Combination of Phospholipid Complex and Submicron Emulsion

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Combination of phospholipid complex and submicron emulsion techniques for improving oral bioavailability and therapeutic efficacy of water-insoluble drug Linghao QIN, Yawei NIU, Yuemin WANG, and Xiaomei CHEN Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01061 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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Molecular Pharmaceutics

Combination of phospholipid complex and submicron emulsion techniques for improving oral bioavailability and therapeutic efficacy of water-insoluble drug

Linghao QIN 1, #, *, Yawei NIU 1, 2, #, Yuemin WANG 1, Xiaomei CHEN 1

1

Department of Pharmaceutics, School of Pharmacy, Guangdong Pharmaceutical University, No.

280, Waihuandong Road, High Education Mega Center, Guangzhou 510006, PR China 2

Guangzhou Hanfang Pharmaceutical Co., LTD., No 134, Jiangnan Dadao Zhong, Guangzhou

510240, PR China

Corresponding Author * E-mail: [email protected]. Tel/Fax: +86-20-39352117

#

These authors contributed equally

ACS Paragon Plus Environment

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ABSTRACT: Water-insoluble drugs can not be absorbed effectively through gastrointestinal tract due to insufficient solubility and often face the problems of low bioavailability and poor therapeutic efficacy. To overcome these biopharmaceutical challenges, lipid-based formulations were suggested and have been researched in recent years. In this study, we used atorvastatin as a model drug to prepare a phospholipid complex pro-drug system to upgrade its lipophilicity and further developed a drug loaded submicron emulsion to improve its in vivo bioavailability. The mean particle size and zeta-potential of submicron emulsion were 122.7 nm and -22.7 mv. Intestinal absorption of atorvastatin from submicron emulsion was significantly improved compared with free drug, and the absorption rate constant (Ka) and apparent permeability coefficients (Papp) increase 2.88-fold and 2.45-fold respectively. After oral administration, atorvastatin plasma concentration of emulsion group was much higher than that of free drug and the area under the curve (AUC) reach to 4.033 mg/L·h (2.58-fold). In vivo pharmacodynamics results revealed that atorvastatin submicron emulsion appeared excellent anti-hyperlipidemia efficacy by reducing the total cholesterol, triglyceride and low density lipoprotein cholesterol (LDL-cholesterol) level and simultaneously increasing the high density lipoprotein cholesterol (HDL-cholesterol) level in comparison with Lipitor. In conclusion, drug-phospholipid complex loaded submicron emulsion was a promising oral delivery system for improving in vivo absorption behavior and therapeutic efficacy for water-insoluble drugs.

KEYWORDS:

phospholipid

complex,

submicron

anti-hyperlipidemia, atorvastatin


emulsion,

oral

absorption,

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1. INTRODUCTION Oral administration, as the non-invasive drug delivery route, is considered to be a safe and effective way for patients to take medicine and it could offer designed drug release profile, low toxicity and good physiological compliance.1 However, more than 40% Active pharmaceutical ingredients (APIs) have lower bioavailability and poor therapeutic effect due to their water insoluble characteristic. In order to overcome this challenge, on the one hand, novel techniques such as nano-extrusion, liquid crystal formulation and nano suspension etc are applied to upgrade the drug solubility or dissolution rate in aqueous medium.2-4 On the other hand, suitable drug carriers systems are selected to improve drug physicochemical properties or transportation behavior to promote oral absorption. Oral delivery system using lipid-based formulations is one of the most useful strategies to develop drug loaded lipid preparations with improved bioavailability and therapeutic benefits. As one of the most widely used lipid-based preparations, submicron emulsion (also referred to as lipid emulsion or lipid microsphere) draws more attention in recent decades.5-8 Submicron emulsion can be defined as a kind of colloidal dispersion system with an average diameter of 200 nm using liquid fat oils as inner core and phospholipids as major emulsifier. Except for being energy and essential fatty acid supplementary sources for postoperative patients, submicron emulsion has already become excellent vehicle for drugs delivery with broad range of applications.9-11 Drug was incorporated into lipophilic core and related degradation or side effect caused by direct contact with body fluid and tissues could be avoided. With increasing lipophilicity, drug permeability and the transmembrane rate would be increased when drug transferred through intestinal mucous cells.12-14 Moreover, oil phase in submicron emulsion



