Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1141 – 1151 nanomedjournal.com
G5 PAMAM dendrimer versus liposome: A comparison study on the in vitro transepithelial transport and in vivo oral absorption of simvastatin Rong Qi, PhD a, b,⁎, Heran Zhang, MS a, b, d , Lu Xu, BS a, b , Wenwen Shen, MS a, b , Cong Chen, BS a, b , Chao Wang, PhD a, b , Yini Cao, BS a, b , Yunan Wang, PhD a, b , Mallory A. van Dongen, PhD e , Bing He, PhD c , Siling Wang, PhD d , George Liu, PhD a, b , Mark M. Banaszak Holl, PhD e , Qiang Zhang, PhD c,⁎⁎ a
Peking University Institute of Cardiovascular Sciences, Peking University, Beijing, China b Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, China c School of Pharmaceutical Sciences, Peking University, Beijing, China d School of Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang, China e Department of Chemistry, University of Michigan, Ann Arbor, MI, USA Received 6 October 2014; accepted 14 February 2015
Abstract This study compared formulation effects of a dendrimer and a liposome preparation on the water solubility, transepithelial transport, and oral bioavailability of simvastatin (SMV). Amine-terminated G5 PAMAM dendrimer (G5-NH2) was chosen to form SMV/G5-NH2 molecular complexes, and SMV-liposomes were prepared by using a thin film dispersion method. The effects of these preparations on the transepithelial transport were investigated in vitro using Caco-2 cell monolayers. Results indicated that the solubility and transepithelial transport of SMV were significantly improved by both formulations. Pharmacokinetic studies in rats also revealed that both the SMV/G5NH2 molecular complexes and the SMV-liposomes significantly improved the oral bioavailability of SMV with the liposomes being more effective than the G5-NH2. The overall better oral absorption of SMV-liposomes as compared to SMV/G5-NH2 molecular complexes appeared to arise from better liposomal solubilization and encapsulation of SMV and more efficient intracellular SMV delivery. © 2015 Elsevier Inc. All rights reserved. Key words: Simvastatin; G5-NH2 PAMAM dendrimer; Liposome; Transepithelial transport; Oral bioavailability
Introduction Oral delivery is the most convenient approach for selfadministration of a wide variety of drugs; however, poor solubility in gastrointestinal fluid and/or low absorption across the intestinal epithelia can result in poor oral bioavailability and therefore unsatisfactory therapeutic effects. Drug delivery
The study was supported by National Natural Science Foundation of China (no. 81270368, 81360054). We have no conflicts of interest to declare. ⁎Correspondence to: R. Qi, 38 Xueyuan Road, Institute of Cardiovascular Sciences, Peking University Health Science Center, Peking University, Beijing 100191, China. ⁎⁎Correspondence to: Q. Zhang, Institute of Cardiovascular Sciences, Peking University Health Science Center, Peking University, Beijing, China. E-mail addresses: [email protected] (R. Qi), [email protected] (Q. Zhang). http://dx.doi.org/10.1016/j.nano.2015.02.011 1549-9634/© 2015 Elsevier Inc. All rights reserved.
systems including liposomes, 1 nanoparticles 2 and polymeric microspheres 3 show great promise to enhance oral absorption and bioavailability of a wide variety of drugs. Simvastatin (SMV), an inhibitor of 3-hydroxy-3-methyl coenzyme A (HMG-CoA) reductase, lowers plasma cholesterol concentration and is used clinically to treat hyperlipidemia and cardiovascular diseases. 4 SMV is poorly soluble in water and has irregular intestinal absorption, which make its oral bioavailability less than 5%. 5 Several methods including self-emulsifying drug delivery system (SEDDS), 6 cyclodextrin encapsulation, 7 nanostructured lipid carrier 8 and lipid nanoparticles, 9 have been used to improve the oral absorption, pharmacokinetics and tissue uptake of SMV. However, these formulations have some disadvantages. In the case of SEDDS, the formulation is thermodynamically unstable, whereas cyclodextrin in high concentrations (N 6 mM/L) induces hemolysis. 10
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Poly(amidoamine) (PAMAM) dendrimers have been studied on their drug and gene delivery carriers 11 because of their favorable properties including good water solubility, low polydispersity, abundant surface groups, and well defined structures. 12 Possible interaction between PAMAM dendrimer molecule and drug including hydrogen bond, electrostatic force, Van-der-Waals force, etc. leads to formation of a “molecular complex”, which is different from “coordination complex” in chemistry. Kulhari et al reported that G4 PAMAM dendrimers improved dissolution and stability of SMV, slowed down its release rate, 13 increased in vivo oral bioavailability, and improved the plasma cholesterol-lowering effect of SMV. 14 However, no in vitro transport and mechanism studies were carried out, and no comparison was made to liposomal formulation of SMV. Liposomes encapsulate hydrophobic drugs within their lipid bilayers to increase solubility and stability of the loaded drugs. 15 The liposomal bilayers are very similar to the structure of cell membranes, and liposomes can deliver encapsulated drug by fusing with cell membranes. 15 Liposomes containing SMV were proved to be more potent than free SMV in inhibiting various cell lines of monocytes/macrophages. One single systemic administration of liposomal SMV significantly suppressed neointimal formation in a rat model of restenosis. 16 In this study, we investigated and compared the ability of generation 5 (G5) PAMAM dendrimer molecular complexes and liposome formulation to provide information of transepithelial absorption and oral bioavailability of SMV. We have selected G5 PAMAM dendrimer because higher degree of branches and larger interior core of G5 material allow for greater surface modification and a higher drug-loading percentage as compared to lower generation dendrimers. This generation also maintains a less congested surface, retains arm flexibility, and is substantially less cytotoxic than higher generations. G5 PAMAM has been shown to be capable of promoting absorption and transepithelial transport of drugs by solubilizing the drugs in their internal void space, interacting with cell membranes, and delivering the loaded drugs into the cells. 11 Therefore, G5-NH2 was chosen in this study to form SMV/G5-NH2 molecular complexes. Previous studies have shown that G0 to G4 PAMAM dendrimers containing either cationic –NH 2 or anionic –COOH termination contribute to the opening of cell tight junctions (TJ) to enhance permeability of drugs. The zoning of occludin-1 protein, a key regulator of TJ assembly and function, becomes blurry and TJ structure is looser after treatment with G2 to G4 PAMAM dendrimers. 17-20 Furthermore, G3-NH 2 PAMAM dendrimers were found to bypass P-glycoprotein (P-gp), a drug efflux transporter, leading to a 200-fold increase of doxorubicin's bioavailability. 21,22 Besides, it was reported that endocytosis pathway of liposomes was dramatically affected by their particle size, 23 and their intracellular uptake was mainly related to clathrin-mediated endocytosis (CME). 24 Therefore, influence of P-gp, TJ and CME on the transepithelial transport of the SMV/G5-NH2 molecular complexes and the SMV-liposomes was probed in vitro in Caco-2 cells. Last of all, the efficacy of the two investigated SMV formulations on the oral bioavailability of SMV was evaluated in vivo in rats.
