Improved Transport and Absorption through Gastrointestinal Tract by

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Improved Transport and Absorption through Gastrointestinal Tract by PEGylated Solid Lipid Nanoparticles Hong Yuan,* Chun-Yan Chen, Gui-hong Chai, Yong-Zhong Du, and Fu-Qiang Hu* College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, P. R. China ABSTRACT: The aim of the present study was to evaluate the potential of PEGylated solid lipid nanoparticle (pSLN) as mucus penetrating particles (MPP) for oral delivery across gastrointestinal mucus. The SLN was prepared by an aqueous solvent diffusion method, subsequently modified with PEG2000−stearic acid (PEG2000−SA) as hydrophilic groups. Surface properties, cytotoxicity, cellular uptake, and transport across Caco-2/HT29 coculture cell monolayers, intestinal absorption, and pharmacokinetics of pSLN were studied compared with that of SLN. Quantitative cellular uptake showed that the internalization of SLN and pSLN was an active transfer process, which would be restrained by several inhibitors of cell activity. Compared with SLN, the permeation ability of pSLN decreased through Caco-2 cell monolayer while it increased through a mucus-secreting Caco-2/HT29 coculture cell monolayer, which indicated that the mucus layer has a significant impact on determining the efficiency of oral nanoformulations. In addition to increasing permeation ability, the stability of the nanoparticles in simulated intestinal fluids was also increased by the PEGylation. Moreover, in vitro everted gut sac technique and the ligated intestinal loops model in vivo also demonstrated that pSLN can rapidly penetrate mucus secretions, whereas the SLN were strongly trapped by highly viscoelastic mucus barriers. The pharmacokinetic studies presented that pSLN exhibited improved absorption efficiency and prolonged blood circulation times with a 1.99-fold higher relative bioavailability compared with SLN. In conclusion, PEGylated solid lipid nanoparticles had advantages in enhancing the bioavailability of oral administration. KEYWORDS: Caco-2 cells, HT29 cells, solid lipid nanoparticles, polyethylene glycol, oral drug delivery

1. INTRODUCTION Solid lipid nanoparticle (SLN),1−3 a colloidal carrier for controlled drug delivery system with a mean diameter ranging from 50 to 1000 nm, has attracted increasing attention in recent years. It has advantages such as good tolerability, low drug leakiness, high oral bioavailability, large-scale production by high pressure homogenization, and less acute and chronic toxicity. Nowadays, oral delivery of SLN is considered as the preferred administration route due to the distribution on a larger surface area, better physical stability, more protection of incorporated drugs from degradation, more constant plasma level, controlled drug release, smaller decrease in bioavailability compared to single-unit systems, and site-specific targeting. An exciting example4 is that the cyclosporin A-loaded SLN showed a sufficiently high oral bioavailability; they kept the plasma drug concentration within the therapeutic window for the Sandimmun Optoral/Neoral microemulsion formulation. Moreover, they could avoid nephrotoxicity when the drug concentration was above 1000 ng/mL due to sustained release. Hence, SLN has been usually used as a promising vehicle for oral delivery of drugs. However, one of the greatest challenges in developing an efficient nanocarrier for oral administration is to overcome the absorption barrier of intestinal mucosa, which consists of intestinal epithelial cells as well as a mucus layer.5 © 2013 American Chemical Society

For example, the routes of oral administration may suffer from the rapid clearing of the drug by the highly viscoelastic and adhesive mucous layer and limited paracellular permeability through the epithelial lining of the gastrointestinal tract.6 Therefore, the most foreign particles can be rapidly trapped by gastrointestinal tracts and eliminated by different mechanisms of mucus clearance.7,8 Based on the aforementioned comments, active research has focused on the preparation of nanocarrier, which could rapidly penetrate mucus secretions and achieve sustained drug delivery to mucosal tissues. Mucus-penetrating particles (MPP) can be actively engineered as a potential nanoparticle carrier by carefully tuning the surface properties and overcoming the low oral bioavailability of drugs.9−11 The search for the most suitable characteristics of MPP led us to investigate the potential of PEGylated SLN as a nanocarrier for the oral drug delivery system. Poly(ethylene glycol) (PEG) is nontoxic, nonimmunogenic, nonantigenic, highly soluble in water, and FDA-approved and has been considered to negligibly interfere with the drug release.12 Drug Received: Revised: Accepted: Published: 1865

November 12, 2012 March 7, 2013 March 15, 2013 March 15, 2013 dx.doi.org/10.1021/mp300649z | Mol. Pharmaceutics 2013, 10, 1865−1873