promoted the bile salts secretion and formation of drug loading micelles which were helpful to upgrade the absorption through gastrointestinal tract. Several studies also indicated that first pass effect of drug can be avoided when drug loaded lipid carriers were digested and uptake via lymphatic transportation.15-17 Atorvastatin is a selective competitive inhibitor of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA). As a member of second-generation statins drugs, atorvastatin can inhibit biosynthesis of cholesterol and HMG-CoA reductase in liver. It can decrease the total cholesterol, triglyceride and low density lipoprotein cholesterol (LDL-cholesterol) levels and increase the high density lipoprotein cholesterol (HDL-cholesterol) level in plasma. At the same time, atorvastatin can significantly relieve the symptoms of homozygous and heterozygote patient with familial hypercholesterolemia or non familial hypercholesterolemia.18 Atorvastatin is a BCSⅡ drug and commonly used as atorvastatin calcium (ATC) in clinical. The absolute bioavailability of ATC is only 14% and the inhibition activity of HMG-CoA reductase was only 30%.19 The main reasons for this phenomenon are poor drug solubility in aqueous solution, easier degradation in acid medium and fast elimination in vivo.20-21 To improve ATC gastrointestinal absorption, Researchers attempted a series of lipid based carrier systems including nanoparticles, self emulsifying system and dry emulsion system.22-24 However, although these lipid carrier systems can promote the drug bioavailability to varying degrees, some drawbacks can not be ignored. The use of large amount of organic solvent and high proportion of co-emulsifier can bring potential safety risk and complicated processes such as dialysis, ultra filtration and freeze-drying are not suitable for large-scale production. Using submicron emulsion system to deliver ATC could be an effective way to avoid these


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defects. Since ATC was both insoluble in water and oil, in order to successfully develop a submicron emulsion system, drug solubility in oily medium should be upgraded. Here, we utilized the phospholipid complex technology to prepare the ATC pro-drug to increase its lipophilicity in oils and further developed a drug loaded submicron emulsion. We hope that the combination of phospholipid complex and submicron emulsion techniques could be useful to prepare lipid based oral drug delivery system and satisfied the requirement of safety, stability, scale-up production and improved bioavailability for water insoluble drugs. Above all, the aims of this article are: (1) to prepare

ATC-phospholipid

complex

loaded

submicron

emulsion

and

investigate

its

physicochemical properties; (2) to study the cellular uptake behavior, endocytosis route and cytotoxicity using Caco-2 cell model; (3) to investigate the in vivo gastrointestinal absorption characteristics and pharmacokinetics feature; and (4) to evaluate the anti-hyperlipidemia efficacy in rats compared with commercial product.

2. MATERIALS AND METHODS 2.1. Materials. Atorvastatin calcium (ATC) was obtained from Jacobson pharma Co. LTD. (Hongkong, China). Purified Ovolecithin and soybean oil were kindly provided by Guangzhou Hanfang

Pharmaceutical

Co.

LTD.

(Guangdong,

China).

Pluronic

F-68

and

Carboxymethylcellulose sodium (CMC-Na) were kindly provided by BASF (Ludwigshafen, Germany). Sodium oleate was purchased from Xi’an Ruixi Biological Technology Co. LTD. (Shanxi, China). Glycerol for injection was obtained from Zhejiang Suichang Huikang Pharmaceutical Co. LTD. (Zhejiang, China). 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), 1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine (DiD), were purchased from Keygen Biotech (Nanjing, China). Fetal bovine serum (FBS), streptomycin (10 µg/mL)



(Penicillin-Streptomycin Solution, 100×), 0.25% Trypsin-EDTA (1×), and Gibco Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Invitrogen (USA). Amicon Ultra-4 Centrifugal Filter (30 kDa) was purchased from Millipore (Boston, USA). Cell lysis buffer and glycerol jelly mounting medium were purchased from Beyotime (Shanghai, China). All other chemicals and reagents were of analytical grade, and used without further purification. Caco-2 cell line (passage: 40-50) was kindly provided by department of pharmacology of Guangdong Pharmaceutical University. The cells were cultured with MEM medium containing 20% FBS under 5% CO2 atmosphere condition at 37 °C. 8 weeks age SD rats (220-250 g) were purchased from the Experimental Animal Center of Southern Medical University of China and were raised under specific pathogen-free (SPF) condition. The animal experiments involved in present study were consistent with the guidelines set by the National Institutes of Health and were approved by the Experimental Animal Ethics Committee of Guangdong Pharmaceutical University. 2.2. Preparation of ATC-phospholipid Complex. ATC-phospholipid complex was prepared according to previous study with modification.25 Briefly, ATC (0.25 mmol) and ovolecithin with 1:1 molar ratio were placed into 100 mL round bottom flask and dissolved by 50 mL anhydrous ethanol. This mixture solution was then stirring for 2 h under nitrogen atmosphere at 40 °C, and the solvent was removed by vacuum rotary evaporation method at 40 °C for 1h. The resulting ATC-phospholipid complex product was further dried using vacuum drying oven and kept in brown exsiccator before use. 2.3. Characterization of ATC-phospholipid Complex. 2.3.1. Solubility and Oil/water Partition Coefficient of ATC-phospholipid Complex. The solubility