Materials and methods Materials SMV (≥ 98%) was purchased from Haizheng Company Ltd (Zhejiang, China). G5-NH2 dendrimer was purchased from Sigma-Aldrich, purified firstly by dialyzing (cut off: 10 KDa) against distilled water (8 media changes), and then centrifuged by Millipore tube (Amicon Ultra, cut off: 5 KDa). The purified G5-NH2 (Mn = 27700 Da by 1HNMR and GPC, PDI = 1.047) was used for the following study. The other G5 PAMAM dendrimers mentioned in the manuscript were all purified and characterized before use according to the same methods as described in G5-NH2 purification. Lecithin (injection class, PC-57 T) and cholesterol were purchased from Kewpie Corporation (Tokyo, Japan) and Damao Chemical Reagent Factory (Tianjin, China), respectively. Caco-2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cyclosporine A (CsA) was purchased from Taishan Pharmaceutical Company Ltd (Guangdong, China). BCA Protein Quantitation Kit was obtained from Thermo Scientific (Waltham, Mass, USA). Rabbit Anti-Human Polyclonal Antibody of occludin-1 (H-27), occludin-1 small interfering RNA (siRNA), non-targeted siRNA and RNase-free water were purchased from Santa Cruz (Santa Cruz, CA, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Chlorpromazine (CPZ) hydrochloride and Cy5 were purchased from Sigma-Aldrich Chemical Company (Saint Louis, MO, USA). 1-Palmitoyl-2-{12-[(7nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero3-phosphoethanolamine (16:0-12:0 NBD PE, NBP) was purchased from Avanti Polar Lipids, Inc (Alabaster, AL, USA). 10% PEG (WM 5000)-conjugated G5-NH2 PAMAM dendrimers (G5-PEG) were synthesized according to a procedure described in our previous publication. 25 Alexa Fluor® 488 labeled G5-NH2 were synthesized in our laboratory according to the previously published procedures. 26 All the other reagents were provided from local suppliers, unless otherwise mentioned. Solubilization studies Aqueous solubility of SMV The apparent solubility of SMV in pH 6.8 phosphate buffer saline (PBS) was measured according to the Pharmacopoeia (China, Version 2010). Briefly, 3 mg SMV was added into 2 mL pH 6.8 PBS and shaken in an incubating shaker (Ronghua SHA-C, China) at 100 rpm and 37 °C. Samples (300 μL) were taken at 24, 36, 42, and 48 h and spun on a centrifuge (Eppendorf 5810R, Germany) at 15048 g for 20 min. The amount of SMV in the supernatant was measured using High Performance Liquid Chromatography (HPLC, Shimadzu LC-15C, Japan) at λ 238 nm with a shim-pack VP-ODS C18 reversed-phase column (150 mm × 4.6 mm, 5 μm), and acetonitrile/water (16/9) as a mobile phase. Effects of G5-NH2 on SMV solubility To assess the concentration-dependent solubilization effect of G5-NH2 on SMV, G5-NH2 at different concentrations of 11.3, 22.6, 45, 90, 200 mg/L (0.391, 0.782, 1.564, 3.128 and 6.256 μM) were prepared by diluting 5.63, 11.3, 22.5, 45.0
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and 112 μL of G5-NH2 stock solution (4 mg/mL) into 2 mL PBS, respectively. SMV (3 mg) was added in the resultant G5-NH2 solutions, respectively, and the saturated SMV solubility in the different concentrations of G5-NH2 was determined by HPLC following the same procedure as described above. Preparation and characterization of SMV/G5-NH2 molecular complexes and SMV-liposomes SMV/G5-NH2 molecular complexes SMV/G5-NH2 complexes were prepared by adding 3 mg SMV into 22.6 mg/L (782 nM) G5-NH2 in 2 mL PBS, dispersing using an ultrasonicator (KQ-100DE CNC ultrasonic washer, Kunshan Ultrasonic Instrument Company, China), and shaking at 100 rpm and 37 °C for 36 h. One milliliter of the mixture was centrifuged at 13800 g, and SMV concentration present in the supernatant was determined by HPLC according to the same procedure as described above. The loading percentage of SMV in the G5-NH2 formulation was calculated by a concentration ratio of SMV and G5-NH2. Size, zeta-potential and polydispersity index (PDI) of the SMV/G5-NH2 complexes were measured by Malvern Zetasizer Nano-ZS (Santa Barbara, CA, USA). SMV-liposomes SMV-liposomes were prepared by using a thin film dispersion method. 27 To prepare lipid films, soybean lecithin (225 mg), cholesterol (25 mg) and SMV (25 mg) were mixed in a ratio of 9:1:1 and dissolved in 3 mL chloroform. The solvent was slowly evaporated in a rotary evaporator at 37 °C for 30 min to achieve a lipid film, which was dried overnight under vacuum and at room temperature. Multilamellar vesicles (MLVs) were prepared by hydrating the lipid film with 6 mL PBS (pH 7.5), and the resultant suspensions were sonicated for 30 min using a probe sonicator to obtain small unilamellar vesicles (SUVs) of SMV-liposomes. Size, zeta-potential and PDI of the SMV-liposomes were measured by Malvern Zetasizer Nano-ZS mentioned above. Drug encapsulation efficiency (EE) in the SMV-liposomes was calculated by a formula as follows:
EEð%Þ ¼
Mtotal −Mfree Mtotal
drug
100%
Mtotal and Mfree drug are the concentrations of total and free SMV in the liposome system, respectively. The detail method was described in the supplemental information. The drug loading percentage in the complexes or the liposomes was calculated as follows: Drug loading% ¼ Mencapsulated
drug =Mcarrier
100%
Mencapsulated drug is the mass of SMV encapsulated in the complexes or liposomes, Mcarrier is the mass of G5-NH2 in the complex system or the lipids in the liposome system.
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Cell culture and cytotoxicity assay Caco-2 cell culture Caco-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, New York, CA, USA) supplemented with 1% non-essential amino acid, 1% L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum. Cells from passage numbers 25-35 were used for the following in vitro experiments. Caco-2 cell monolayers Caco-2 cell monolayers were obtained by seeding 0.5 mL of Caco-2 cells onto polycarbonate membrane filters (3.0 μm pore size, 1.12 cm 2 growth area) inside Transwell cell culture chambers (Corning Costar, Cambridge, MA) at a density of 4 × 10 5 cells/cm 2 and were cultured according to the procedure as described above. After 21 days, Caco-2 cell monolayers were confirmed by determining transepithelial electrical resistance (TEER) using a Millicell-ERS system (MilliCell Corporation, Billerica, MA, USA), and the cells whose TEER value were around 350–400 (Ω ∙ cm 2 ) were used for transepithelial transport studies. Cytotoxicity Caco-2 cells were seeded on 96-well plates at a density of 5 × 10 4 cells/mL in 200 μL DMEM medium per well, and were incubated at 37 °C in 5% CO2 until 80% confluent. The effects of SMV, SMV/G5-NH2 complexes, and SMV-liposomes on the viability of Caco-2 cells were studied by MTT assay. Briefly, after incubating the cells with different concentrations of each sample for 6 h, MTT assays were performed according to a protocol in the instructions of the cell enzyme activity kit (Sigma, agent at Beijing, China). Optical density (OD) of the plates was read on a microplate reader (Bio-Rad Model 550, Hercules, CA, USA) at 490 nm. The cell viability was analyzed with untreated control cells defined as 100%. Cytotoxicity of CsA was performed according to the same procedure as described above and the experimental concentration of CsA was set as 20 μM (cell viability N 90%, data not shown). Transepithelial transport of SMV/G5-NH2 molecular complexes and SMV-liposomes in Caco-2 cells Effects of P-gp pump on transepithelial transport of the two formulations Transepithelial transport studies of SMV in the apical (A) to the basolateral (B) direction (A→B) were conducted on Caco-2 cell monolayers as follows. SMV, SMV/G5-NH2 complexes, or SMV-liposomes were added into the A side in 500 μL HBSS (Hank's Balanced Salt Solution, pH 7.4, with Ca 2 + and Mg 2 +), and 1.5 mL of HBSS was added into the B side of the Millicell-ERS system. In the case of the transepithelial transport of SMV from the B side to the A side of the Caco-2 cell monolayers (B→A), the same procedure was followed except that 1.5 mL of the SMV sample was added to the B side and 500 μL of HBSS was added to the A side. The Transwell plate was incubated at 37 °C and 200 μL sample solution in the B or A side of the monolayers was taken at 1, 2, 3, and 4 h with equal volume of HBSS supplemented for the transport of A→B or B→A, respectively. Concentrations of SMV from the collected
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samples were then analyzed by HPLC. Three repeated experiments were conducted for each sample, and the TEER of Caco-2 monolayers was determined after the transport experiment to ensure integrity of the monolayers. CsA was employed to investigate influence of the P-gp efflux pump on the transepithelial transport of SMV. Briefly, 20 μM CsA was added to the A or B side of the Caco-2 cell monolayers according to experimental purpose. After incubating the cells with CsA for 30 min, the transepithelial transport of SMV was measured according to the procedure described above. To identify the effects of P-gp on the transepithelial transport of SMV and the two formulations, apparent permeability coefficient (Papp) and efflux ratio (ER) were calculated according to the following formula: Papp ¼
ðδQ=δtÞ cm s−1 ðA C 0 Þ
Where δQ/δt is the amount of drug transported across Caco-2 cell monolayer per second (mol · s − 1), A is the area of the polycarbonate film (1.13 cm 2), and C0 is the initial drug concentration in the donor side of Caco-2 cell monolayer. ER ¼ Papp ðB→AÞ=Papp ðA→BÞ Effect of occludin-1 protein on transepithelial transport of the two formulations Determination of occludin-1 expression. Expression of occludin-1 protein was detected by Western blot (WB) using a first antibody of rabbit anti-human polyclonal antibody (1:400) and a second antibody of goat anti-rabbit antibody (1:3000). GAPDH (1:1500 dilutions for the first antibody and 1:3000 dilutions for the second antibody) was used as an internal control. Effect of G5-NH2 on the expression of occludin-1 protein. Caco-2 cells were seeded on 6-well plates at a density of 5 × 10 5 cells/ well, and cultured to 80% confluence. The cells were then incubated with G5-NH2 for 4 h. After the solution of G5-NH2 was removed, the cells were rinsed and cell lysates were collected. WB was performed to determine the effect of G5-NH2 on the expression of occludin-1 protein. Downregulation of occludin-1 expression by siRNA. Occludin-1 was knocked down by using Lipofectamine 2000 to transfect a specific occludin-1 siRNA into Caco-2 cells. Briefly, Caco-2 cells were seeded on 6-well plates at a density of 5 × 10 5 cells/well, and cultured to 50% confluence. RNA interference (RNAi) procedure was performed according to the manufacturer's protocol using the following volumes and concentrations for each well: 100 pmol occludin-1 siRNA was diluted into 250 μL of Opti-MEM medium, and 5 μL of Lipofectamine 2000 was gently mixed with 250 μL of Opti-MEM medium for 5 min. The diluted siRNA and Lipofectamine 2000 were mixed gently and incubated at room temperature for 30 min. The mixture was then added into each well and the cells were incubated for 6 h. Finally, 1 mL of complete medium was added to each well and the cells were harvested after a 48 h incubation to detect the expression of occludin-1.