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2.2. Preparation of SLN and pSLN. SLN and pSLN were prepared by the solvent diffusion method in an aqueous system reported in our previous study.20 Briefly, a 60 mg mixture of monostearin and PEG2000−SA (the weight percent of PEG2000− SA in the mixture was 0%, 5%, 10%, 20%) were dissolved in 3 mL of ethanol in a water bath at 70 °C. DOX (3 mg, only for DOX loaded SLN and pSLN) and ODA-FITC (6 mg, only for ODA-FITC labeled SLN and pSLN) were dissolved in 3 mL of DMSO and 3 mL of ethanol, respectively. Then, the DOX solution (1 mg/mL) or ODA-FITC solution (2 mg/mL) was premixed with solid lipid solution containing PEG2000−SA at different ratios. The resultant organic solution was quickly dispersed into 60 mL of poloxamer 188 solution (0.1%, w/v) under mechanical stirring at 400 rpm for 5 min in 70 °C in water bath. The pre-emulsion (melted lipid droplet) was then cooled to room temperature. Finally, the obtained SLN or pSLN were concentrated by dialysis using a dialysis membrane (MWCO: 3.5 KDa, Spectrum Laboratories, Laguna Hills, CA) against 10% polyvinylpyrrolidone K30 solution for 48 h. The final SLN or pSLN was redispersed in poloxamer 188 solution (0.1%, w/v), and the concentration of SLN or pSLN was controlled as 10 mg/mL. The SLN containing 0%, 5%, 10%, and 20% PEG2000−SA was termed as SLN, pSLN-5%, pSLN10%, and pSLN-20%, respectively. 2.3. Characterization of SLN and pSLN. 2.3.1. Determination of Particle Size and Zeta Potential. The hydrodynamic diameters of SLN and pSLN suspension (1 mg/mL) in poloxamer 188 solution (0.1%, w/v) were determined with a Zetasizer analyzer (3000HS, Malvern Instruments Ltd., UK). The zeta potential of SLN and pSLN with the same concentration was detected in deionized water solution by the Zetasizer. 2.3.2. TEM Observation. The morphological examinations of the SLN and pSLN were performed by transmission electron microscopy (TEM) (JEOL JEM-1230, Japan). The samples were placed on copper grids with films and then stained with 2% (w/v) phosphotungstic acid for viewing by TEM. 2.3.3. Measurement of Drug Entrapment Efficiency and Drug Loading. The DOX content was measured by fluorescence spectrophotometer (F-2500, HITACHI Co., Japan). The excitation wavelength, emission wavelength and slit openings were set at 505, 565, and 5 nm, respectively. The SLN dispersion was destroyed by adding 100-fold DMSO in 80 °C water bath for 10 min. This solution was then cooled down to room temperature and centrifuged for 15 min at 20 000 rpm. The DOX content in DMSO (C1, μg/mL) was determined. The entrapment efficiency (EE, %) and drug loading (DL, %) of DOX in the SLN were then calculated from eqs 1 and 2, respectively.

delivery vehicles modified with low molecular weight (MW) PEG are capable of (a) sterically stabilizing particles and avoiding plasma protein binding, thereby reducing the elimination by the reticuloendothelial system and resulting in prolonging the half-life of drug in circulation; (b) reducing immunogenicity; (c) enhancing permeability and retention effect in tumor tissue.13−15 Moreover, it is also shown that low MW PEG and high surface coverage of PEG could increase the hydrophilic of SLN and minimize mucoadhesion by reducing hydrophobic or electrostatic interactions. As such, modification of nanoparticles with 2 kDa PEG provided rapid mucuspenetrating transport properties, while unmodified nanoparticles were strongly trapped by highly viscoelastic and adhesive gastrointestinal mucosa.16 Therefore, it is essential to search for the transport and absorption properties of PEGylated SLN in mucus gel of small intestine circumstances. In the present study, the pSLN was prepared to imitate mucus penetrating particles (MPP). The chemical conjugates of otcadecylamine and fluorescein isothiocyanate (ODA-FITC) and doxorubicin (DOX) were used as a fluorescence marker and a model drug, respectively. The main purpose of this study was to evaluate their efficiency as oral vehicles of hydrophobic drugs and investigate the influence of mucus on the penetrating effect. Two intestinal models were used for the absorption studies of pSLN. Mucus-secreting Caco-2/HT29 coculture cell monolayers model, which simulated the intestinal epithelium, were applied for the in vitro evaluations of penetrating effect and the influence of mucus gel. HT29 was used to provide the ability to examine the effect of mucus gel layer in coculture cell monolayers model contributed by the mucus-secreting properties.17,18 The in vitro everted gut sac technique is also frequently used to study the permeability and absorption kinetics of drugs, which enable the determination of drug content absorbed through the intestinal mucous membrane independently of other factors such as transit time.19 Finally, the pharmacokinetic studies of the pSLN were carried out in male SD rats. In all, we sought to test whether MPP based on PEG modification could enhance the bioavailability of oral administration.