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of ATC and the ATC-phospholipid complex was determined in water, various buffers (pH=1.2, 4.5 and 6.8) and oily medium at room temperature. To determine the oil/water partition coefficient, samples were dissolved in equal volume of n-octanol/buffer system and kept stirring for 24 h at 37 °C. The aqueous phase solution was separated and centrifuged at 4000 rpm for 10 min, and upper layer was collected for HPLC analysis to calculate the drug concentration. 2.3.2. FT-IR, DSC and X-ray Diffraction Analysis. The properties of ATC-phospholipid complex were characterized by several methods. Firstly, four samples including free drug (ATC), ATC-phospholipid physical mixture (ATC+PC), ATC-phospholipid complex (ATC-PC) and phospholipids were prepared. For FT-IR analysis, samples were compressed into tablets with KBr, and were scanned from 400-4000 cm-1. For X-ray diffraction analysis, samples were examined under graphite monochromatized Cu Kα radiation over the range 10-45 ° (2θ) at 40 kV and 40 mA. For DSC analysis, AI2O3 as control, these four samples were conducted form 30 °C to 300 °C with the heating rate of 10 °C/min under N2 atmosphere. 2.4. Preparation of ATC Submicron Emulsion. ATC-phospholipid complex was used as pro-drug to prepare ATC loaded submicron emulsion by high-pressure homogenization method. Firstly, 0.3% (w/v) ATC-phospholipid complex (calculated as ATC) was dissolved in 5% (w/v) soybean oil with stirring at 70 °C, and this solution was used as oily phase. 1.4% (w/v) egg lecithin, 1.0% (w/v) F-68, 0.8% (w/v) Tween-80, 0.05% (w/v) sodium oleate and 2.25% glycerin were dispersed in double distilled water as aqueous phase with agitation at the same temperature. Secondly, the oily phase solution was added into aqueous solution with vigorous agitation at 10000 rpm for 10min at 70 °C to obtain O/W coarse emulsion. The pH of submicron emulsion was adjusted to 7.0-7.5, and then the coarse solution was subjected to high pressure homogenization (AH-2010,



ATS Engineering Inc, Canada) at 1200 bar for 7 times. The final drug loaded submicron emulsion was filed into vials under N2 protection and sterilized at 121 °C for 15 min by autoclaving. The DiD labeled submicron emulsion was prepared using the same method described above except that the fluorescence labeled submicron emulsion was sterilized by filtration through 0.22 µm microporous membrane. 2.5. Characterization of ATC Submicron Emulsion. 2.5.1. Droplet Size and Zeta Potential. The mean article size, size distribution, polydispersity index (PDI) and zeta potential of drug loaded submicron emulsion were measured using Zetasizer Nano ZSP (Malvern Instruments, UK). Each test sample was diluted with double distilled water (1: 500 v/v) and repeated in triplicate. 2.5.2. Morphology. The structure of ATC submicron emulsion was examined with a transmission electron microscope (TEM). A drop of submicron emulsion sample was deposited onto a glow-discharged carbon-coated grid, and then stained by 1% uranyl acetate. The grid was subsequently dried and visualized under a HITACHI H-7650 microscope. 2.5.3. ATC Content and Loading Efficiency Assay. ATC concentration was calculated with HPLC method. Briefly, 0.1 mL ATC submicron emulsion was diluted to 25 mL with methanol under sonication to ensure complete drug extraction. The solution was filtrated through 0.45 µm microporous membrane, and 20 µL was analyzed by HPLC system (Chromaster with 5110 pump, 5210 Autosampler, 5310 Column Oven, 5410 UV detector, Hitachi, Japan). Drug loading efficiency (LE%) was also measured with the equation LE%=Min/Mtotal×100% (Where, Min = the amount of ATC in oily phase and emulsifier layer, Mtotal = the total amount of ATC in submicron emulsion).


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2.6. In Vitro Release. ATC submicron emulsion in vitro dissolution test was conducted by a dialysis method. Briefly, 2 mL sample was sealed in a dialysis bag (Spectra/Por membranes, MWCO: 3500, Spectrum USA), and placed in a beaker containing 900 mL PBS (pH=6.8) at 37 °C. Under magnetic stirring condition, 5 mL sample was collected and replaced with equal volume fresh PBS at each time point (0.5, 1, 2, 3, 4, 6, 8, 12, 16, 20 and 24h) to calculate drug released percentage. 2.7. Storage Stability Study. The long-term storage stability was carried out at 25 °C for 6 months. At setting time intervals (0, 1, 2, 3 and 6 month), the particle size, zeta potential, pH, drug content and LE% were determined to evaluate the ATC submicron emulsion stability. 2.8. MTT Assay. MTT assay was conducted to identify the biocompatibility of ATC submicron emulsion. Caco-2 cells were seeded at the density of 104 cells per well in 96-well plate with 100 µL DMEM. When 80% cells confluence was observed, the growth media were replaced with equal volume of test samples (ATC solution or ATC emulsion) with various concentrations for 24 h incubation. Then, the media were refreshed and were incubated with 20 µL MTT (0.5 mg/mL in PBS) for another 4 h. Finally, the culture medium was replaced by 100 µL DMSO, and the absorbance was measured at 490 nm with a microplate reader (BioRad, USA). 2.9. Caco-2 Cellular Uptake Studies. To evaluate the uptake efficiency of ATC submicron emulsion, Caco-2 cells were plated in 12 well plates at an appropriate dilution and allowed to attach for 24 h. Free ATC (10 µg/mL, DMEM solution) and ATC submicron emulsion (10 µg/mL, DMEM solution) samples were added and incubated for 12 h at 37 °C. At presetting time point (1, 2, 4, 6 and 12h), the test samples was discarded and cells was washed twice with PBS. Then the Caco-2 cells were split using 100 µL cell lysis buffer and mixed with 900 µL methanol under