In this study, two concentrations of occludin-1 siRNA (100 pmol, 150 pmol) and three time points (24, 48, and 60 h) were investigated to optimize downregulation efficiency. In addition, a non-specific siRNA was used as a control in parallel experiments to monitor the specificity of occludin-1 siRNA to the expression of occludin-1. Effect of occludin-1 downregulation on transepithelial transport of the two SMV formulations. After the Caco-2 cell monolayers were formed in the Transwell plate, the complete medium was discarded and the cells were rinsed with HBSS. The cells were then treated with 100 pmol occludin-1 siRNA or non-specific control siRNA for 6 h, followed by 48 h of incubation with complete medium. After that, the transepithelial transport of SMV, SMV/G5-NH2 complexes, and SMV-liposomes were performed according to the procedure described above. Effect of CPZ on the cellular uptake of Cy5/G5-NH2 molecular complexes and Cy5-liposomes Chlorpromazine (CPZ), a typical inhibitor of clathrinmediated endocytosis (CME), was used to investigate the effect of CME on the cellular uptake of G5-NH2 complexes and liposomes. To analyze the amount of drug internalized into the Caco-2 cells, Cy5, a hydrophobic fluorescent substance, was used to simulate SMV. Cy5/G5-NH2 complexes and Cy5liposomes were prepared following the preparing procedures of SMV/G5-NH2 complexes and SMV-liposomes described above, except for replacing the SMV with Cy5. Briefly, Caco-2 cells were seeded onto six-well plates with a cell density of 1 × 10 5 per well and incubated in an atmosphere of 5% CO2 at 37 °C for 24 h. When the cells were grown to 70-80% confluence in the complete medium, 10 μg/mL CPZ (30 μM) in DMEM was added to pre-incubate with cells for 30 min at 37 °C. Then, Cy5/ G5-NH2 complexes and Cy5-liposomes were added into the wells and incubated with the cells for 3 h at 37 °C, respectively. The cells were rinsed for three times with PBS, and fluorescence intensity of Cy5 within the cells was determined by flow cytometry (FACS Calibur, BD Company, USA), and the results were compared with that without CPZ treatment. In vivo pharmacokinetics studies Male Sprague Dawley (SD) rats with a body weight of 150 g were obtained from the animal department of Peking University Health Science Center (Beijing, China). The Laboratory Animal Care Principles (NIH publication no. 85-23, revised 1996) were followed, and the experimental protocol was approved by Animal Care Committee, Peking University Health Science Center (LA2010-059). All rats were raised under a 12-hour light/ dark cycle with free access to food and water. To determine the formulation effects of the G5-NH2 complexes and the liposomes on the pharmacokinetics of SMV, 12 rats were randomly assigned to three groups (4 rats in each group) and a single dose of SMV, SMV/G5-NH2 complexes or SMV-liposomes was orally administrated at an equal SMV dose of 140 mg/kg body weight. It was reported previously that peak time (Tmax) of SMV was at 2 h and its half-life (t1/2) was less than 12 h after oral administration of a
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concentration-dependent. Figure 1, B illustrates that the solubility of SMV increased rapidly for concentrations of G5-NH2 below 22.6 mg/L (782 nM), but increased more slowly between 22.6 mg/L and 90 mg/L (3.128 μM). At the concentration of 22.6 mg/L G5-NH2 increased aqueous SMV solubility by about a factor of 2.93, and from 853 to 2500 μg/L. Therefore, given the cytotoxicity of this cationic dendrimer, 22.6 mg/L of G5-NH2, which equals to 100 μM charge concentration of G5-NH2, was chosen to prepare SMV/G5-NH2 complexes for the following experiments. Characterization of SMV/G5-NH2 molecular complexes and SMV-liposomes Particle size, PDI, zeta potential and encapsulation efficiency of SMV/G5-NH2 complexes and SMV-liposomes are shown in Table 1. The results indicate that the particle size of the SMV/ G5-NH2 complexes was 7.8 ± 0.5 nm, and SMV loading percentage (SMV mass/G5-NH2 mass, g/g) was 11.1 ± 0.1%. In comparison with the SMV/G5-NH2 complexes, the SMVliposomes had higher encapsulation efficiency (84.7 ± 1.4%) and greater particle size (111.4 ± 2.9 nm). The concentration of the encapsulated SMV in the liposomes was 141.7 ± 1.4 mg/L, which was about 57 times higher than that of the SMV/G5-NH2 complexes (2.5 ± 0.4 mg/L). Figure 1. Effects of incubation time and G5-NH2 concentrations on water solubility of SMV. Effect of incubation time on apparent solubility of SMV in pH 6.8 PBS (A). Effect of G5-NH2 concentrations on water solubility of SMV (B).
single dose of SMV, 28 thus blood samples from each group were collected at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.75, 5.5, 7, and 10 h. SMV blood concentrations were measured by HPLC. Statistical analysis All experiments were repeated three times, and data are presented as mean ± SD. Statistical analysis was performed using unpaired two-tailed t-tests and one-way ANOVA test. P b 0.05 was considered statistically significant.
Results
Cytotoxicity To study cytotoxicity of SMV/G5-NH2 complexes and SMV-liposomes, SMV concentrations were chosen from 5 to 100 μM based on previous literature reports. 21,29 Figure 2 illustrates the expected decrease in cell viability with increasing SMV concentrations in the SMV/G5-NH2 complexes and the SMVliposomes. SPSS (Statistical Product and Service Solutions) analysis indicates that cell viability in Figure 2, A was above 90% when SMV concentrations in the G5-NH2 complexes were below 10 μM, and the cytotoxicity was significant at SMV concentration N 20 μM. Figure 2, B indicates that the cytotoxicity of the SMV-liposomes was less than that of the SMV/G5-NH2 complexes at equivalent SMV concentrations of 20 and 100 μM. After overall consideration, 10 μM was selected as the concentration of SMV in the G5-NH2 complexes and the liposomes for the following in vitro transepithelial transport studies.
Solubility of SMV
Transepithelial transport of SMV/G5-NH2 molecular complexes and SMV-liposomes in Caco-2 cells
As shown in Supplementary Figure 1 for determination of SMV concentration by HPLC, retention time (tR) of SMV was 9.8 min with a good linear relationship at SMV concentrations from 8.83 μg/L to 4.52 mg/L in the chromatographic conditions. The apparent solubility of SMV at pH 6.8 in PBS detected by HPLC is shown in Figure 1, A. The results indicated that solubility of SMV in aqueous solvent is quite low and time-dependent. The extent of solubility increased dramatically in the first 24 h, and gave the greatest saturated value (853 μg/L) at 36 h. Therefore, 36 h was chosen as the time point to evaluate the solubilization effects of G5-NH2. It has been reported that solubilization effects of PAMAM dendrimers on certain poorly water-soluble drugs are
Papp has been employed to characterize transepithelial transport capacity of drugs in Caco-2 cells. 9,30,31 Figure 3, A indicates the quantities of SMV transported across the Caco-2 cell monolayers over a time course of 4 h. Compared with free SMV, the transported SMV concentrations from either the SMV-liposomes or the SMV/G5-NH2 complexes were significantly higher at all of the investigated time points (P b 0.05), which demonstrated that the two formulations significantly improved transepithelial transport of SMV in the Caco-2 cells. At both the 1 h (P b 0.05) and 2 h (P b 0.01) time point, the transepithelial transport of SMV for the liposomes was significantly greater than that for the G5-NH2 complexes, which might contribute to better SMV absorption of the SMV-liposomes.
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Table 1 Encapsulation efficiency, particle size, PDI and zeta potential of the two SMV formulations. Sample
Encapsulation efficiency (%)
Drug loading (g/g, %)
Average drug concentrations (mg/L)
Average particle size (nm)
PDI
Zeta potential (mV)
SMV/G5-NH2 complexes SMV-liposomes
84.7 ± 1.7
11.1 ± 0.1 8.5 ± 0.2
2.5 ± 0.4 141.7 ± 1.4
7.8 ± 0.5 111.4 ± 2.9
0.323 0.260
1.6 ± 0.1 − 4.54 ± 0.9
Effects of P-gp on transepithelial transport of SMV/G5-NH2 molecular complexes and SMV-liposomes Figure 3, B shows efflux ratios (ER) of SMV, SMV/CsA, SMV/G5-NH2 complexes and SMV-liposomes over a time course of 4 h in Caco-2 cells. The ER measured for SMV was approximately 2, which indicated that the excretion rate of SMV was greater than its absorption rate, and its transport was modulated by the P-gp efflux pump. After inhibition of the P-gp pump by addition of 20 μM CsA, ER of SMV decreased to approximately 1. The expected strong inhibitory effect of CsA on the P-gp pump resulted in a decreased ER and thus an increased absorption transport of SMV. The G5-NH2 complexes similarly inhibited the P-gp pump with the ER value of SMV decreasing from initial value of 2.5 to 0.75-1 at 2 h (P b 0.01). The decrease in ER suggests that the enhanced absorption of SMV in this case rose from inhibitory effect of G5-NH2 on the P-gp pump. Compared with the G5-NH2 complexes, the SMV-liposomes had no inhibitory effect on the P-gp pump, and presented a higher ER value (Figure 3, B), but the ER value of the SMV-liposomes could be decreased about 72-90% by the addition of 20 μM CsA (data not shown). The results support the hypothesis that the net transepithelial transport of the SMV-liposomes was modulated by P-gp.
Effect of occludin-1 knockdown on transport of SMV/G5-NH2 molecular complexes and SMV-liposomes As is shown in Figure 4, C, transepithelial transport of SMV over a time course of 4 h increased significantly (P b 0.05, P b 0.01) upon occludin-1 knockdown in the Caco-2 cells. It was also seen that there was a significant increase in SMV transport comparing the SMV/G5-NH2 with SMV/siRNA (P b 0.05, P b 0.01). Figure 4, D shows that the downregulation of occludin-1 did not contribute to further enhancement of the transport of the SMV-liposomes (P N 0.05, SMV-liposomes vs SMV-liposomes/siRNA). Effect of CPZ on the cellular uptake of Cy5/G5-NH2 molecular complexes and Cy5-liposomes As shown in Figure 5, with an equal concentration of Cy5 and at the absence of CPZ, the liposome formulation more significantly increased intracellular delivery of Cy5 and resulted in a 2.4-fold increase of the Cy5 fluorescence intensity than G5-NH2 complex did (P b 0.0001). After pre-treating the cells with CPZ, internalization of the Cy5-liposomes in the Caco-2 cells was significantly inhibited and resulted in a 60% decrease of the Cy5 fluorescence intensity in the cells (P b 0.0001). The endocytosis of the Cy5/G5-NH2 complexes was not obviously influenced by CPZ. Effects of the two formulations on oral bioavailability of SMV
Effects of occludin-1 on transepithelial transport of SMV/G5NH2 molecular complexes and SMV-liposomes Effect of G5-NH2 on expression of occludin-1 protein Figure 4, A shows that the expression of occludin-1 protein on Caco-2 cells was downregulated about 40% after the cells were incubated with G5-NH2 for 4 h. This result suggests that G5-NH2 can interrupt and open TJ through downregulating the expression of occludin-1 protein. Downregulation of occludin-1 expression by siRNA To better evaluate effects of occludin-1 protein on promoting the transepithelial transport of SMV, the protein expression has to be reduced by over 70%. Figure 4, B shows the timedependent knockdown efficiency of a dose of 100 pmol siRNA on the expression of occludin-1 protein. At 60 h, occludin-1 expression was only 20-25% of the initial value, which met the requirements of siRNA experiments. When siRNA concentration increased to 150 pmol, the knockdown efficiency was the best, yet excess Lipofectamine 2000 had significant cytotoxicity. Thus, siRNA at 100 pmol with a 60 h incubation time was set for siRNA experiments.
