore diameter; Corning Costar, USA) to establish in vitro transcellular models. DMEM (0.5 mL) was added to the apical side (AP) of the Transwell® insert, and 1.5 mL of DMEM was added to the basolateral side (BL). Cells were cultivated for 21 days. During this period, the medium was replaced every two days, and the transepithelial electrical resistance (TEER) was measured by an Epithelial Volt-Ohm Meter (Millicell® ERS2, Millipore, USA) to corroborate the integrity of the cell monolayers. Monolayers with TEER values greater than 450 Ω cm2 were used to evaluate the transport of TMS-NLCs, and the TEER values of the cell monolayers were measured again to ensure their integrity upon ending the transport of TMS-NLCs.

Wtotal − Wfree × 100% Wtotal Wtotal − Wfree DL(%) = × 100% Wlipid EE(%) =

where Wtotal, Wfree and Wlipid are the total amounts of TMS, the unincorporated TMS and the amount of lipid in the formulation, respectively.

2.8.2. Permeability of TMS-NLCs across Caco-2 cell monolayers The permeability of TMS across the Caco-2 cell monolayers was evaluated by comparing API with TMS-NLCs. For apical to basolateral (AP→BL) side transport, experiments were conducted at 4 ℃ or 37 ℃ by adding TMS (0.5 mL, 10 μg/mL) to the AP side and 1.5 mL of HBSS to the BL side. For basolateral to apical (BL→AP) side transport, 0.5 mL of HBSS was added to the AP side and 1.5 mL of 10 μg/mL TMS was added to the BL side. Samples were collected from the BL or AP side after 4 h of incubation, and the TMS concentration was analyzed by HPLC. To evaluate the contribution of TMS-NLCs to the inhibition of P-gp, cell monolayers were pretreated with 100 μM verapamil for 1 h, and TMS-NLCs were subsequently added to the AP or BL sides for 4 h in the presence of verapamil for the bidirectional transport assay. The apparent permeability coefficients (Papp, cm/s) for both AP→BL and BL→AP were calculated using the following equation:

2.6. In vitro drug release studies In vitro drug release was performed over 72 h using the dialysis bag technique. Briefly, a cellulose dialysis bag (MWCO of 100 kD, 16 mm, and 0.79 mL/cm) containing 3 mL of TMS-NLCs or the corresponding active pharmaceutical ingredient (API) of TMS was firmly tied, immersed in 500 mL of simulated gastric or intestinal fluids at 37 ℃, and stirred at 100 rpm with a magnetic stirrer. At each time interval, samples (1 mL) were withdrawn and replaced with the same volume of fresh simulated GI fluids, and the drug concentrations were analyzed by HPLC.

2.7. Pharmacokinetics study

Papp = All animal experiments were performed according to the Guidelines for Animal Experimentation with approval of the Institutional Animal Care and Use Committee at Nanjing Agricultural University. Piglets (4 weeks old and 16.34 ± 2.54 kg) were purchased from the Jiangsu Academy of Agricultural Sciences (Nanjing, China). Ten piglets were randomly divided into two groups for the pharmacokinetics study. The orally administered doses of API and TMS-NLCs were a single dose of 20 mg/kg body weight (BW). Blood samples (4 mL) were collected from each animal via the precaval vein before dosing (0 min) and after predetermined time points (10 min, 20 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, and 96 h) into heparinized tubes, and samples were immediately centrifuged at 1152 g for 10 min to separate plasma as supernatants. Prior to analysis, 0.2 mL of the plasma sample was mixed with 0.28 mL of methanol and vortexed for 1 min. The mixture was centrifuged at 2352 g for 10 min, and the supernatant was then collected. Methanol (0.28 mL) was used twice to extract TMS in the sediment. NaOH (1 μL, 1 M) was added to adjust the pH of the combined supernatant, and then, trichloromethane (0.5 mL) was added. The mixture was vortexed for 1 min and centrifuged at 2352 g for 10 min. The trichloromethane layer was subsequently collected. Additional trichloromethane (0.2 mL) was added to the residue, and the procedure was repeated. The chloroform fractions were pooled and dried under nitrogen at 50 ℃, and the residue was resuspended with 0.2 mL of mobile phase by vortexing for 1 min. Samples were analyzed by HPLC after filtering.