votex for 1 min. The resulting solution was centrifuged at 10000 rpm for 10 min, and upper solution was directly collected for HPLC analysis. To evaluate the uptake behavior of ATC submicron emulsion, Caco-2 cells were plated in Laboratory-Tek chambered cover glasses. DiD labeled submicron emulsion sample was added and incubated for 12 h at 37 °C. At presetting time point (3, 6 and 12h), the distribution of submicron emulsion inside cells was analyzed. The cells was washed with PBS and fixed with 0.5% paraformaldehyde PBS solution for 10 min. Cell nuclei were stained using a 300 nM DAPI solution for 10 min, and confocal images were acquired on a Zeiss LSM 510 NLS confocal system (Carl Zeiss, NJ). To investigate the uptake route of ATC submicron emulsion, various test conditions and different inhibitors were used to conduct the experiment. Caco-2 cells were treatment with emulsion samples at 4 °C and 37 °C for 6 h to study the impact of temperature on cellular uptake. To study the effect of inhibitors on submicron emulsion uptake, the cells were treated with sodium azide (0.1% w/v) for 1 h, chlorpromazine (10 µg/mL) for 30 min, sucrose (450 mM) for 1 h, ammonium chloride (50 mM) for 30 min or genistein (20 µg/mL) for 1 h at 37 °C with 5% CO2. Inhibitor solutions were removed and all pretreated cells were washed twice with 1 mL PBS. Only fresh medium and ATC submicron emulsions were added and cultured for 6 h. The Caco-2 cells were collected and the ATC absorption amount was calculated by HPLC method. 2.10. In Situ Single Pass Intestinal Perfusion (SPIP) of ATC Submicron Emulsion. This SPIP model was employed to assess ATC flux across rat different intestinal and colon segments.26 Briefly, experimental rats were injected sodium pentobarbital (30 mg/kg) and fixed on the surgery table using infrared light to maintain body temperature. The selecting intestinal (the duodenum, jejunum,


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ileum) and colon segments were first washed with saline solution and equilibrated with Krebs-Ringer’s (K-R) buffer (0.35 g of KCl, 7.8 g of NaCl, 1.37 g of NaHCO3, 0.22 g of NaH2PO4, 0.02 g of MgCl, and 1.48 g of glucose in 1000 mL of double distilled water) at a flow rate of 0.5 mL/min for 30 min. Then different intestinal segments were treated with K-R buffer containing 20 µg/mL phenol red and 20 µg/mL ATC submicron emulsion or free ATC. Perfusate flow rate was maintained at 0.2 mL/min. The length and width of the intestinal segments were precisely measured at the end of the experiment. The collected samples were centrifuged at 10000 rpm for 5 min, and the supernatant was diluted with mobile phase for analysis by HPLC method described above. The phenol red content was determined using a UV-vis detector at 558 nm. The apparent permeability coefficients (Papp) and absorption rate constant (Ka) were calculated by the following equations:

K a = ( lnX 0 - lnX t ) / t X t = ( CPR −in / CPR −out ⋅V ) ⋅ Ct Papp =Q ⋅ ln(

X0 ) / 2πrl Xt

Where X0 was the initial amount of ATC in perfusate solution, Xt was the residual amount of ATC in perfusate solution at time of t, CPR-in was the initial concentration of phenol red in perfusate solution, CPR-out was the concentration of phenol red in perfusate solution at time of t, V was the initial volume of perfusate solution, Q was the flow rate, r and l were the radius and length of the intestinal segment. 2.11. Pharmacokinetics Study of ATC Submicron Emulsion after Oral Administration. SD rats were fed a standard diet and made to fast for 24 h prior to experiment. The rats were randomly divided into two groups (Lipitor group and ATC submicron emulsion group). For oral



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administration, two test groups received Lipitor suspension (suspended in 5% CMC-Na) and ATC submicron emulsion at a dose of 8 mg/kg. At each predetermined time point (0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12 and 20 h), blood samples were collected and centrifuged at 4000 rpm for 10 min to separate serum. To prepare analysis sample, 180 µL serum, 20 µL ethylparaben (3 µg/mL, as internal standard) and 1800 µL methanol were added into a tube, and this mixture was vortexed for 3 min. 200 µL supernatant solution was collected after centrifugation at 8000 rpm for 10 min. The solvent was evaporated under N2 atmosphere at 40 °C, and redissolved with 100 µL mobile phase buffer for next analysis. ATC concentration in analysis samples were determined by HPLC apparatus with C18 chromatographic column (5 µm). The mobile phase was ammonium acetate buffer (pH4.5) and acetonitrile (60:40), and the column temperature was maintained 40 °C. The flow rate was 1.1 mL/min. Detection wavelength was 244 nm. 2.12. In vivo Therapeutic Efficacy of ATC-submicron Emulsion. In