2. EXPERIMENTAL SECTION 2.1. Materials. Monostearin and polyvinylpyrrolidone K30 were purchased from Chemical Reagent Co., Ltd. (Shanghai, China). Polyethylene glycol monostearate (PEG2000−SA, Mw = 2000) was supplied by Tci Development Co., Ltd. (Shanghai, China). Otcadecylamine (ODA) was purchased from Fluka, USA. Doxorubicin hydrochloride (DOX·HCl) was a gift from Hisun Pharm Co., Ltd. (China). Chlorpromazine, cytochalasin D, nystatin, filipin III, poly-D-lysine hydrobromide, 4′,6diamidino-2-phenylindole (DAPI), and fluorescein isothiocyanate (FITC) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Poloxamer 188 was purchased from Shenyang Jiqi Pharmaceutical Co., Ltd. (China). N-2Hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) was purchased from Sigma Saint Quentin Fallavier (France). The BCA protein assay kit was purchased from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Dulbecco’s modified Eagle’s medium (DMEM, high-glucose), fetal bovine serum (FBS), nonessential amino acids, 0.25% trypsinEDTA solution, and antibiotic-antimycotic were purchased from Gibco BRL (USA). All other chemicals were analytical or chromatographic grade.

EE =

C1 × V × 100% Wa

(1)

DL =

C1 × V × 100% W0 + (C1 × V )

(2)

where Wa and W0 denote the charged amount of DOX (mg) and weight of lipid (mg) added in the system, and V represents the total volume of DMSO solution (mL). 2.3.4. In Vitro Release. The test of drug release behaviors from SLN and pSLN were performed by the dialysis bag method. Phosphate buffer (PBS, pH 7.4) was used as dissolution medium. A sample of 1 mL of DOX loaded SLN 1866

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confluenced HT29 cells and treated for different times:21 (I) sodium azide (0.1% w/v) for 1 h, (II) 50 mM of ammonium chloride for 30 min, (III) 5 μg/mL Nystatin or 1 μg/mL filipin for 30 min, (IV) 30 μM of Cytochalasin D for 30 min, and (V) 10 μg/mL of chlorpromazine for 30 min. After the treatments, the cells were treated with fresh DMEM containing SLN/DOX and pSLN-10%/DOX (10 μg/mL of DOX equivalent) for further 4 h. The cellular uptake percentage of DOX was calculated from the following equation:

and pSLN (0.5 mg/mL of DOX equivalent) was poured into the dialysis bag (MWCO: 3.5 KDa) and placed in a plastic tube containing 15 mL of dissolution media. The plastic tube was placed into a thermostatic shaker (HZQ-C; Haerbin Dongming Medical Instrument Factory, Haerbin, China) at 37 °C at a rate of 60 times per min. At predetermined time intervals, the dissolution medium in the plastic tube was completely removed for analysis before the addition of fresh dialysis medium. The drug concentration was determined by fluorescence spectrophotometer. 2.3.5. Stability of SLN and pSLN in Simulated Intestinal Fluids. The concentrated SLN and pSLN-10% were resuspended in 20 mL of intestinal fluids (USP33 pH 6.8, without pancreatin) to make the final concentration at 1 mg/mL. The mixture was shaken in a thermostatic shaker at the rate of 60 times per min under 37 °C. The periodic samples were then taken at 0, 2, 4, and 8 h after the test. The average particle size was determined by Zetasizer. 2.4. Cell Culture and Evaluation. 2.4.1. Caco-2 and HT29 Cell Culture. Caco-2 and HT29 cells, both obtained from Institute of Biochemistry and Cell Biology (Shanghai, China), were cultured in DMEM supplemented with 10% (v/v) FBS, penicillin (100 U/mL), streptomycin (100 U/mL), and 1% nonessential amino acids (NEAA) with the environmental condition maintained at 37 °C in an atmosphere of 5% CO2/ 95% O2 with 90% relative humidity. Caco-2 and HT29 cells were resuspended with 100:0, 90:10, 75:25, 50:50, and 0:100 ratios, seeded at a density of 6.0 × 105 cells/cm2 and cultured on polycarbonate filter membranes with a pore size of 0.4 μm and a surface area of 1.12 cm2 (Costar Transwell, Millipore Corp., Bedford, MA, USA). The culture medium was changed every other day in the initial two weeks and every day in the following week. The integrity of the cell monolayer was checked after seeding 21 days by measuring the transepithelial electrical resistance (TEER) values. The TEER value was measured by a Millicell-ERS volt-ohmmeter (Millipore Co., USA). The intrinsic resistance (Ω·cm2) of the system (insert alone) was subtracted from the total resistance (cell monolayer plus insert, Ω·cm2) to yield the monolayer resistance(Ω·cm2). 2.4.2. Cytotoxicity of SLN and pSLN. The measurements of cytoxicity of SLN and pSLN against Caco-2 cells were performed by MTT assay. Briefly, 1 × 104 cells/well were seeded in a 96-well plate (Nalge Nunc International, Naperville, IL, USA) and allowed to adhere for 24 hs. After being treated with serial concentrations of SLN and pSLN (50−500 μg/mL), cells were incubated for further 48 h, and then 20 μL of MTT (5 mg/mL) was added in each well for further 4 h. After removing the unreduced MTT and medium, 200 μL of DMSO were then added to each well to dissolve the formazan crystals with 20 min of shaking before absorption was measured at 570 nm in a micro plate reader (Bio-Rad, model 680, USA). 2.4.3. Cellular Uptake Investigation. For quantitative study, Caco-2 and HT29 cells were seeded at a density of 1 × 105 cells/mL into a 24-well culture plate (Nalge Nunc International, Naperville, IL, USA) and allowed to attach for 24 h until confluence. The medium was then removed and treated with fresh DMEM containing SLN/DOX and pSLN/DOX (10 μg/ mL of DOX equivalent) at 37 °C for 4 h. At certain time point, cells were washed twice with PBS and harvested. After repeated freezing and thawing, the cell lysate was centrifuged at 10 000 rpm for 5 min, and the protein content inside the cell was measured using the Micro BCA protein assay kit. For the effects of endocytosis inhibitors, the different inhibitor was added to