dQ dt *AC 0

where dQ/dt, A and C0 are the transport rate, the surface area of the filter membrane, and the initial concentration of the tested drug, respectively. The efflux ratio (ER) value was obtained from the ratio of Papp (BL→AP) to Papp (AP→BL) as follows:

ER =

Papp (BL → AP ) Papp (AP → BL)

2.8.3. Transport mechanism of TMS-NLCs across Caco-2 cell monolayers Lucifer Yellow (0.5 mL, 225 μM) and propranolol (0.5 mL, 100 μM) were added to the AP side of the Transwell® insert cell monolayers and coincubated with TMS-NLCs for 4 h at 37 °C. The tracer concentrations in the opposite side (BL) were quantified by a Multiskan Spectrum (Infinite M200 Pro, Tecan, Switzerland) for fluorescence analysis. The monolayers were pretreated with different endocytosis inhibitors for 1 h, namely chlorpromazine (30 μM), MβCD (2 mM), dynasore (80 μM), and EIPA (50 μM). TMS-NLCs were subsequently added to the AP side and coincubated with endocytosis inhibitors for 4 h. Finally, the transport fluids were collected from the BL side for HPLC analysis. TMS-NLCs without any endocytosis inhibitors were used as the control group. In addition, the transport fluids from the control group were collected and negatively stained with 2% phosphotungstic acid to analyze the morphology of the nanoparticles using TEM after transcellular transport. 3

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Fig. 1. Characterization of optimized TMS-NLCs. (A) The morphology of TMS-NLCs was determined by transmission electron microscopy (TEM). Scale bar was 100 nm. (B) The size distribution histogram was obtained via size analysis of several TEM images of particles. The mean diameter was 120.77 nm. (C) The hydrodynamic diameter and (D) zeta potential of TMS-NLCs in aqueous solution were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano and were 283.03 nm and -30.04 mV, respectively. (E) DSC thermograms and (F) FT-IR spectra for TMS-NLCs, TMS, SA, SA + TMS and sucrose were analyzed. Table 1 Characterization of the prepared TMS-NLCs. Formulation code

Distribution size (nm)

hydrodynamic diameter (nm)

Polydispersity index (PDI)

zeta potential (mV)

Entrapment efficiency (EE, %)

Drug loading (DL, %)

TMS-NLCs

120.77 ± 22.96

283.03 ± 6.64

0.219 ± 0.027

−30.04 ± 1.36

93.46 ± 0.50

9.23 ± 0.08

for evaluating the stability of colloidal systems [19]. In the current study, the HD of optimized TMS-NLCs was 283.03 ± 6.64 nm (Fig. 1A), suggesting they are suitable for oral administration. In addition, the PDI of the TMS-NLCs was less than 0.3, indicating high homogeneity in the particle population [20]. Zeta potential (ZP) is also a key factor for evaluating the stability of colloidal dispersions. In general, a high ZP, especially with an absolute value greater than 30 mV, allows nanoparticles to stably disperse due to electric repulsion among nanoparticles [21]. The ZP of TMS-NLCs was -30.04 ± 1.36 mV (Fig. 1B), indicating its good stability. In addition, drug loading (DL) and entrapment efficiency (EE) were determined by HPLC with values of 9.23 ± 0.08% and 93.46 ± 0.50%, respectively (Table 1). These data indicated that NLCs exhibit potential as ideal carriers of TMS. The morphology of TMS-NLCs was observed by transmission electron microscopy (TEM). As shown in Fig. 1C, TMS-NLCs were welldispersed and spherically shaped with smooth surfaces. The mean distribution size of TMS-NLCs was 120.77 ± 22.96 nm (Table 1 and

2.9. Statistical analysis All measurements were performed in triplicate, and the results are expressed as mean values ± standard deviation (SD). The results were statistically depicted using GraphPad Prism 6 (GraphPad InStat Software, USA) and OriginPro 8 (Originlab, USA). Pharmacokinetic parameters were calculated by a noncompartmental model with WinNonLin 5.2 (Pharsight, USA). Statistical analyses were performed using one-way analysis of variance (ANOVA) in SPASS 18 (SPASS company, USA). Differences were considered significant at *P < 0.05 and **P < 0.01.