vivo

anti-hyperlipidemia

efficacy was performed based on previous literature.27 Briefly, experimental SD rats were randomly divided into five groups (5 rats per group) including normal diet group (negative control), high fat diet group (positive control), blank emulsion group (blank control), Lipitor treatment group and ATC emulsion treatment group. The test cycle was eight weeks. For normal diet group, high fat diet group, blank emulsion group, rats were fed with normal diet, high fat diet (65% normal diet, 10% lard, 1% cholesterol, 10% egg-yolk powder, 10% sugar and 0.5% bile salt) and mixture of 90% normal diet and 10% blank emulsion respectively. For Lipitor treatment group and ATC emulsion treatment group, rats were fed with high fat diet throughout the test cycle. At the beginning of fourth week, rats were treated with ATC formulations (Lipitor, 3mg/kg/day or


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ATC emulsion, 1.5 mg/kg/day) and stopped at the end of sixth week. Blood samples were collected at the end of 2, 4, 6 and 8 weeks, and various biochemical indexes (total cholesterol, serum triglyceride, LDL-cholesterol and HDL-cholesterol) were tested to analyze the rat physiological variation. 2.13. Statistical Analysis. Data in this study were shown as mean ± standard deviation (SD). Statistical comparisons were calculated by using Student’s t test, and p < 0.05 was taken to indicate statistical difference.

3. RESULTS AND DISCUSSION 3.1. Characterization of ATC-phospholipid Complex. Previous studies have shown that the API-phospholipid complex can significantly increase the drug solubility and permeability,28 and also improve the drug stability in gastrointestinal (GI) tract and therapeutic efficacy in vivo.29 In this study, ATC was an insoluble drug both in aqueous and oily medium. To successfully prepare a drug loaded submicron emulsion, the drug solubility in the oil phase should be upgraded. Therefore, we developed a kind of atorvastatin calcium phospholipid complex system, and investigated its physicochemical properties. ATC-phospholipid complex was prepared using a simple solvent evaporation method and the optimal reaction parameters are determined by single factor test (Figure s1). To substantiate the association of ATC with phospholipids FT-IR, X-ray diffraction and DSC analysis were performed on various samples. Results showed that the characteristic absorption peaks of ATC sample (3669 cm-1, 3365 cm-1) and phospholipid sample (2852 cm-1, 2920 cm-1) could be clearly observed in FT-IR spectrum. However these peaks shifted in the spectrum of phospholipid complex sample and no new absorption peaks were observed suggesting that there was only weak interaction



between ATC and phospholipid. (Figure s3). Based on X-ray diffraction patterns, ATC exhibited sharp crystalline peaks at 2θ=16.8°, 19.2° and 21.4° which showed a typical characteristic of molecule crystalline behavior. Compared with physical mixture, the crystalline peaks of ATC in phospholipid complex sample disappeared and replaced by a wide diffraction peak from 15 ° to 25 ° indicated that ATC was no longer crystalline but existed as amorphous structure (Figure s4). The DSC (Figure s2) and UV absorption data (Figure s5) also well demonstrated the interaction between ATC and phospholipids and formation of ATC phospholipid complex. The solubility of free ATC and ATC-phospholipid complex in various medium was investigated and the results were shown in Table 1. It can be seen that the solubility of ATC-phospholipid complex in different pH buffer (1.2, 4.5 and 6.8) saline increased dramatically and much higher than that of free ATC. The solubility increased by 130-fold and 30-fold when the pH value was 4.5 and 6.8, which means that the drug absorption may increase due to higher solubility in intestinal fluids within the pH range of 5 to 7. Compared with free ATC, the oil/water partition coefficient (logP) of ATC-phospholipid complex in different buffer saline also significantly increased (Figure 1), and therefore improved drug lipo-solubility in soybean oil by about 120-fold suggesting its good permeability to pass through intestinal epithelial cells. 3.2. Preparation and Characterization of ATC Submicron Emulsion. Considering commercial emulsion product and characteristics of ATC loaded submicron emulsion, in this study, soybean oil was selected as oil phase, lecithin as the emulsifier, tween-80 and poloxamer F-68 as co-emulsifier, and sodium oleate as surface charge regulator. To optimize the formula of the drug loaded submicron emulsion, an L16 (45) orthogonal design experiment was conducted with the influence factors described above, and the particle size, PDI and stability constant (Ke) were used as the