DOX uptake percentage(%) = C t/ C t0 × 100%

(3)

where Ct and Ct0 are the intracellular DOX concentration and initial DOX concentration corrected by intracellular protein concentration at t time and t0 time, respectively. 2.4.4. Permeation of DOX through Caco-2 and Caco-2/ HT29 Cell Monolayers. For the transport assay of SLN/DOX and pSLN/DOX across the coculture cell monolayer (100:0, 90:10, 75:25), the TEER of the monolayer was measured after the culture medium was replaced by transport medium HBSS solution (0.5 mL to apical side and 1.5 mL to basolateral side) and equilibrating for 15 min. 0.5 mL of SLN/DOX and pSLN/ DOX (5 μg/mL of DOX equivalent) HBSS solution was applied to the apical side followed by addition of 1.5 mL of HBSS solution to the basolateral side after the transport medium was discarded. At certain time intervals, solution in basolateral side was collected and rapidly replaced with equivalent fresh HBSS solution. The content of SLN/DOX and pSLN/DOX was detected by fluorescence spectrometer. Before and after the experiment was performed, the integrity of the monolayers was assessed by means of TEER measurements. The apparent permeability coefficient (Papp) was calculated from the measurement of the transfer rate of DOX across Caco-2 cells from upper to lower compartments of the transwell diffusion cells: Papp(cm/s) =

dQ A × C 0 × dt

(4)

where dQ is the total amount of permeated DOX (μg), A is the surface area of the porous membrane (A = 1.12 cm2), C0 is the initial DOX concentration in the upper compartment (5 μg/ mL), and dt is the time of experiment (s). 2.5. Intestinal Absorption of SLN and pSLN by in Vitro Everted Gut Sac. All of the animal studies were done according to the guidelines of the local Institutional Animal Ethical Care Committee (IAEC). Male SD rats (250 ± 20 g body weight) were fasted for 16−24 h. Water was allowed ad libitum. The rats were anesthetized with an intraperitoneal injection of 10% chloral hydras. The abdominal cavity was cut opened with a midline incision (2−3 cm) to isolate the desired segment. The excised intestinal segments were immediately flushed with ice-cold Krebs−Ringer (K-R) culture solution to clean the intestinal contents and remove the underlying mesenterium. The intestinal segments (10 cm) were tied at one end, everted with a smooth glass rod, and blotted dry. Then 2 mL of K-R culture solution was injected into the serosa compartment (i.e., a receiver compartment). At time equal to zero, the everted intestinal sac was suspended in the predetermined volume (40 mL) of mucosal solution containing the SLN/FITC or pSLN-10%/FITC (100 μg/mL). The experimental solution (i.e., the donor compartment) was continually aerated with 5% CO2 and 95% O2 and maintained 1867

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Table 1. Properties of Blank and DOX Loaded SLN and pSLNa blank SLN carriers SLN pSLN-5% pSLN-10% pSLN-20% a

size (nm) 280.2 270.7 192.1 178.5

± ± ± ±

0.23 0.84 1.59 0.76

DOX loaded SLN

ζ (mV) −24.5 −20.5 −20.3 −20.4

± ± ± ±

0.9 0.5 0.8 1.3

PI 0.34 0.32 0.32 0.30

± ± ± ±

size (nm) 0.03 0.01 0.02 0.02

229.9 160.1 154.0 152.7

± ± ± ±

24.9 11.2 8.3 8.6

ζ (mV) −21.8 −17.8 −16.8 −14.7

± ± ± ±

6.1 6.5 0.9 0.7

PI 0.29 0.28 0.27 0.26

± ± ± ±

0.01 0.02 0.01 0.02

EE%

DL%

76.17 71.82 71.32 70.33

3.63 3.42 3.40 3.35

PI: presents the polydispersity index of the particle size. ζ: zeta potential. Data represent the mean standard deviation (n = 3).