Three groups of rats were treated with a single dose of SMV, SMV/G5-NH2 complexes, or SMV-liposomes by gavage, and plasma concentrations of SMV in the rats were evaluated by HPLC. In the chromatographic conditions, retention times of lovastatin (LV, an internal standard) and SMV were 19.6 and 27.5 min, respectively, and the endogenous components from the blank plasma did not interfere with the analysis of LV and SMV (Supplementary Figure 3). Linear relationship was good within the concentration range of SMV from 0.01656 mg/L to 1.060 mg/L. The maximum plasma concentrations (Cmax) of SMV from the SMV/G5-NH2 complexes and the SMVliposomes treated groups were 2.2 (P b 0.05) and 3.8 (P b 0.01) times higher, respectively, than the free SMV treated group (Figure 6). The key parameters of pharmacokinetics and relative bioavailability of the SMV/G5-NH2 complexes and the SMVliposomes are shown in Table 2. The results demonstrate that both the G5-NH2 complexes and the liposome formulation dramatically improved the oral bioavailability of SMV (P b 0.01), with a 2.5 times improvement for the SMV/ G5-NH2 complexes and a 3.7 times improvement for the SMV-liposomes, as compared to free SMV.
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Figure 2. Viability of Caco-2 cells after the cells were treated with different concentrations of the SMV/G5-NH2 (A) or the SMV-liposomes (B). ⁎⁎P b 0.01, ⁎P b 0.05 compared with control group. n = 3.
The Tmax of the SMV/G5-NH2 complexes was greater than SMV, which was consistent with the literature 14 and demonstrated the slower drug release rate of the SMV/G5-NH2 as compared with free SMV. There was no significant difference among the t1/2 of SMV, SMV/G5-NH2 complexes, and SMV-liposomes, indicating that neither the SMV/G5-NH2 complex nor the liposome significantly altered the metabolism rate of SMV.
Discussion At the beginning of this study, effects of terminal groups and charges of the G5 PAMAM dendrimers on water solubility and transepithelial transport of SMV were investigated. Although there is a paper demonstrating that PEGylated G4-NH2 had better solubilization effect on SMV than non-PEGylated one, 13 we found in our experimental conditions that G5-NH2 (positive charge), compared with G4.5-COOH (negative charge), G5-OH (neutral) and PEGylated G5-NH2 (10% PEG 5000, positive charge), showed the greatest improvement in water solubility and in vitro transepithelial transport of SMV (Supplementary Figure 4). Therefore, G5-NH2 was chosen in this study to form SMV/G5-NH2 complexes. In Table 1, the encapsulation efficiency of SMV/G5-NH2 complexes could not be calculated exactly, since excess amount of SMV (3 mg) was added to saturate the solution. The drug loading percentage of SMV/G5-NH2 complexes (11.1 ± 0.1%, g/g) was close to and even higher than that of SMV-liposomes
Figure 3. SMV concentrations transported across the Caco-2 cell monolayers (A) and efflux ratios in the cells (B). (A) SMV concentrations transported across the Caco-2 cell monolayers were calculated after the cells were treated with SMV, SMV/G5-NH2 complexes or SMV-liposomes for 4 h. ⁎P b 0.05, ⁎⁎P b 0.01 for SMV/G5-NH2 vs SMV; #P b 0.05, ##P b 0.01, ###P b 0.001 for SMV-liposomes vs SMV; $P b 0.05, $$P b 0.01 for SMV-liposomes vs SMV/G5-NH2. n = 3. (B) Efflux ratios of SMV, SMV/CsA, SMV/G5-NH2 complexes, and SMV-liposomes in Caco-2 cells. ⁎P b 0.05, ⁎⁎P b 0.01, compared with SMV. n = 3.
(8.5 ± 0.2%, g/g), but less molecules of G5-NH2 carrier (22.6 mg/L or 782 nM) in the complex system than lipid carrier (soybean lecithin and cholesterol 41.7 mg/mL) in the liposomes resulted in a much lower encapsulated SMV concentration in the G5-NH2 complexes (2.5 mg/L) than in the SMV-liposomes (141.7 mg/L). Under physiological conditions, the TJ gap among Caco-2 cells is less than 10 nm, and can only be opened up to 20-30 nm under abnormal physiological conditions. 32 Since free SMV is a small molecule, downregulating the expression of occludin-1 and opening the TJ gap could increase the intercellular transport of SMV, therefore, improve its intestinal absorption (Figure 4, C). The particle size of SMV/G5-NH2 complexes was less than 10 nm (Table 1), indicating the possibility of transport through the opened TJ gap, and in turn contributing to the observed increase of SMV transport in the G5-NH2 complexes (Figure 3, A). However, more significant increase in SMV transport comparing the SMV/G5-NH2 with SMV/siRNA (P b 0.05, P b 0.01) indicates that G5-NH2 promoted the transport of SMV through mechanisms in addition to intercellular pathway of interrupting
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Figure 4. Effects of occludin-1 on the transepithelial transport of the two formulations across Caco-2 cell monolayers. (A) Effects of G5-NH2 on the expression of occludin-1 protein in Caco-2 cells detected by Western blot. (B) Expression of occludin-1 in Caco-2 cells after the cells were treated with different doses of occludin-1 siRNA and at different incubation time. (C and D) Effects of occludin-1 knockdown on transepithelial transport of SMV/G5-NH2 complexes (C), and SMV-liposomes (D). ⁎P b 0.05, ⁎⁎P b 0.01 for SMV/siRNA vs SMV; #P b 0.05, ##P b 0.01, ###P b 0.005 for SMV/G5-NH2 or SMV-liposomes vs SMV/siRNA. $P b 0.05, $$P b 0.01, $$$P b 0.005 for SMV/G5-NH2/siRNA or SMV-liposomes/siRNA vs SMV/siRNA. n = 3.
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Figure 5. Effects of chlorpromazine (CPZ) on the intracellular uptake of Cy5/G5-NH2 complexes and Cy5-liposomes in Caco-2 cells. (A) Cy5 fluorescence in the cells delivered by the Cy5/G5-NH2 complexes or the Cy5-liposomes without or with CPZ pre-treatment. (B and C) Overlay of Cy5 fluorescence from the Cy5/G5-NH2 complexes (B) and the Cy5-liposomes (C) before (green line) and after (pink line) treating the cells with CPZ. (D) Histogram of the mean fluorescence of Cy5. ⁎⁎⁎⁎P b 0.0001 for Cy5-liposomes vs Cy5/G5-NH2 complexes without CPZ treatment. ####P b 0.0001 for Cy5-liposomes before vs after treating the cells with CPZ.
occludin-1 and opening TJ. This observation is consistent with both the enhanced solubilization effect and P-gp inhibition, which also contribute to increased SMV transport. Because the particle size of the SMV-liposomes (111.4 ±2.9 nm) was much greater than the width of the TJ gap (less than 30 nm), the
SMV-liposomes could not transport through intercellular pathway. Moreover, the transepithelial transport of the SMV-liposomes was significantly higher than that of SMV/siRNA (P b 0.005), which indicates that the liposomes promoting the transport of SMV were through an intracellular pathway (Figure 4, D).
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R. Qi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1141–1151 Table 2 Main pharmacokinetics parameters of SMV and the two SMV formulations. Parameters
SMV
SMV/G5-NH2 complexes
SMV-liposomes
Cmax (μg/L) Tmax (h) T1/2 (h) AUC0→10 (μg/L ∙ h) AUC0→∞ (μg/L ∙ h) Fr (%)
165.46 ± 41.0 2.00 ± 0.0 1.84 ± 0.2 453.41 ± 60.1
356.66 ± 65.5⁎ 3.00 ± 0.0 1.84 ± 0.4 1153.07 ± 54.5⁎⁎
627.72 ± 119.0⁎⁎, # 2.50 ± 0.0 1.79 ± 0.6 1686.87 ± 154.7⁎⁎
536.12 ± 101.7
1229.11 ± 67.5⁎⁎
1819.33 ± 256.5⁎⁎
100
254.3
372
Figure 6. Plasma concentrations of SMV following an intragastric administration of a single dose (equivalent to 140 mg SMV/kg body weight) of free SMV, SMV/G5-NH2 complexes or SMV-liposomes in rats.
⁎ P b 0.05 ⁎⁎ P b 0.01 for SMV/G5-NH2 complexes or SMV-liposomes vs SMV. # P b 0.05 SMV-liposomes vs SMV/G5-NH2 complexes.