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index to evaluate the formulations (Table s1-s2). According to Table 2, R value of factor E was highest indicating that oil phase amount was the most important factor. Poloxamer F-68 and sodium oleate had less significant effects on the emulsion stability, and lecithin was similar in importance to tween-80. Based on the R values, factors that influenced the formation of drug loaded submicron emulsion could be ranked by importance of E > D > B > C > A, and the ATC submicron emulsion optimal formula was A4B2C4D3E1. Droplet size, size distribution and surface zeta potential of ATC submicron emulsion were analyzed by laser light scanning instrument. The drug loaded emulsion had a mean particle size of 122.7 nm with narrow distribution and small PDI (Figure 2B). TEM images showed that the submicron emulsion particle was spherical and about 150 nm in diameter (Figure 2A), which was consistent with the particle size test results. When the percentage of sodium oleate increased to 0.05%, the value of zeta potential decreased to -28.1mv which was useful to maintain the emulsion stability due to higher electrostatic repulsion forces between dispersed particles. 3.3. In vitro Release of ATC from Submicron Emulsion. Dialysis method was performed to compare the in vitro dissolution profiles of free ATC and ATC submicron emulsion in pH 6.8 PBS, and the results were presented in Figure 3. As can be seen, the free ATC showed the fast release pattern and almost 100% drug was released within 4 h. On the contrary, drug loaded submicron emulsion showed a prolonged release behavior and only 31.7% of ATC could be detected in dissolution medium. In addition, sustained release of ATC from submicron emulsion could be maintained up to 24h. Different mathematical models including first order, Higuchi and Ritger-Peppas models were also used to describe the dissolution curve (Table s3). The release behavior of ATC from submicron emulsion was best fitted with Ritger-Peppas equation with



highest linearity (R=0.977) indicating that the drug release mechanism was the combination of diffusion and corrosion. 3.4. Stability Study. As non-homogeneous dispersion system, particle size increase or drug crystal precipitation and chemical degradation may occur during preparation process or storage to affect the drug safety and therapeutic efficacy. In order to verify the stability of ATC submicron emulsion, prepared samples was subjected to a long term stability tests at 25°C in 6 months. As shown in Table 3, a slight increase of mean particle size form 122.7 nm to 155.5 nm can be observed, which caused by droplets aggregation in submicron emulsion. The absolute value of surface charge was still above 25 mv in test period time indicating that higher repulsion forces is useful to maintain the emulsion stability. Some earlier studies reported that phospholipids hydrolysis can release free fatty acid and induce the pH reduction in aqueous phase. Similar results also can be found in this research that pH value decreased about 0.3 units. Significant drug degradation or entrapment efficiency changes can not be found. ATC content and drug loading efficiency were 99% and 97.8% respectively. Based on above results, it can be concluded that ATC submicron emulsion was stable within 6 months. 3.5. In vitro Cytotoxicity of ATC Submicron Emulsion. Standard MTT assay was performed to determine the cytotoxicity of ATC submicron emulsion using Caco-2 cell line and the IC50 was calculated to compare the biocompatibility of free ATC and ATC loaded emulsion. Based on Figure 4, cytotoxicity of both free ATC and ATC submicron emulsion showed a concentration dependent manner. The IC50 of free ATC and ATC emulsion was 101.7 µg/mL and 86.8 µg/mL. It was interesting to note that emulsion formulation showed a relatively higher cytotoxicity than free ATC when drug concentration exceed 60 µg/mL. Previous studies reported that ATC was a


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P-glycoprotein (P-gp) substrate, and P-gp was highly expressed in Caco-2 cell line.30 When Caco-2 cells were treated with free ATC, P-gp as efflux pumps can expulse drug out of cells and resulted in cytotoxicity reduction. To prove this hypothesis, cells were co-incubated with ATC (80 µg/mL) and verapamil (20 mM), the widely used P-gp inhibitor, to observe the change tendency of cytotoxicity. The results showed that the cytotoxicity of free ATC in verapamil test group was higher and significantly different from the normal test group suggesting that when P-gp on cell membrane was inhibited, the efflux effect of free drug was suppressed. However, the drug loaded submicron emulsion was not affected by P-gp, and therefore exhibited higher cytotoxicity compared with the free drug. 3.6. Cellular Uptake Studies of ATC Submicron Emulsion. Cellular uptake of ATC submicron emulsion was investigated to obtain further insights. Caco-2 cells were treated with ATC or ATC loaded emulsion samples for presetting time, and drug content absorbed was quantitatively analyzed by HPLC method. As can be seen in Figure 5, for both two test samples, ATC absorption was positively correlated with incubation time. Free ATC showed a quick uptake response by Caco-2 cells in the first 2 h compared to ATC loaded emulsion. But after 2 h, ATC level inside cells for submicron emulsion group was higher than that for free drug group. The average uptake rate of free ATC and ATC loaded emulsion within 12 h was 1.9×10-3 µg/h and 4.3×10-3 µg/h. This result was consistent with previous cytotoxicity data that compared with free ATC Caco-2 cells will uptake more ATC submicron emulsion in the same incubation time. Confocal laser scanning microscopy (CLSM) was used to observe the location of submicron emulsion in Caco-2 cells at different time points (Figure 6). The cell nucleus was stained by DAPI to be blue spots, while emulsion emitted red fluorescence. With increasing incubation time, more