at 37 °C. Aliquots of 0.5 mL of serosal solution were taken out from the receiver compartment at different time intervals (0, 15, 30, 45, 60, 75, 90, 105, 120 min) and rapidly supplemented with equivalent fresh K-R culture solution. After sampling, the length and width of the intestinal segments were measured. Then uptake per unit area was calculated as follows: The absorption per unit area = The absorption of Dox (μg) in the sacs/The intestinal segments area (cm2). 2.6. Validation of the Everted Rat Gut Sacs by Ligated Intestinal Loops Model in Vivo. The in vivo uptake of SLN/ FITC and pSLN-10%/FITC were evaluated using the ligated intestinal loops model. Male SD rats were anesthetized with an intraperitoneal injection of 10% chloral hydras, and then 2 cm sections of jejunum from small intestinal loop were made and washed with K-R culture solution (37 °C). SLN/FITC and pSLN-10%/FITC K-R culture solution (100 μg/mL, 0.5 mL) with equal fluorescent intensity was injected into the loop and then ligated at both ends. After 2 h, the rats were sacrificed, and the section of each loop was removed, extensively washed using K-R culture solution. Subsequently, the small intestinal tissuesections were placed in tissue freezing medium for cryostat sections with controlled temperature (−15 ± 1 °C). Five micrometer-thick sections were prepared by Cryotome (CM1990, LEICA, Germany) and mounted on the polylysine-coated slide glasses. The tissue sections were visualized using inverted two-photon confocal microscopy (IX81-FV1000, Olympus, Japan). 2.7. Pharmacokinetic Studies. The pharmacokinetic study was performed using male SD rats (200 ± 10 g). The rats were fasted 16 h before experiment but had free access to water and were divided at random into four sets (three rats per set). The first group received DOX·HCl (12 mg/kg) by tail intravenous injection. The other groups received DOX (12 mg/ kg) in different formulations by oral gavage: (I) DOX·HCl, (II) SLN/DOX, (III) pSLN/DOX. After administration, 0.5 mL of blood samples were taken at different time intervals. Prior to analysis, an equal volume of acetonitrile was added to a 0.5 mL blood sample for deproteinization. The mixture was vortexed for 3 min and centrifuged at 8000 rpm for 10 min. Then, DOX concentrations in these samples were measured. 2.8. Statistical Analysis. Data was expressed as means of three or six separate experiments. Differences between groups were assessed using unpaired two-tailed Student’s t test, and a p-value 0.05), which might contribute to the near uniform distribution of the drug in lipid nanoparticles. Hence, the PEG would be considered to negligibly interfere with the drug release. After 72 h, the cumulative drug released percentage reached 80%. The corrosion of lipid materials might be one of the drug release mechanisms.24

3. RESULTS AND DISCUSSION 3.1. Properties of Blank and DOX Loaded SLN and pSLN. In the present study, pSLN was prepared by mixing different amounts of PEG2000−SA. As shown in Table 1, the size of SLN was larger than that of pSLN. By increasing the amount of PEG, the absolute value of zeta potential of SLN decreased from about 20 to 15 mV, which could contribute to 1868