Supplementary Figure 5 indicates that both the liposome and the G5-NH2 could be internalized together with fluorescence Cy5 into the Caco-2 cells. It was reported that the endocytosis of the liposomes was mainly mediated by clathrin. 24 CME is associated with the assembly of “clathrin”, a coat protein on the intracellular face of the plasma membrane. CPZ can pharmacologically inhibit CME involving loss of the complexes of clathrin and AP2 adaptor. 33 The significant inhibition (60%) in the uptake of Cy5-liposomes by CPZ indicates that drug delivery mechanism of the liposomes in Caco-2 cells was mediated by clathrin, which was consistent with the literature. 23 The significantly higher (2.4-fold) intracellular Cy5 delivery by the liposomes than the G5-NH2 complexes (Figure 5) explained well the greater transepithelial transport and absorption of the SMV-liposomes than the SMV/G5-NH2 complexes. Kulhari et al found that SMV/G4-NH2 complexes improved the Cmax of SMV by a factor of 1.65. 13,14 Consistent with their results, we also found that the SMV/G5-NH2 complexes enhanced solubility and oral absorption of SMV, and resulted in a 2.2 times increase of the Cmax of SMV. The absorption enhancement provided by the G5-NH2 complexes resulted from the combined effects of an increase in solubility, inhibition of P-gp, and interruption of TJ. Moreover, it was noted in our work that the SMV-liposomes significantly improved the Cmax of SMV by a factor of 3.8, and oral bioavailability by a factor of 3.7 times (Table 2), indicating the SMV-liposomes provided greater improvement in in vivo oral absorption of SMV than the SMV/G5-NH2 complexes did. Better solubilization and encapsulation of SMV and more efficient intracellular delivery of SMV appear to be the greatest contributors to the improved absorption observed for the SMV-liposomes, as compared to the G5-NH2 complexes. These findings indicate that both SMV/G5-NH2 molecular complexes and SMV-liposomes are effective in promoting the in vitro transport and in vivo bioavailability of SMV and provide two complementary strategies for the improvement of oral administration of this drug.
References
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2015.02.011.
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G5 PAMAM dendrimer versus liposome: A comparison study on the in vitro transepithelial transport and in vivo oral absorption of simvastatin Rong Qi, PhD a, b,⁎, Heran Zhang, MS a, b, d , Lu Xu, BS a, b , Wenwen Shen, MS a, b , Cong Chen, BS a, b , Chao Wang, PhD a, b , Yini Cao, BS a, b , Yunan Wang, PhD a, b , Mallory A. van Dongen, PhD e , Bing He, PhD c , Siling Wang, PhD d , George Liu, PhD a, b , Mark M. Banaszak Holl, PhD e , Qiang Zhang, PhD c,⁎⁎ a
Peking University Institute of Cardiovascular Sciences, Peking University, Beijing, China b Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, China c School of Pharmaceutical Sciences, Peking University, Beijing, China d School of Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang, China e Department of Chemistry, University of Michigan, Ann Arbor, MI, USA Received 6 October 2014; accepted 14 February 2015
Abstract This study compared formulation effects of a dendrimer and a liposome preparation on the water solubility, transepithelial transport, and oral bioavailability of simvastatin (SMV). Amine-terminated G5 PAMAM dendrimer (G5-NH2) was chosen to form SMV/G5-NH2 molecular complexes, and SMV-liposomes were prepared by using a thin film dispersion method. The effects of these preparations on the transepithelial transport were investigated in vitro using Caco-2 cell monolayers. Results indicated that the solubility and transepithelial transport of SMV were significantly improved by both formulations. Pharmacokinetic studies in rats also revealed that both the SMV/G5NH2 molecular complexes and the SMV-liposomes significantly improved the oral bioavailability of SMV with the liposomes being more effective than the G5-NH2. The overall better oral absorption of SMV-liposomes as compared to SMV/G5-NH2 molecular complexes appeared to arise from better liposomal solubilization and encapsulation of SMV and more efficient intracellular SMV delivery. © 2015 Elsevier Inc. All rights reserved. Key words: Simvastatin; G5-NH2 PAMAM dendrimer; Liposome; Transepithelial transport; Oral bioavailability
Introduction Oral delivery is the most convenient approach for selfadministration of a wide variety of drugs; however, poor solubility in gastrointestinal fluid and/or low absorption across the intestinal epithelia can result in poor oral bioavailability and therefore unsatisfactory therapeutic effects. Drug delivery
The study was supported by National Natural Science Foundation of China (no. 81270368, 81360054). We have no conflicts of interest to declare. ⁎Correspondence to: R. Qi, 38 Xueyuan Road, Institute of Cardiovascular Sciences, Peking University Health Science Center, Peking University, Beijing 100191, China. ⁎⁎Correspondence to: Q. Zhang, Institute of Cardiovascular Sciences, Peking University Health Science Center, Peking University, Beijing, China. E-mail addresses: [email protected] (R. Qi), [email protected] (Q. Zhang). http://dx.doi.org/10.1016/j.nano.2015.02.011 1549-9634/© 2015 Elsevier Inc. All rights reserved.
systems including liposomes, 1 nanoparticles 2 and polymeric microspheres 3 show great promise to enhance oral absorption and bioavailability of a wide variety of drugs. Simvastatin (SMV), an inhibitor of 3-hydroxy-3-methyl coenzyme A (HMG-CoA) reductase, lowers plasma cholesterol concentration and is used clinically to treat hyperlipidemia and cardiovascular diseases. 4 SMV is poorly soluble in water and has irregular intestinal absorption, which make its oral bioavailability less than 5%. 5 Several methods including self-emulsifying drug delivery system (SEDDS), 6 cyclodextrin encapsulation, 7 nanostructured lipid carrier 8 and lipid nanoparticles, 9 have been used to improve the oral absorption, pharmacokinetics and tissue uptake of SMV. However, these formulations have some disadvantages. In the case of SEDDS, the formulation is thermodynamically unstable, whereas cyclodextrin in high concentrations (N 6 mM/L) induces hemolysis. 10
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Poly(amidoamine) (PAMAM) dendrimers have been studied on their drug and gene delivery carriers 11 because of their favorable properties including good water solubility, low polydispersity, abundant surface groups, and well defined structures. 12 Possible interaction between PAMAM dendrimer molecule and drug including hydrogen bond, electrostatic force, Van-der-Waals force, etc. leads to formation of a “molecular complex”, which is different from “coordination complex” in chemistry. Kulhari et al reported that G4 PAMAM dendrimers improved dissolution and stability of SMV, slowed down its release rate, 13 increased in vivo oral bioavailability, and improved the plasma cholesterol-lowering effect of SMV. 14 However, no in vitro transport and mechanism studies were carried out, and no comparison was made to liposomal formulation of SMV. Liposomes encapsulate hydrophobic drugs within their lipid bilayers to increase solubility and stability of the loaded drugs. 15 The liposomal bilayers are very similar to the structure of cell membranes, and liposomes can deliver encapsulated drug by fusing with cell membranes. 15 Liposomes containing SMV were proved to be more potent than free SMV in inhibiting various cell lines of monocytes/macrophages. One single systemic administration of liposomal SMV significantly suppressed neointimal formation in a rat model of restenosis. 16 In this study, we investigated and compared the ability of generation 5 (G5) PAMAM dendrimer molecular complexes and liposome formulation to provide information of transepithelial absorption and oral bioavailability of SMV. We have selected G5 PAMAM dendrimer because higher degree of branches and larger interior core of G5 material allow for greater surface modification and a higher drug-loading percentage as compared to lower generation dendrimers. This generation also maintains a less congested surface, retains arm flexibility, and is substantially less cytotoxic than higher generations. G5 PAMAM has been shown to be capable of promoting absorption and transepithelial transport of drugs by solubilizing the drugs in their internal void space, interacting with cell membranes, and delivering the loaded drugs into the cells. 11 Therefore, G5-NH2 was chosen in this study to form SMV/G5-NH2 molecular complexes. Previous studies have shown that G0 to G4 PAMAM dendrimers containing either cationic –NH 2 or anionic –COOH termination contribute to the opening of cell tight junctions (TJ) to enhance permeability of drugs. The zoning of occludin-1 protein, a key regulator of TJ assembly and function, becomes blurry and TJ structure is looser after treatment with G2 to G4 PAMAM dendrimers. 17-20 Furthermore, G3-NH 2 PAMAM dendrimers were found to bypass P-glycoprotein (P-gp), a drug efflux transporter, leading to a 200-fold increase of doxorubicin's bioavailability. 21,22 Besides, it was reported that endocytosis pathway of liposomes was dramatically affected by their particle size, 23 and their intracellular uptake was mainly related to clathrin-mediated endocytosis (CME). 24 Therefore, influence of P-gp, TJ and CME on the transepithelial transport of the SMV/G5-NH2 molecular complexes and the SMV-liposomes was probed in vitro in Caco-2 cells. Last of all, the efficacy of the two investigated SMV formulations on the oral bioavailability of SMV was evaluated in vivo in rats.