3. Results and discussion 3.1. Characterization of TMS-NLCs It is believed that the HD is one of the most important parameters 4

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Fig. 2. Storage stability and gastrointestinal stability of TMS-NLCs. (A) Hydrodynamic diameter, (B) polydispersity index and (C) zeta potential changes during storage for 90 d at 4, 25 and 40 ℃ are shown. (D) Hydrodynamic diameter, (E) polydispersity index and (F) zeta potential changes during storage for 7 d in simulated gastric (pH 1.23) and intestinal (pH 6.8) fluids are shown.

migration of peaks (74.18 ℃→71.89 ℃ and 106.12 ℃→77.67 ℃) was due to the interaction of solid lipids with drugs that altered their melting behavior. The endothermic peak of TMS disappeared, while that of the cryoprotectant still existed at 191.86 ℃, indicating that TMS is an amorphous structure or almost completely dissolved in carrier materials consisting of the TMS-NLC formulation [25]. In addition, the peak of SA at 74.18 ℃ disappeared, suggesting that lyophilization may influence crystallization of solid lipids. FT-IR analysis were performed for pure TMS, SA, sucrose, the lipid/ drug physical mixture and freeze-dried TMS-NLCs. As shown in Fig. 1F, the absorption peaks for pure TMS were as follows: OeH stretching vibration around at 3460 cm−1; NeH (piperidyl) stretching vibration around 3140 cm−1; CeH (methyl) antisymmetric stretching vibration around 2940 cm−1; C]O (inner ester) stretching vibrations at 1750 cm−1 and 1690 cm−1; C]C (inner ester) stretching vibration at 1590 cm−1; C–O (inner ester or pyranyl) stretching vibration at 1080 cm−1; and C–H (pyranyl or piperidyl) flexural vibration at 980 cm−1. These peaks were consistent with a previous report [26]. The absorptions peaks for the TMS-NLC powder were characteristic of those for TMS as follows: NeH (piperidyl) stretching vibration around 3140 cm−1; the C]C (inner ester) stretching vibration at 1590 cm-1 disappeared; and the C]O (inner ester) stretching vibrations at 1750 cm−1 and 1690 cm-

Fig. 1D), which was smaller than the HD as determined by DLS. Importantly, DLS does not directly measure the diameter of nanoparticles but measures the fluctuation of the scattered light intensity caused by Brownian motion to indirectly reflect particle size [22]. In addition, HD measurements are performed by DLS in an aqueous state, indicating that highly hydrated lipid nanospheres had larger diameters than nonhydrated ones [23]. To perform the DSC and FT-IR analyses, freeze-dried powders of TMS-NLCs were acquired using 10% sucrose as the cryoprotectant via a lyophilization technique. Thermograms of pure TMS, SA, sucrose, the lipid/drug physical mixture and freeze-dried TMS-NLCs were analyzed by DSC (Fig. 1E). DSC monitors and quantifies the physical state of dispersed nanoparticles via the absence of these endotherms, even minute thermal amounts in the sample. As shown in Fig. 1E, TMS had a weak endothermic peak at 106.12 ℃. The DSC curve of SA presented a flat profile with a sharp endothermal peak at 74.18 ℃, which is characteristic of a typical crystal structure. Similarly, the thermograms for sucrose had an intense peak at 190.96 ℃. The SA and TMS physical mixture had two peaks at 71.89 ℃ and 77.67 ℃, but the intensities of the two peaks were lower than those of pure lipid or drug. The reduced crystallinity may be attributed to both the components contained in the formulation and preparation technology of TMS-NLCs [24]. The 5

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Table 2 Main pharmacokinetic parameters of TMS-NLCs and API in piglets. Parameters

API

TMS-NLCs

Tmax (h) Cmax (μg/mL) Tlast (h) t1/2 (h) λz (1/h) Vd/F CL/F MRT (h) AUC (μg·h/mL)

1.30 ± 0.71 1.56 ± 0.14 28.80 ± 6.57 11.27 ± 2.11 0.09 ± 0.02 24008.35 ± 6894.42 1594.95 ± 241.64* 8.09 ± 0.90 10.72 ± 1.15

3.40 ± 0.55** 1.54 ± 0.29 45.60 ± 11.49* 11.68 ± 2.68 0.07 ± 0.03 20159.66 ± 5249.61 1217.41 ± 237.18 10.60 ± 1.73* 15.26 ± 2.45*