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for DOX·HCl while the drug content for pSLN-20%/DOX was also 4.2-fold higher. Furthermore, the result of quantitative cellular uptake of SLN in HT29 cells was weaker than that in Caco-2, which may contribute to the secretion of mucin molecules by HT29. The secretion of mucin molecules results in a visible mucus layer that forms a diffusion barrier to the lipophilic marker molecule.27 Moreover, contrasting the results of SLN, the cellular uptake of pSLN-10%/DOX and pSLN/ DOX-20% in HT29 cells was higher than that in Caco-2. The property difference between two carriers was that pSLN-(10%, 20%) has a better hydrophilicity, which may be beneficial to mucus secretion penetration. 3.3.3. Effect of PEG on DOX Permeation Across Intestinal Cell Models. Figure 4B shows the TEER values of various Caco-2/HT29 proportions of monolayers. The TEER values of the Caco-2 monoculture (450 ± 5 Ω·cm2) were much higher than that of the HT29 monoculture (144 ± 8.5 Ω·cm2). In the cocultures, the TEER value decreases as the proportion of HT29 increases as other authors have already noted.28 In the 75:25 proportion, the TEER excess of 250 Ω·cm2, but in the other monolayers there are very considerable decreases. Therefore, we selected the proportion of 100:0, 90:10, and 75:25 for subsequent investigation. Three different ratios of Caco-2/HT29 monolayers were selected to study the transport efficiency of SLN and pSLN. With the mucus-secreting cells of HT29, cell monolayer model could provide a better simulation of natural conditions. For each model, the model drug was administered in 5 formulations, which included SLN/DOX, three different ratios of pSLN/DOX and DOX·HCl. Starting with the simplest intestinal model, Caco-2 cell monolayer was exposed to nanoparticles in the delivery forms described. As shown in Figure 4C, the largest amount of drug transport was facilitated by the SLN, and PEGylation appeared to decrease the permeation with different degrees, which may be caused by the increase of hydrophilicity. The permeation of SLN and pSLN through Caco-2/HT29 cell monolayer was also shown in Figure 4C, and the incorporation of mucus-secreting cells in the culture seemed to alter the transport situation. The apparent permeability coefficient (Papp) of DOX through Caco-2/HT29 (75:25) cell monolayer was increased by 5-fold when delivered by pSLN-10%, while just 3.9-fold by SLN. The results showed an increase in Papp of pSLN/DOX as the proportion of HT29 increases, especially for the pSLN/DOX-10%. However, the incorporation of HT29 only had little effect on SLN/DOX, and the Papp of which even experienced a little decrease at the proportion of 75:25. A vast amount of literature has shown that modifying nanoparticles with 2 kDa PEG provided rapid mucus-penetrating transport properties while unmodified

Figure 2. In vitro release profiles of DOX from different formulations.

3.3. Cell Evaluation. 3.3.1. Cytotoxicity of SLN. Figure 3A shows the cytotoxic effect of SLN and pSLN incubated with Caco-2 cells by MTT assay. From the graph, the 50% inhibition concentration (IC50) can be calculated at around 300 μg/mL. Moreover, the cytotoxicity was decreased with the increasing ratios of PEG. This result demonstrated that SLN and pSLN had low cytotoxicity, and the cell survival rate was approximately 100% when the concentration of SLN was 100 μg/mL which were used for further experiments. The cytoxicity of SLN was also confirmed by the transepithelial electrical resistance (TEER) profiles (Figure 3B). Duizer et al25 have demonstrated a good correlation between TEER values and the monolayer integrity. Only monolayers of which the tight junction (TJ) was close-up were used for the transport experiments and data processing. At the first time (at 2 h) of the TEER profile, the SLN system presented a parallel decrease of the TEER in comparison with the control. With the increase of time, the TEER value declined more obviously than the control. The change of the TEER profiles implicated that SLN was responsible for the modulation of the cell junction integrity only after 2 h. 3.3.2. Cell Uptake Investigation. Using Caco-2 and HT29 as model cells, the drug contents internalized into cells were quantitively evaluated after the cells were incubated with different formulations for 4 h. Since DOX base was insoluble in water and can hardly release in culture medium, so it could be used as model drug to indicate the uptake of the nanoparticles. As shown in Figure 4A, the uptake percentage in Caco-2 cells was about 11.92% for SLN/DOX while just 3.98% for pSLN20%/DOX, which experienced a sharp decline. The result demonstrated that the internalization ability of nanoparticles decreased by increasing the amount of PEG fraction on SLN surface, which could improve the hydrophilicity of SLN.26 Meanwhile, a weak decline of cellular uptake from SLN to pSLN-20% in HT29 cells was also found. The results of SLN/ DOX cellular uptake by HT29 were 5.2-fold higher than that

Figure 3. (A) Cytotoxic effect of SLN and pSLN incubated with Caco-2 cells. (B) TEER vs time profiles observed for SLN and the control (n = 3). 1869

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Figure 4. (A) Cellular uptake percentages of DOX from different formulations: (I) SLN/DOX; (II) pSLN-5%/DOX; (III) pSLN-10%/DOX; (IV) pSLN-20%/DOX; (V) DOX·HCl. Significant differences were against the data of SLN/DOX. (B) TEER values in the various proportions of Caco2/HT29 assayed. (C) Papp of DOX from different formulations in the various Caco-2/HT29 monolayers. (I) SLN/DOX; (II) pSLN-5%/DOX; (III) pSLN-10%/DOX; (IV) pSLN-20%/DOX; (V) DOX·HCl. (D) Effect of endocytotic inhibitors on cellular uptake against HT29 cell calculated considering the value of control groups as 100%: (I) sodium azide; (II) ammonium chloride; (III) filipin; (IV) cytochalasin D; (V) chlorpromazine; (VI) nystatin; all of the data represent the mean ± standard deviation (n = 3). Significant difference against control.*: P < 0.05, #: P < 0.01.