Materials and methods Materials SMV (≥ 98%) was purchased from Haizheng Company Ltd (Zhejiang, China). G5-NH2 dendrimer was purchased from Sigma-Aldrich, purified firstly by dialyzing (cut off: 10 KDa) against distilled water (8 media changes), and then centrifuged by Millipore tube (Amicon Ultra, cut off: 5 KDa). The purified G5-NH2 (Mn = 27700 Da by 1HNMR and GPC, PDI = 1.047) was used for the following study. The other G5 PAMAM dendrimers mentioned in the manuscript were all purified and characterized before use according to the same methods as described in G5-NH2 purification. Lecithin (injection class, PC-57 T) and cholesterol were purchased from Kewpie Corporation (Tokyo, Japan) and Damao Chemical Reagent Factory (Tianjin, China), respectively. Caco-2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cyclosporine A (CsA) was purchased from Taishan Pharmaceutical Company Ltd (Guangdong, China). BCA Protein Quantitation Kit was obtained from Thermo Scientific (Waltham, Mass, USA). Rabbit Anti-Human Polyclonal Antibody of occludin-1 (H-27), occludin-1 small interfering RNA (siRNA), non-targeted siRNA and RNase-free water were purchased from Santa Cruz (Santa Cruz, CA, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Chlorpromazine (CPZ) hydrochloride and Cy5 were purchased from Sigma-Aldrich Chemical Company (Saint Louis, MO, USA). 1-Palmitoyl-2-{12-[(7nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero3-phosphoethanolamine (16:0-12:0 NBD PE, NBP) was purchased from Avanti Polar Lipids, Inc (Alabaster, AL, USA). 10% PEG (WM 5000)-conjugated G5-NH2 PAMAM dendrimers (G5-PEG) were synthesized according to a procedure described in our previous publication. 25 Alexa Fluor® 488 labeled G5-NH2 were synthesized in our laboratory according to the previously published procedures. 26 All the other reagents were provided from local suppliers, unless otherwise mentioned. Solubilization studies Aqueous solubility of SMV The apparent solubility of SMV in pH 6.8 phosphate buffer saline (PBS) was measured according to the Pharmacopoeia (China, Version 2010). Briefly, 3 mg SMV was added into 2 mL pH 6.8 PBS and shaken in an incubating shaker (Ronghua SHA-C, China) at 100 rpm and 37 °C. Samples (300 μL) were taken at 24, 36, 42, and 48 h and spun on a centrifuge (Eppendorf 5810R, Germany) at 15048 g for 20 min. The amount of SMV in the supernatant was measured using High Performance Liquid Chromatography (HPLC, Shimadzu LC-15C, Japan) at λ 238 nm with a shim-pack VP-ODS C18 reversed-phase column (150 mm × 4.6 mm, 5 μm), and acetonitrile/water (16/9) as a mobile phase. Effects of G5-NH2 on SMV solubility To assess the concentration-dependent solubilization effect of G5-NH2 on SMV, G5-NH2 at different concentrations of 11.3, 22.6, 45, 90, 200 mg/L (0.391, 0.782, 1.564, 3.128 and 6.256 μM) were prepared by diluting 5.63, 11.3, 22.5, 45.0
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and 112 μL of G5-NH2 stock solution (4 mg/mL) into 2 mL PBS, respectively. SMV (3 mg) was added in the resultant G5-NH2 solutions, respectively, and the saturated SMV solubility in the different concentrations of G5-NH2 was determined by HPLC following the same procedure as described above. Preparation and characterization of SMV/G5-NH2 molecular complexes and SMV-liposomes SMV/G5-NH2 molecular complexes SMV/G5-NH2 complexes were prepared by adding 3 mg SMV into 22.6 mg/L (782 nM) G5-NH2 in 2 mL PBS, dispersing using an ultrasonicator (KQ-100DE CNC ultrasonic washer, Kunshan Ultrasonic Instrument Company, China), and shaking at 100 rpm and 37 °C for 36 h. One milliliter of the mixture was centrifuged at 13800 g, and SMV concentration present in the supernatant was determined by HPLC according to the same procedure as described above. The loading percentage of SMV in the G5-NH2 formulation was calculated by a concentration ratio of SMV and G5-NH2. Size, zeta-potential and polydispersity index (PDI) of the SMV/G5-NH2 complexes were measured by Malvern Zetasizer Nano-ZS (Santa Barbara, CA, USA). SMV-liposomes SMV-liposomes were prepared by using a thin film dispersion method. 27 To prepare lipid films, soybean lecithin (225 mg), cholesterol (25 mg) and SMV (25 mg) were mixed in a ratio of 9:1:1 and dissolved in 3 mL chloroform. The solvent was slowly evaporated in a rotary evaporator at 37 °C for 30 min to achieve a lipid film, which was dried overnight under vacuum and at room temperature. Multilamellar vesicles (MLVs) were prepared by hydrating the lipid film with 6 mL PBS (pH 7.5), and the resultant suspensions were sonicated for 30 min using a probe sonicator to obtain small unilamellar vesicles (SUVs) of SMV-liposomes. Size, zeta-potential and PDI of the SMV-liposomes were measured by Malvern Zetasizer Nano-ZS mentioned above. Drug encapsulation efficiency (EE) in the SMV-liposomes was calculated by a formula as follows:
EEð%Þ ¼
Mtotal −Mfree Mtotal
drug
100%
Mtotal and Mfree drug are the concentrations of total and free SMV in the liposome system, respectively. The detail method was described in the supplemental information. The drug loading percentage in the complexes or the liposomes was calculated as follows: Drug loading% ¼ Mencapsulated
drug =Mcarrier
100%
Mencapsulated drug is the mass of SMV encapsulated in the complexes or liposomes, Mcarrier is the mass of G5-NH2 in the complex system or the lipids in the liposome system.
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Cell culture and cytotoxicity assay Caco-2 cell culture Caco-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, New York, CA, USA) supplemented with 1% non-essential amino acid, 1% L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum. Cells from passage numbers 25-35 were used for the following in vitro experiments. Caco-2 cell monolayers Caco-2 cell monolayers were obtained by seeding 0.5 mL of Caco-2 cells onto polycarbonate membrane filters (3.0 μm pore size, 1.12 cm 2 growth area) inside Transwell cell culture chambers (Corning Costar, Cambridge, MA) at a density of 4 × 10 5 cells/cm 2 and were cultured according to the procedure as described above. After 21 days, Caco-2 cell monolayers were confirmed by determining transepithelial electrical resistance (TEER) using a Millicell-ERS system (MilliCell Corporation, Billerica, MA, USA), and the cells whose TEER value were around 350–400 (Ω ∙ cm 2 ) were used for transepithelial transport studies. Cytotoxicity Caco-2 cells were seeded on 96-well plates at a density of 5 × 10 4 cells/mL in 200 μL DMEM medium per well, and were incubated at 37 °C in 5% CO2 until 80% confluent. The effects of SMV, SMV/G5-NH2 complexes, and SMV-liposomes on the viability of Caco-2 cells were studied by MTT assay. Briefly, after incubating the cells with different concentrations of each sample for 6 h, MTT assays were performed according to a protocol in the instructions of the cell enzyme activity kit (Sigma, agent at Beijing, China). Optical density (OD) of the plates was read on a microplate reader (Bio-Rad Model 550, Hercules, CA, USA) at 490 nm. The cell viability was analyzed with untreated control cells defined as 100%. Cytotoxicity of CsA was performed according to the same procedure as described above and the experimental concentration of CsA was set as 20 μM (cell viability N 90%, data not shown). Transepithelial transport of SMV/G5-NH2 molecular complexes and SMV-liposomes in Caco-2 cells Effects of P-gp pump on transepithelial transport of the two formulations Transepithelial transport studies of SMV in the apical (A) to the basolateral (B) direction (A→B) were conducted on Caco-2 cell monolayers as follows. SMV, SMV/G5-NH2 complexes, or SMV-liposomes were added into the A side in 500 μL HBSS (Hank's Balanced Salt Solution, pH 7.4, with Ca 2 + and Mg 2 +), and 1.5 mL of HBSS was added into the B side of the Millicell-ERS system. In the case of the transepithelial transport of SMV from the B side to the A side of the Caco-2 cell monolayers (B→A), the same procedure was followed except that 1.5 mL of the SMV sample was added to the B side and 500 μL of HBSS was added to the A side. The Transwell plate was incubated at 37 °C and 200 μL sample solution in the B or A side of the monolayers was taken at 1, 2, 3, and 4 h with equal volume of HBSS supplemented for the transport of A→B or B→A, respectively. Concentrations of SMV from the collected
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samples were then analyzed by HPLC. Three repeated experiments were conducted for each sample, and the TEER of Caco-2 monolayers was determined after the transport experiment to ensure integrity of the monolayers. CsA was employed to investigate influence of the P-gp efflux pump on the transepithelial transport of SMV. Briefly, 20 μM CsA was added to the A or B side of the Caco-2 cell monolayers according to experimental purpose. After incubating the cells with CsA for 30 min, the transepithelial transport of SMV was measured according to the procedure described above. To identify the effects of P-gp on the transepithelial transport of SMV and the two formulations, apparent permeability coefficient (Papp) and efflux ratio (ER) were calculated according to the following formula: Papp ¼
ðδQ=δtÞ cm s−1 ðA C 0 Þ
Where δQ/δt is the amount of drug transported across Caco-2 cell monolayer per second (mol · s − 1), A is the area of the polycarbonate film (1.13 cm 2), and C0 is the initial drug concentration in the donor side of Caco-2 cell monolayer. ER ¼ Papp ðB→AÞ=Papp ðA→BÞ Effect of occludin-1 protein on transepithelial transport of the two formulations Determination of occludin-1 expression. Expression of occludin-1 protein was detected by Western blot (WB) using a first antibody of rabbit anti-human polyclonal antibody (1:400) and a second antibody of goat anti-rabbit antibody (1:3000). GAPDH (1:1500 dilutions for the first antibody and 1:3000 dilutions for the second antibody) was used as an internal control. Effect of G5-NH2 on the expression of occludin-1 protein. Caco-2 cells were seeded on 6-well plates at a density of 5 × 10 5 cells/ well, and cultured to 80% confluence. The cells were then incubated with G5-NH2 for 4 h. After the solution of G5-NH2 was removed, the cells were rinsed and cell lysates were collected. WB was performed to determine the effect of G5-NH2 on the expression of occludin-1 protein. Downregulation of occludin-1 expression by siRNA. Occludin-1 was knocked down by using Lipofectamine 2000 to transfect a specific occludin-1 siRNA into Caco-2 cells. Briefly, Caco-2 cells were seeded on 6-well plates at a density of 5 × 10 5 cells/well, and cultured to 50% confluence. RNA interference (RNAi) procedure was performed according to the manufacturer's protocol using the following volumes and concentrations for each well: 100 pmol occludin-1 siRNA was diluted into 250 μL of Opti-MEM medium, and 5 μL of Lipofectamine 2000 was gently mixed with 250 μL of Opti-MEM medium for 5 min. The diluted siRNA and Lipofectamine 2000 were mixed gently and incubated at room temperature for 30 min. The mixture was then added into each well and the cells were incubated for 6 h. Finally, 1 mL of complete medium was added to each well and the cells were harvested after a 48 h incubation to detect the expression of occludin-1.