DiD labeled emulsion particles were continuously entered cells and could be observed around cell nucleus, indicating that submicron emulsion can efficiently transfer into cells. Endocytosis can internalize macromolecules or particles into cell and target them to specific organelles. It plays an important role for nano-size particle transportation. Earlier report indicated that there are four types of endocytosis including phagocytosis, clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis.31 Here we test the endocytosis route of ATC-emulsion using various pathway inhibitors and different conditions (Figure 7). Results showed that both temperature reduction and metabolic inhibitor could decrease the nanoparticles uptake proving that the absorption of ATC emulsion was an active transportation process. Hypertonic solution could inhibit fluid-phase endocytosis of nanoparticle.32 When cells incubated with sucrose solution, emulsion particle uptake decreased to 48.7%. The existence of chlorpromazine as CME inhibitor and genistein as CvME inhibitor could both suppressed the ATC emulsion uptake to 38.1% and 62.3% respectively. All above result suggested that endocytosis plays a significant role for ATC submicron emulsion absorption. 3.7. In Situ Intestinal Perfusion of ATC Submicron Emulsion in Rats. Good permeability was essential for poorly soluble drugs absorption through gastrointestinal tract. Here, the oral absorption of ATC submicron emulsion in different rat intestinal segments was investigated using the in situ single-pass perfusion method. Firstly, the physical stability of ATC emulsion in K-R solution was determined, and the data showed that within test time period particle size increasing or drug content decreasing could not be observed (Figure s6). The results of Papp and Ka obtained in different intestinal segments are shown in Figure 8. As can be seen, there was no significant difference for Ka and Papp of free ATC group in all test intestinal segments. However, compared


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with free ATC, ATC emulsion exhibited markedly higher Papp and Ka value in all test intestinal segments (p < 0.05). For ATC emulsion, the absorption rate constant in duodenum, jejunum, ileum and colon segment compared with free ATC increased 1.67, 1.73, 1.44 and 2.88-fold and the apparent permeability coefficients increased 1.76, 1.82, 1.66 and 2.45-fold. It was important to note that in all four test intestinal segments, the Papp value and Ka value of ATC emulsion in the colon site are significantly higher than those of other intestinal segments which mean that ATC submicron emulsion displayed intestinal segment-dependent absorption characteristic. Lymphatic transportation played an important role for nanoparticles absorption33-34 and we also investigate this process to evaluate the transfer behavior from various intestinal segments. Compared with free ATC, ATC submicron emulsion markedly enhanced the drug transfer amount into lymph fluid especially in colon. After 2h no significant drug concentration variation could be observed for ATC solution, but higher drug concentration could be detected in lymph fluid from all test intestinal segments for ATC submicron emulsion group (Figure s7). Drug intestinal transportation amount to lymph fluid could be ranked as colon > ileum > jejunum ≈ duodenum. 3.8. Pharmacokinetics of ATC Submicron Emulsion in Rats. Pharmacokinetics and in vivo bioavailability of marketed product (Lipitor) and ATC submicron emulsion after oral administration at a dose of 8 mg/kg were studied. The drug concentration-time curves are shown in Figure 9, and the pharmacokinetic parameters are summarized in Table 4. As can be seen, the ATC concentration from submicron emulsion system was significantly higher than that from ATC commercial product. The plasma peak concentration of ATC emulsion was 0.924 mg/L and was over 1.73-fold than that of Lipitor (0.535 mg/L). Moreover, the AUC of ATC emulsion was 4.033 mg/L·h and increased 2.58-fold compared with Lipitor (1.561 mg/L·h), suggesting that submicron



emulsion delivery system was the effective approach for improving the intestinal absorption and bioavailability. 3.9. In vivo Efficacy Evaluation. As a competitive inhibitor of HMG-CoA reductase, ATC is a widely used drug for clinical treatment of hyperlipidemia. In this study, compared with commercial product (Lipitor), we used total cholesterol, triglyceride, LDL-cholesterol and HDL-cholesterol as index to examine the anti-hyperlipidemia efficacy of ATC submicron emulsion in vivo. Firstly a hyperlipidemia animal model was established by feeding the high fat diet to SD rats. The results showed that the total cholesterol and triglyceride levels in rats increased significantly after feeding high fat diet for four weeks. The plasma cholesterol concentration reached to 75.7 mg/dL, which was 2.5 times of normal diet fed group (Figure 10A). The plasma triglyceride was 87.7 mg/dL and was 2.29 times of normal diet fed group (Figure 10B). When two ATC preparations (Lipitor and ATC-emulsion) were given to treat hyperlipidemia, plasma cholesterol level significantly declined. Lipitor treated group decreased to 67.3 mg/dL and ATC-emulsion treated group decreased to 53.7 mg/dL (p < 0.05). Plasma triglyceride level also showed varying degrees of decline trend and fell to 82.7 mg/dL and 65.8 mg/dL (p < 0.05) for Lipitor treated group and ATC-emulsion treated group respectively. When drug administration stopped from the end of sixth week to the end of eighth week, the plasma cholesterol and triglyceride level in two treatment group all increased. But the cholesterol and triglyceride concentration for ATC-emulsion treatment group were still lower than that of Lipitor treatment group which means that ATC-emulsion could effectively control the plasma cholesterol and triglyceride level. It was important to note that ATC-emulsion treated group was given a dose of only 1.5 mg/kg/day which means that compared with Lipitor, ATC submicron emulsion has good