3.4. Influence of PEGylation of SLN on the Intestinal Absorption. To evaluate the stability of SLN and pSLN-10%, the nanoparticles were incubated in simulated intestinal fluids. As reflected by the data in Table 2, the extent of aggregation of

nanoparticles were strongly trapped by highly viscoelastic and adhesive gastrointestinal mucosa.16 Furthermore, the Papp of pSLN-20% was lower than pSLN-10%, indicating that appropriate PEGylation is important to convert nanoparticles from mucoadhesive to mucoinert. Specifically the degree of surface modification is required for rapid mucus penetrating particles, so we selected the pSLN-10% for subsequent investigation. 3.3.4. Effect of Endocytotic Inhibitors in HT29 Cells. Exposures of cells to different inhibitors were investigated to explicit the possible permeation route of SLN and pSLN. After treated with different types of inhibitors, both NPs had significantly reduced uptake (p < 0.05) (Figure 4D). Generally, cytochalasin D exhibited the most significant inhibition on the uptake of the SLN and pSLN, indicating that both nanoparticles were internalized into the cells through macropinocytosis and phagocytosis. The results also implied that macropinocytosis constitutes a major endocytotic process in HT29 cells. When treated with ammonium chloride and chlorpromazine, SLN had significantly reduced uptake compared to pSLN. Chlorpromazine was used as an inhibitor for clathrin mediated uptake, and ammonium chloride treatment increases the pH of acidic intracellular organelles to inhibit endocytosis. The similar situation occurred when treated with filipin and nystatin, which bind cholesterol and inhibit caveloae/lipid raft mediated endocytosis.29 Furthermore, the uptake of both NPs was significantly decreased with the addition of sodium azide, an active transport inhibitor. These results indicated that the active transport processes, involving adsorptive endocytosis, might play an important role on the uptake of both nanoparticles. The mechanism investigations also revealed that the different inhibitors exhibited a stronger effect on the SLN than pSLN, which implied that the paracellular pathway might be another possible route for the permeation of the PEG modified SLN delivery system.

Table 2. Size Changes of SLN and pSLN after Incubation with Simulated Intestinal Fluids for Different Time Intervals carriers

0h

2h

4h

SLN pSLN-10%

280.2 ± 0.23 150.23 ± 5.19

462.33 ± 0.1 155.2 ± 5.05

472.77 ± 1.59 156.03 ± 2.27

SLN was greater than that of pSLN after incubation for 4 h. Although remained at the nanometer level, the size of SLN was about 3-fold larger than that of pSLN, which indicates PEG can prevent SLN from agglomerating. The important interaction of PEGylation SLN could easily be understood by the recognized repellent effect of PEG modified surfaces.30 Taking aggregation into consideration, the stabilizing effect of the PEGylation in the GI tract could be regarded as an important quality for improving the absorption of pSLN. Moreover, the PEG chains located at the periphery of the nanoparticles will reduce the degradation of the lipid core by lipolysis. It could also be presumed that the protein-repellent properties of the PEGylated nanoparticles would stabilize the nanoparticles in the digestive fluids. The intestinal absorptive behavior of SLN and pSLN-10% was studied in the gut sac model from three different intestines for 2 h (Figure 5). The results showed that the apparent permeability coefficient (Papp) of SLN from three different intestines (duodenum, jejunum, ileum) was about 1.91 × 10−5, 2.09 × 10−5, and 2.75 × 10−5 cm/s. The correlation between the drug absorption and the incubation time was approximately linear when SLN added was less than 100 μg/mL. Furthermore, the absorptive profile of pSLN also exhibited time dependence while the Papp values were significantly enhanced (2.56 × 10−5, 1870

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Figure 5. Absorptive characteristics of SLN and pSLN-10% in the everted rat gut sac system from three different intestines: (A) duodenum, (B) jejunum, and (C) ileum. All of the data represent the mean ± standard deviation (n = 4).

Figure 6. Distribution of SLN and pSLN-10% in villi with different magnifications. Blue fluorescence refers to the nucleus of small intestinal cells, and green fluorescence refers to nanoparticles.

2.46 × 10−5, 3.56 × 10−5 cm/s). It is indicated that pSLN were able to diffuse nearly unimpeded through the mucus barriers, whereas SLN was trapped. The results also were closely related to permeation observed using Caco-2/HT29 (75:25) cell monolayer, which suggests that the presence of mucus-secreting cells is important in the vitro evaluation of delivery systems. 3.5. Validation of the Everted Rat Gut Sacs by Ligated Intestinal Loops Model in Vivo. To get a visualization of the permeation of SLN and pSLN-10% in villi, double fluorescent labeling with nucleus-labeled DAPI (blue) and FITC-labeled SLN (green) were used in the in vivo uptake study. After being injected into loop, nanoparticles may (1) remain in the intestinal lumen; (2) adhere to mucin fiber and trapped in mucus; (3) penetrate through the mucus layer for possible entry to the underlying epithelial.31 As shown in Figure 6, pSLN could permeate deeply into villi, suggesting the latent enhancing absorption ability of PEGylation. It could be interpreted that the efficient modification of PEG to the particle surface allowed nanoparticles to rapidly penetrate through highly viscoelastic small intestinal mucus by moving through openings between mucin mesh fibers.32 On the other hand, the green signals of SLN were only presented in the distal end and inside edge of villi, which might be strongly trapped by highly viscoelastic mucus barriers of the gastrointestinal (GI)