Table 3 Permeability and efflux rate (ER) of API and TMS-NLC transport into Caco-2 cell monolayer models. Formuation code

Verapamil

Papp(×10−6 cm/s) AP→BL

API TMS-NLCs

– + – +

0.31 0.53 0.57 0.49

± ± ± ±

Efflux rate (ER)

BL→AP 0.08 0.04* 0.03** 0.03*

0.95 0.61 0.83 0.70

± ± ± ±

0.06 0.05** 0.03* 0.08**

3.04 1.15 1.45 1.44

good storage stability of TMS-NLCs. The harsh gastrointestinal environment is an obstacle that affects the stability of nanoparticles for oral absorption [27]. In addition, particle size plays an important role in gastrointestinal absorption, and it has been reported that a particle size less than 300 nm is suitable for intestinal transport [24]. To test whether TMS-NLCs as an oral preparation would not be degraded before reaching its absorption sites, the pH stability of TMS-NLCs was measured in simulated gastric fluids with pepsin (pH 1.2) and intestinal fluids with trypsin (pH 6.8). As shown in Fig. 2F, the ZP of TMS-NLCs varied with the pH change of simulated gastrointestinal (GI) fluids it was reversed from anionic to cationic when in an acidic pH environment, which may help nanoparticles escape lysosomal degradation in the cytoplasm [28]. In contrast, there was no significant change in HD (Fig. 2D). Similarly, the PDI values were both less than 0.2 in simulated gastric and intestinal fluids (Fig. 2E). Together, these results indicated the TMS-NLCs stably exist in simulated GI fluids without being destroyed.

Fig. 3. In vitro drug release profiles of TMS-NLCs and API in simulated gastric (A) and intestinal (B) fluids.

3.3. In vitro drug release of TMS-NLCs

1 were found but with slightly altered positions (1710 cm−1 and 1640 cm−1, respectively). These data indicate that TMS is almost completely encapsulated in the NLCs by interacting with the lipid matrix.

The in vitro release behaviors of TMS-NLCs or API in simulated GI fluids were analyzed at 37 ℃ over 72 h using the dialysis bag technique. The release rate of TMS-NLCs (59.96 ± 0.64%) was significantly lower than that of API (81.94 ± 0.71%) in simulated gastric fluids (Fig. 3A), and the release tendency was similar in simulated intestinal fluids when comparing TMS-NLCs (23.03 ± 0.22%) to API (85.23 ± 1.65%) (Fig. 3B) after cumulative release over 72 h. Generally, crystallization by cold water enriches the drug in the outer layers of nanoparticles during the preparation process of NLCs, thus causing an initial burst release [29]. However, TMS-NLCs displayed a slow and sustained release without a burst release behavior in both simulated gastric fluids and intestinal fluids, which may be attributed to the high EE of TMSNLCs (Table 1).

3.2. Stability evaluation of TMS-NLCs

3.4. Pharmacokinetics study of TMS-NLCs and API in piglets

Stability is an important factor to ensure the safety and effectiveness of pharmaceutical preparations. TMS-NLCs were first stored at 4 ℃, 25 ℃ and 40 ℃ for 90 days, and the HD, PDI and ZP of the TMS-NLCs were then determined after different storage times. The results showed no significant changes during the storage period (Fig. 2A-C), indicating the

TMS-NLCs and API showed a similar pharmacokinetics behavior to that of TMS in piglets according to their drug concentration-time (C-T) curves (Fig. 4). The maximal plasma concentration (Cmax) and peak time (Tmax) of TMS were directly obtained from the C-T curves [30]. Table 2 summarizes the main pharmacokinetic parameters of TMS as

Fig. 4. Drug concentration-time (C-T) curves of TMS-NLCs and API in piglets.