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absorption feature in vivo and therefore could maintain plasma lipids level under the condition of low administration dose. Comparison of plasma LDL-cholesterol and HDL-cholesterol, it was clearly found that the plasma LDL-cholesterol and HDL-cholesterol concentration could keep stable when rats were given regular diet or regular diet with blank submicron emulsion. However, when rats were fed with high fat diet, LDL-cholesterol level in plasma showed a significant upward trend and HDL-cholesterol level showed significant reduction. During four weeks, LDL-cholesterol increased from 10.1 to 42.1 mg/dL (Figure 10C), while HDL-cholesterol decreased from 18.9 to 11.1 mg/dL (Figure 10D). When rats were given ATC formulations (Lipitor or ATC-emulsion) for treatment of hyperlipidemia, plasma concentrations of LDL-cholesterol rapidly decreased to 28.9 and 22.1 mg/dL and HDL-cholesterol levels increased to 14.3 mg/dL and 15.1 mg/dL respectively. According to above data, it was clear to conclude the fact that ATC submicron emulsion was more efficient for anti-hyperlipidemia therapy in vivo.

4. CONCLUSION In this paper, a novel type of atorvastatin phospholipid complex loaded submicron emulsion was prepared. This combined drug carrier system exhibited numerous benefits in terms of small particle size, uniform size distribution, higher drug loading efficiency, good physicochemical stability and sustained release profile. Cell viability and uptake studies showed that atorvastatin submicron emulsion had good cellular transfer behavior compared with free drug. In situ perfusion results indicated that drug loaded submicron emulsion system significantly increased the apparent permeability coefficients and absorption rate constant and therefore resulted in good drug absorption and higher drug plasma concentration after oral administration. Experiment of



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hyperlipidemia treatment in SD rats displayed that atorvastatin submicron emulsion could effectively regulate the plasma cholesterol, triglyceride, LDL-cholesterol and HDL-cholesterol level which demonstrated that the combination of phospholipid complex and submicron emulsion technology was an efficient way to enhance the bioavailability and therapeutic efficacy for water-insoluble drugs.

ACKNOWLEDGMENTS This

work

was

financially

supported

by

Guangdong

Natural

Science

Foundation

(2014A030310362), Science and Technology Program of Guangzhou (201508010036) and the IAR Collaborative Innovation Project of Guangzhou (201605131249066).

SUPPORTING INFORMATION Optimization of reaction parameters for ATC-phospholipid complex (Figure s1). DSC thermograms (Figure s2), FT-IR spectra (Figure s3) and X-ray diffraction patterns (Figure s4) of ATC, phospholipid, physical mixture and ATC-phospholipid complex. UV-vis absorbance feature of ATC-phospholipid complex (Figure s5). The stability of ATC loaded emulsion in K-R solution (Figure s6). ATC concentration in lymph fluid transported from different intestinal segments (Figure s7). Factors and levels of L16 (45) orthogonal test (Table s1). Evaluation index of L16 (45) orthogonal design (Table s2). Mathematical modeling on drug release form ATC loaded emulsion (Table s3).

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Figure legends Figure 1. Oil/water partition coefficient (Log P) of ATC and ATC phospholipid complex in water and buffer saline. Data are shown as mean ± SD, n=3. ***, p < 0.001 versus ATC group. Figure 2. Characterization of ATC loaded submicron emulsion. (A) Transmission electron microscopy (TEM) image. (B) Particle size distribution. Figure 3. In vitro dissolution of ATC and ATC-emulsion using dialysis method in pH 6.8 PBS. Data are shown as mean ± SD, n=3. Figure 4. (A) Cytotoxicity of blank emulsion, ATC and ATC submicron emulsion on Caco-2 cell treated for 24 h. (B) The effect of P-gp inhibitor verapamil on cellular uptake of ATC or ATC submicron emulsion. Data are shown as mean ± SD, n=3. *, p < 0.05 versus normal group. Figure 5. Quantitative analysis of ATC absorption by Caco-2 cells after incubation for different time. Data are shown as mean ± SD, n=3. *, p < 0.05 versus ATC group. Figure 6. CLMS images of DiD-labeled submicron emulsion in Caco-2 cells after incubation for 3 h, 6 h and 12 h. (nuclei were stained by DAPI, Bar = 20 µm). Figure 7. Endocytosis pathways study of ATC submicron emulsion using different test conditions or incubating with various inhibitors. Data are shown as mean ± SD, n = 3. *, p < 0.05; **, p



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Combination of Phospholipid Complex and Submicron Emulsion

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