tract. Furthermore, SLN may be efficiently removed from mucosal surfaces as mucus is cleared. This implies that pSLN experiences the low viscosity interstitial fluid between mucus mesh elements and, thus, are able to rapidly penetrate the mucus secretion.33 3.6. In Vivo Pharmacokinetics of SLN/DOX. To further verify the above experiments, the pharmacological effects of SLN/DOX and pSLN-10%/DOX were evaluated on male SD rats. As illustrated in Figure 7, in the case of oral administration of DOX·HCl solution and SLN/DOX, the plasma DOX concentrations reached peak levels, Cmax, at 1 and 1.5 h, respectively. However, in the case of pSLN/DOX-10%, peak concentration was reached at 0.5 and 4 h, which not only indicated that pSLN was uptake quickly from the gastrointestinal tract into systematic circulation but also presented bimodal distribution. Furthermore, when DOX·HCl was administered by the intravenous route, the drug plasma concentration rapidly decreased with time. The mean residence time (MRT) was 1.34 h. The MRT of DOX delivered in different nanoparticle formulations was calculated to be 22.87 h for SLN and 33.02 h in case of pSLN, which is strongly higher than DOX·HCl by the i.v. route. As such, the plasma levels of DOX loaded pSLN was characterized by a slow decrease and remained high and constant in time. The performance of 1871

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internalized by active transport processes, and slight differences in uptake mechanisms exist. At last, the relative bioavailability of SLN was significantly improved by modification of PEG, suggesting that PEG was a potent mucus-penetrating agent and could be used as a promising nanoparticle modification for oral drug delivery. Overall, all of these observations underline the importance of the PEGylated SLN system to overcome the mucus barrier and deliver poorly absorbed drugs to mucosal surfaces and enhance the transport. However, still little is known about the mucosal drug delivery after oral administration in vivo. This work about transport properties of pSLN across mucus-secreting Caco-2/ HT29 cell monolayers and intestinal tissue would provide some useful suggestions to further research on oral administration.

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Figure 7. Plots of plasma DOX concentration against the time after oral administration with different dosage form of DOX. Indicated values are the means (SD) of three parallel animals.

Corresponding Author

*Tel. (Fax): 86-571-88208439. E-mail address: yuanhong70@ zju.edu.cn (H.Y.) and [email protected] (F.-Q.H.).

PEGylation to prolong the therapeutic plasma levels of drug is particularly interesting, which would provide a more consistent therapeutic effect. The key pharmacokinetic parameters were shown in Table 2. The results demonstrated that the relative bioavailability of pSLN/DOX-10% was 1.99-fold and 7.52-fold higher than nonmodified SLN and DOX·HCl, which contributed to the improved transport efficiency and prolonged blood circulation times by PEGylation. In a word, the vivo pharmacological and pharmacokinetic results coincided with the enhanced absorption and transport of SLN in the small intestinal model. It is demonstrated that significant fractions of pSLN were capable of penetrating highly viscoelastic mucus layers in the GI tract, while unmodified SLN was strongly trapped and efficiently cleared from the mucosal tissue.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We appreciate the financial support from the National Basic Research Program of China (973 Program) under Contract 2009CB930300 and Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents.

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Tmax (h) Cmax(μg/mL) AUC0‑t (μg/mL·h) MRT (h) Ke (h−1) relative bioavailability

DOX·HCl (iv)

DOX·HCl (oral)

0.08 9.93 7.91

1 1.46 4.71

1.42 2.08

5.25 0.32 100%

SLN/DOX (oral)

pSLN-10%/DOX (oral)

1.5 1.90 17.79

0.5, 4 2.26 35.40

22.87 0.2 377.71%

33.02 0.07 751.59%

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Table 3. Pharmacokinetic Parameters of SLN/DOX, pSLN10%/DOX, and DOX·HCla PK

AUTHOR INFORMATION

a PK: pharmacokinetics parameters. Tmax: the time when peak plasma DOX concentration was reached. C max : peak plasma DOX concentration. MRT: the mean residence time. Ke: the elimination rate constant of DOX in the systemic circulation. AUC0‑t: area under the plasma DOX concentration time curve.

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