6

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Fig. 5. Pathway of TMS-NLC transport into Caco-2 cell monolayers. (A) Effect of TMSNLCs on Papp of Lucifer Yellow. “+” and “-” were treated with/without TMS-NLCs, respectively. (B) The Papp of TMS-NLCs and API at 4 ℃ and 37 ℃ was detected. (C) The effect of various endocytosis inhibitors on the Papp of TMS-NLCs was determined. CHL: chlorpromazine, MβCD: methyl-β-cyclodextrin, EIPA: 5(N-ethyl-N-isopropyl)-amiloride, ns: no significance, *P < 0.05, and **P < 0.01. (D) After 4 h of transcellular transport, TMS-NLCs from the AP→BL side transport fluids were collected to observe the nanoparticle morphology by TEM. Scale bar was 200 nm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

transcellular pathway [33]. Therefore, Lucifer Yellow and propranolol are used as paracellular and transcellular transport markers, respectively [34]. The Papp values of Lucifer Yellow and propranolol transported separately without TMS-NLCs were (0.30 ± 0.11) ×10−6 cm/s and (9.99 ± 0.30) ×10−6 cm/s, respectively. After coincubation with TMS-NLCs, the permeability of propranolol was significantly increased, while there was no significant difference for Lucifer Yellow. These results confirm that TMS-NLCs transport across intestinal barriers mainly through the transcellular pathway rather than the paracellular pathway (Fig. 5A). It is well-known that low temperature is a normal inhibitor of cell metabolism and that the pinocytic/endocytic uptake of tracer is inactivated at 4 ℃ [35]. The Papp of API and TMS-NLCs at 4 ℃ were lower than that at 37 ℃ (Fig. 5B), and the intracellular concentrations were even below the detection limit of HPLC. These data suggest that NLCs mainly enter cells by an energy-dependent active transport route. It has been reported that nanoparticles are taken up by cells through active transport by endocytosis, including macropinocytosis, clathrin-mediated endocytosis and caveolae/lipid raft-mediated endocytosis [36]. Therefore, the Caco-2 cell monolayers were pretreated with different endocytosis inhibitors to evaluate the endocytosis mechanism involved in TMS-NLC transport across cell monolayers. As shown in Fig. 5C, there were no significant changes in the Papp of TMS when TMS-NLCs were coincubated with CHI or EIPA, while the Papp of TMS was decreased in the presence of MβCD or dynasore, indicating that TMS-NLCs are mainly internalized by Caco-2 cells via the caveolae/lipid raftmediated endocytosis pathway. After transcellular transport, AP→BL side transport fluids were collected to observe the morphology of TMSNLCs. Smooth and spherically shaped particles were observed by TEM, with a mean distribution size of 108.81 ± 24.22 nm, which was not significantly different compared to the distribution size prior to transcellular transport. However, the amount of nanoparticles was decreased due to cell monolayer barriers (Fig. 5D). Taken together, these data indicate that NLCs have potential to escape the efflux of P-gp and avoid lysosome degradation of intestinal barriers, thereby allowing them to

calculated by a noncompartmental model analysis with WinNonLin 5.2. After oral administration, API and TMS-NLCs similarly reached a maximal plasma concentration (Cmax). The peak times (Tmax) of TMSNLCs and API were significantly different, with values of 3.4 h and 1.3 h, respectively. The last time point of measurable concentration (Tlast) and the mean residence time (MRT) of TMS-NLCs were significantly longer than those of API, indicating that the TMS-NLC formulation had an obvious sustained-release effect. In addition, AUCTMSNLCs was 1.42-fold higher than AUCAPI, suggesting that TMS-NLCs may significantly improve the oral bioavailability of TMS in piglets. 3.5. In vitro cell experiments 3.5.1. Evaluation of TMS permeability across Caco-2 cell monolayers Table 3 shows that the efflux rate (ER) of API in Caco-2 cell monolayers was 3.04, which was greater than the value of 1.5 reported in FDA regulations [31], indicating that TMS may be a substrate of Pgp. After incubation with verapamil, a typical P-gp inhibitor [32], the Papp value was significantly increased from (0.31 ± 0.08)×10−6 cm/s to (0.53 ± 0.04)×10−6 cm/s and the ER value of API was reduced to 1.15, further confirming that TMS is the substrate of P-gp. Similarly, the Papp value was improved to (0.57 ± 0.03)×10−6 cm/s by TMS-NLCs, which was significantly different from that of API, suggesting that NLCs improve the permeability of TMS in Caco-2 cell monolayers. However, there was no significant difference in either Papp of ER of TMS-NLCs regardless of the presence or absence of verapamil, indicating that TMSNLCs escape the efflux of P-gp. Taken together, these data indicate that TMS-NLCs not only increase the transport of TMS across Caco-2 cell monolayers but simultaneously decrease the efflux of P-gp, thereby enhancing the intracellular permeability of TMS. 3.5.2. Transport mechanism of TMS-NLCs across Caco-2 cell monolayers Lucifer Yellow cannot permeate into the lipid barrier of cell membranes due to its low permeability. By contrast, propranolol is a highly lipophilic drug and can easily permeate into cell monolayers through a 7

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be successfully transported across intestinal epithelial cells in an intact form.