In this study, two concentrations of occludin-1 siRNA (100 pmol, 150 pmol) and three time points (24, 48, and 60 h) were investigated to optimize downregulation efficiency. In addition, a non-specific siRNA was used as a control in parallel experiments to monitor the specificity of occludin-1 siRNA to the expression of occludin-1. Effect of occludin-1 downregulation on transepithelial transport of the two SMV formulations. After the Caco-2 cell monolayers were formed in the Transwell plate, the complete medium was discarded and the cells were rinsed with HBSS. The cells were then treated with 100 pmol occludin-1 siRNA or non-specific control siRNA for 6 h, followed by 48 h of incubation with complete medium. After that, the transepithelial transport of SMV, SMV/G5-NH2 complexes, and SMV-liposomes were performed according to the procedure described above. Effect of CPZ on the cellular uptake of Cy5/G5-NH2 molecular complexes and Cy5-liposomes Chlorpromazine (CPZ), a typical inhibitor of clathrinmediated endocytosis (CME), was used to investigate the effect of CME on the cellular uptake of G5-NH2 complexes and liposomes. To analyze the amount of drug internalized into the Caco-2 cells, Cy5, a hydrophobic fluorescent substance, was used to simulate SMV. Cy5/G5-NH2 complexes and Cy5liposomes were prepared following the preparing procedures of SMV/G5-NH2 complexes and SMV-liposomes described above, except for replacing the SMV with Cy5. Briefly, Caco-2 cells were seeded onto six-well plates with a cell density of 1 × 10 5 per well and incubated in an atmosphere of 5% CO2 at 37 °C for 24 h. When the cells were grown to 70-80% confluence in the complete medium, 10 μg/mL CPZ (30 μM) in DMEM was added to pre-incubate with cells for 30 min at 37 °C. Then, Cy5/ G5-NH2 complexes and Cy5-liposomes were added into the wells and incubated with the cells for 3 h at 37 °C, respectively. The cells were rinsed for three times with PBS, and fluorescence intensity of Cy5 within the cells was determined by flow cytometry (FACS Calibur, BD Company, USA), and the results were compared with that without CPZ treatment. In vivo pharmacokinetics studies Male Sprague Dawley (SD) rats with a body weight of 150 g were obtained from the animal department of Peking University Health Science Center (Beijing, China). The Laboratory Animal Care Principles (NIH publication no. 85-23, revised 1996) were followed, and the experimental protocol was approved by Animal Care Committee, Peking University Health Science Center (LA2010-059). All rats were raised under a 12-hour light/ dark cycle with free access to food and water. To determine the formulation effects of the G5-NH2 complexes and the liposomes on the pharmacokinetics of SMV, 12 rats were randomly assigned to three groups (4 rats in each group) and a single dose of SMV, SMV/G5-NH2 complexes or SMV-liposomes was orally administrated at an equal SMV dose of 140 mg/kg body weight. It was reported previously that peak time (Tmax) of SMV was at 2 h and its half-life (t1/2) was less than 12 h after oral administration of a
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concentration-dependent. Figure 1, B illustrates that the solubility of SMV increased rapidly for concentrations of G5-NH2 below 22.6 mg/L (782 nM), but increased more slowly between 22.6 mg/L and 90 mg/L (3.128 μM). At the concentration of 22.6 mg/L G5-NH2 increased aqueous SMV solubility by about a factor of 2.93, and from 853 to 2500 μg/L. Therefore, given the cytotoxicity of this cationic dendrimer, 22.6 mg/L of G5-NH2, which equals to 100 μM charge concentration of G5-NH2, was chosen to prepare SMV/G5-NH2 complexes for the following experiments. Characterization of SMV/G5-NH2 molecular complexes and SMV-liposomes Particle size, PDI, zeta potential and encapsulation efficiency of SMV/G5-NH2 complexes and SMV-liposomes are shown in Table 1. The results indicate that the particle size of the SMV/ G5-NH2 complexes was 7.8 ± 0.5 nm, and SMV loading percentage (SMV mass/G5-NH2 mass, g/g) was 11.1 ± 0.1%. In comparison with the SMV/G5-NH2 complexes, the SMVliposomes had higher encapsulation efficiency (84.7 ± 1.4%) and greater particle size (111.4 ± 2.9 nm). The concentration of the encapsulated SMV in the liposomes was 141.7 ± 1.4 mg/L, which was about 57 times higher than that of the SMV/G5-NH2 complexes (2.5 ± 0.4 mg/L). Figure 1. Effects of incubation time and G5-NH2 concentrations on water solubility of SMV. Effect of incubation time on apparent solubility of SMV in pH 6.8 PBS (A). Effect of G5-NH2 concentrations on water solubility of SMV (B).
single dose of SMV, 28 thus blood samples from each group were collected at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.75, 5.5, 7, and 10 h. SMV blood concentrations were measured by HPLC. Statistical analysis All experiments were repeated three times, and data are presented as mean ± SD. Statistical analysis was performed using unpaired two-tailed t-tests and one-way ANOVA test. P b 0.05 was considered statistically significant.
Results
Cytotoxicity To study cytotoxicity of SMV/G5-NH2 complexes and SMV-liposomes, SMV concentrations were chosen from 5 to 100 μM based on previous literature reports. 21,29 Figure 2 illustrates the expected decrease in cell viability with increasing SMV concentrations in the SMV/G5-NH2 complexes and the SMVliposomes. SPSS (Statistical Product and Service Solutions) analysis indicates that cell viability in Figure 2, A was above 90% when SMV concentrations in the G5-NH2 complexes were below 10 μM, and the cytotoxicity was significant at SMV concentration N 20 μM. Figure 2, B indicates that the cytotoxicity of the SMV-liposomes was less than that of the SMV/G5-NH2 complexes at equivalent SMV concentrations of 20 and 100 μM. After overall consideration, 10 μM was selected as the concentration of SMV in the G5-NH2 complexes and the liposomes for the following in vitro transepithelial transport studies.
Solubility of SMV
Transepithelial transport of SMV/G5-NH2 molecular complexes and SMV-liposomes in Caco-2 cells
As shown in Supplementary Figure 1 for determination of SMV concentration by HPLC, retention time (tR) of SMV was 9.8 min with a good linear relationship at SMV concentrations from 8.83 μg/L to 4.52 mg/L in the chromatographic conditions. The apparent solubility of SMV at pH 6.8 in PBS detected by HPLC is shown in Figure 1, A. The results indicated that solubility of SMV in aqueous solvent is quite low and time-dependent. The extent of solubility increased dramatically in the first 24 h, and gave the greatest saturated value (853 μg/L) at 36 h. Therefore, 36 h was chosen as the time point to evaluate the solubilization effects of G5-NH2. It has been reported that solubilization effects of PAMAM dendrimers on certain poorly water-soluble drugs are
Papp has been employed to characterize transepithelial transport capacity of drugs in Caco-2 cells. 9,30,31 Figure 3, A indicates the quantities of SMV transported across the Caco-2 cell monolayers over a time course of 4 h. Compared with free SMV, the transported SMV concentrations from either the SMV-liposomes or the SMV/G5-NH2 complexes were significantly higher at all of the investigated time points (P b 0.05), which demonstrated that the two formulations significantly improved transepithelial transport of SMV in the Caco-2 cells. At both the 1 h (P b 0.05) and 2 h (P b 0.01) time point, the transepithelial transport of SMV for the liposomes was significantly greater than that for the G5-NH2 complexes, which might contribute to better SMV absorption of the SMV-liposomes.
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Table 1 Encapsulation efficiency, particle size, PDI and zeta potential of the two SMV formulations. Sample
Encapsulation efficiency (%)
Drug loading (g/g, %)
Average drug concentrations (mg/L)
Average particle size (nm)
PDI
Zeta potential (mV)
SMV/G5-NH2 complexes SMV-liposomes
84.7 ± 1.7
11.1 ± 0.1 8.5 ± 0.2
2.5 ± 0.4 141.7 ± 1.4
7.8 ± 0.5 111.4 ± 2.9
0.323 0.260
1.6 ± 0.1 − 4.54 ± 0.9
Effects of P-gp on transepithelial transport of SMV/G5-NH2 molecular complexes and SMV-liposomes Figure 3, B shows efflux ratios (ER) of SMV, SMV/CsA, SMV/G5-NH2 complexes and SMV-liposomes over a time course of 4 h in Caco-2 cells. The ER measured for SMV was approximately 2, which indicated that the excretion rate of SMV was greater than its absorption rate, and its transport was modulated by the P-gp efflux pump. After inhibition of the P-gp pump by addition of 20 μM CsA, ER of SMV decreased to approximately 1. The expected strong inhibitory effect of CsA on the P-gp pump resulted in a decreased ER and thus an increased absorption transport of SMV. The G5-NH2 complexes similarly inhibited the P-gp pump with the ER value of SMV decreasing from initial value of 2.5 to 0.75-1 at 2 h (P b 0.01). The decrease in ER suggests that the enhanced absorption of SMV in this case rose from inhibitory effect of G5-NH2 on the P-gp pump. Compared with the G5-NH2 complexes, the SMV-liposomes had no inhibitory effect on the P-gp pump, and presented a higher ER value (Figure 3, B), but the ER value of the SMV-liposomes could be decreased about 72-90% by the addition of 20 μM CsA (data not shown). The results support the hypothesis that the net transepithelial transport of the SMV-liposomes was modulated by P-gp.