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4. Conclusion In the current study, tilmicosin-loaded nanostructured lipid carriers (TMS-NLCs) were successfully prepared by a high shear-ultrasonic homogenization technique. The acquired TMS-NLCs had a HD of 283.03 ± 6.64 nm, PDI less than 0.3 and ZP of -30.04 mV, thus exhibiting good storage stability and gastrointestinal stability. In addition, TMS-NLCs had an excellent loading capacity and entrapment efficiency, and TMS-NLCs demonstrated a sustained in vitro release behavior without an initial burst release. TMS-NLCs remarkably improved the oral bioavailability of TMS in piglets. TMS-NLCs not only enhanced the permeability of TMS but also decreased the efflux of P-gp toward TMS according to an in vitro cellular transport study in Caco-2 cell monolayers. Interestingly, TMS-NLCs were internalized by Caco-2 cells via the caveolae/lipid raft-mediated endocytosis pathway and were transported into basolateral fluid in an intact form. Taken together, NLCs may be used as TMS delivery carriers to improve the oral adsorption of TMS in piglets via enhancing cellular permeability and decreasing P-gp efflux. CRediT authorship contribution statement Qian Zhang: Investigation, Writing - original draft. Haifeng Yang: Methodology, Investigation. Benazir Sahito: Investigation. Xinyu Li: Visualization. Lin Peng: Software, Validation. Xiuge Gao: Data curation. Hui Ji: Software. Liping Wang: Conceptualization. Shanxiang Jiang: Conceptualization. Dawei Guo: Conceptualization, Supervision, Funding acquisition, Writing - review & editing. Declaration of Competing Interest These authors declare no conflict of interest. Acknowledgements This work was supported by the National Key Research and Development Program of China (2016YFD0501306), the Fundamental Research Funds for the Central Universities (Y0201800847) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] E.E. Ose, In vitro antibacterial properties of EL-870, a new semisynthetic macrolide antibiotic, J. Antibiot. 40 (2) (1987) 190–194. [2] A. Al-Qushawia, A. Rassoulia, F. Atyabib, et al., Preparation and characterization of three tilmicosin-loaded lipid nanoparticles: physicochemical properties and in-vitro antibacterial activities, Iran. J. Pharm. Res. 15 (4) (2016) 663–676. [3] P.J. Blackall, T. Asakawa, R.J. Graydon, et al., In-vitro antibacterial properties of tilmicosin against Australian isolates of Pasteurella multocida and Actinobacillus pleuropneumoniae from pigs, Aust. Vet. J. 72 (1) (2010) 35–36. [4] X.J. Chen, T. Wang, M.M. Lu, et al., Preparation and evaluation of tilmicosin-loaded hydrogenated castor oil nanoparticle suspensions of different particle sizes, Int. J. Nanomed. Nanosurg. 9 (1) (2014) 2655–2664. [5] B. Li, S.Y. Gong, X.Z. Zhou, et al., Determination of antibacterial agent tilmicosin in pig plasma by LC/MS/MS and its application to pharmacokinetics, Biomed. Chromatogr. 31 (3) (2016) 1–9. [6] J.C. Xiong, Q.Q. Zhu, Y.X. Zhao, et al., Tilmicosin enteric granules and premix to pigs: antimicrobial susceptibility testing and comparative pharmacokinetics, J. Vet, Pharmacol. Ther. 42 (2019) 336–345. [7] Z. Xu, J. Wang, W. Shen, et al., Study on the extraction equilibrium of tilmicosin between the aqueous and butyl acetate phases, Chem. Eng. Commun. 193 (4) (2006) 427–437. [8] C. Han, C.M. Q I, B.K. Zhao, et al., Hydrogenated castor oil nanoparticles as carriers for the subcutaneous administration of tilmicosin: in vitro and in vivo studies, J. Vet. Pharmacol. Ther. 32 (2) (2009) 116–123.

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