Effect of occludin-1 knockdown on transport of SMV/G5-NH2 molecular complexes and SMV-liposomes As is shown in Figure 4, C, transepithelial transport of SMV over a time course of 4 h increased significantly (P b 0.05, P b 0.01) upon occludin-1 knockdown in the Caco-2 cells. It was also seen that there was a significant increase in SMV transport comparing the SMV/G5-NH2 with SMV/siRNA (P b 0.05, P b 0.01). Figure 4, D shows that the downregulation of occludin-1 did not contribute to further enhancement of the transport of the SMV-liposomes (P N 0.05, SMV-liposomes vs SMV-liposomes/siRNA). Effect of CPZ on the cellular uptake of Cy5/G5-NH2 molecular complexes and Cy5-liposomes As shown in Figure 5, with an equal concentration of Cy5 and at the absence of CPZ, the liposome formulation more significantly increased intracellular delivery of Cy5 and resulted in a 2.4-fold increase of the Cy5 fluorescence intensity than G5-NH2 complex did (P b 0.0001). After pre-treating the cells with CPZ, internalization of the Cy5-liposomes in the Caco-2 cells was significantly inhibited and resulted in a 60% decrease of the Cy5 fluorescence intensity in the cells (P b 0.0001). The endocytosis of the Cy5/G5-NH2 complexes was not obviously influenced by CPZ. Effects of the two formulations on oral bioavailability of SMV
Effects of occludin-1 on transepithelial transport of SMV/G5NH2 molecular complexes and SMV-liposomes Effect of G5-NH2 on expression of occludin-1 protein Figure 4, A shows that the expression of occludin-1 protein on Caco-2 cells was downregulated about 40% after the cells were incubated with G5-NH2 for 4 h. This result suggests that G5-NH2 can interrupt and open TJ through downregulating the expression of occludin-1 protein. Downregulation of occludin-1 expression by siRNA To better evaluate effects of occludin-1 protein on promoting the transepithelial transport of SMV, the protein expression has to be reduced by over 70%. Figure 4, B shows the timedependent knockdown efficiency of a dose of 100 pmol siRNA on the expression of occludin-1 protein. At 60 h, occludin-1 expression was only 20-25% of the initial value, which met the requirements of siRNA experiments. When siRNA concentration increased to 150 pmol, the knockdown efficiency was the best, yet excess Lipofectamine 2000 had significant cytotoxicity. Thus, siRNA at 100 pmol with a 60 h incubation time was set for siRNA experiments.
Three groups of rats were treated with a single dose of SMV, SMV/G5-NH2 complexes, or SMV-liposomes by gavage, and plasma concentrations of SMV in the rats were evaluated by HPLC. In the chromatographic conditions, retention times of lovastatin (LV, an internal standard) and SMV were 19.6 and 27.5 min, respectively, and the endogenous components from the blank plasma did not interfere with the analysis of LV and SMV (Supplementary Figure 3). Linear relationship was good within the concentration range of SMV from 0.01656 mg/L to 1.060 mg/L. The maximum plasma concentrations (Cmax) of SMV from the SMV/G5-NH2 complexes and the SMVliposomes treated groups were 2.2 (P b 0.05) and 3.8 (P b 0.01) times higher, respectively, than the free SMV treated group (Figure 6). The key parameters of pharmacokinetics and relative bioavailability of the SMV/G5-NH2 complexes and the SMVliposomes are shown in Table 2. The results demonstrate that both the G5-NH2 complexes and the liposome formulation dramatically improved the oral bioavailability of SMV (P b 0.01), with a 2.5 times improvement for the SMV/ G5-NH2 complexes and a 3.7 times improvement for the SMV-liposomes, as compared to free SMV.
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Figure 2. Viability of Caco-2 cells after the cells were treated with different concentrations of the SMV/G5-NH2 (A) or the SMV-liposomes (B). ⁎⁎P b 0.01, ⁎P b 0.05 compared with control group. n = 3.
The Tmax of the SMV/G5-NH2 complexes was greater than SMV, which was consistent with the literature 14 and demonstrated the slower drug release rate of the SMV/G5-NH2 as compared with free SMV. There was no significant difference among the t1/2 of SMV, SMV/G5-NH2 complexes, and SMV-liposomes, indicating that neither the SMV/G5-NH2 complex nor the liposome significantly altered the metabolism rate of SMV.
Discussion At the beginning of this study, effects of terminal groups and charges of the G5 PAMAM dendrimers on water solubility and transepithelial transport of SMV were investigated. Although there is a paper demonstrating that PEGylated G4-NH2 had better solubilization effect on SMV than non-PEGylated one, 13 we found in our experimental conditions that G5-NH2 (positive charge), compared with G4.5-COOH (negative charge), G5-OH (neutral) and PEGylated G5-NH2 (10% PEG 5000, positive charge), showed the greatest improvement in water solubility and in vitro transepithelial transport of SMV (Supplementary Figure 4). Therefore, G5-NH2 was chosen in this study to form SMV/G5-NH2 complexes. In Table 1, the encapsulation efficiency of SMV/G5-NH2 complexes could not be calculated exactly, since excess amount of SMV (3 mg) was added to saturate the solution. The drug loading percentage of SMV/G5-NH2 complexes (11.1 ± 0.1%, g/g) was close to and even higher than that of SMV-liposomes
Figure 3. SMV concentrations transported across the Caco-2 cell monolayers (A) and efflux ratios in the cells (B). (A) SMV concentrations transported across the Caco-2 cell monolayers were calculated after the cells were treated with SMV, SMV/G5-NH2 complexes or SMV-liposomes for 4 h. ⁎P b 0.05, ⁎⁎P b 0.01 for SMV/G5-NH2 vs SMV; #P b 0.05, ##P b 0.01, ###P b 0.001 for SMV-liposomes vs SMV; $P b 0.05, $$P b 0.01 for SMV-liposomes vs SMV/G5-NH2. n = 3. (B) Efflux ratios of SMV, SMV/CsA, SMV/G5-NH2 complexes, and SMV-liposomes in Caco-2 cells. ⁎P b 0.05, ⁎⁎P b 0.01, compared with SMV. n = 3.
(8.5 ± 0.2%, g/g), but less molecules of G5-NH2 carrier (22.6 mg/L or 782 nM) in the complex system than lipid carrier (soybean lecithin and cholesterol 41.7 mg/mL) in the liposomes resulted in a much lower encapsulated SMV concentration in the G5-NH2 complexes (2.5 mg/L) than in the SMV-liposomes (141.7 mg/L). Under physiological conditions, the TJ gap among Caco-2 cells is less than 10 nm, and can only be opened up to 20-30 nm under abnormal physiological conditions. 32 Since free SMV is a small molecule, downregulating the expression of occludin-1 and opening the TJ gap could increase the intercellular transport of SMV, therefore, improve its intestinal absorption (Figure 4, C). The particle size of SMV/G5-NH2 complexes was less than 10 nm (Table 1), indicating the possibility of transport through the opened TJ gap, and in turn contributing to the observed increase of SMV transport in the G5-NH2 complexes (Figure 3, A). However, more significant increase in SMV transport comparing the SMV/G5-NH2 with SMV/siRNA (P b 0.05, P b 0.01) indicates that G5-NH2 promoted the transport of SMV through mechanisms in addition to intercellular pathway of interrupting
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Figure 4. Effects of occludin-1 on the transepithelial transport of the two formulations across Caco-2 cell monolayers. (A) Effects of G5-NH2 on the expression of occludin-1 protein in Caco-2 cells detected by Western blot. (B) Expression of occludin-1 in Caco-2 cells after the cells were treated with different doses of occludin-1 siRNA and at different incubation time. (C and D) Effects of occludin-1 knockdown on transepithelial transport of SMV/G5-NH2 complexes (C), and SMV-liposomes (D). ⁎P b 0.05, ⁎⁎P b 0.01 for SMV/siRNA vs SMV; #P b 0.05, ##P b 0.01, ###P b 0.005 for SMV/G5-NH2 or SMV-liposomes vs SMV/siRNA. $P b 0.05, $$P b 0.01, $$$P b 0.005 for SMV/G5-NH2/siRNA or SMV-liposomes/siRNA vs SMV/siRNA. n = 3.
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Figure 5. Effects of chlorpromazine (CPZ) on the intracellular uptake of Cy5/G5-NH2 complexes and Cy5-liposomes in Caco-2 cells. (A) Cy5 fluorescence in the cells delivered by the Cy5/G5-NH2 complexes or the Cy5-liposomes without or with CPZ pre-treatment. (B and C) Overlay of Cy5 fluorescence from the Cy5/G5-NH2 complexes (B) and the Cy5-liposomes (C) before (green line) and after (pink line) treating the cells with CPZ. (D) Histogram of the mean fluorescence of Cy5. ⁎⁎⁎⁎P b 0.0001 for Cy5-liposomes vs Cy5/G5-NH2 complexes without CPZ treatment. ####P b 0.0001 for Cy5-liposomes before vs after treating the cells with CPZ.
occludin-1 and opening TJ. This observation is consistent with both the enhanced solubilization effect and P-gp inhibition, which also contribute to increased SMV transport. Because the particle size of the SMV-liposomes (111.4 ±2.9 nm) was much greater than the width of the TJ gap (less than 30 nm), the
SMV-liposomes could not transport through intercellular pathway. Moreover, the transepithelial transport of the SMV-liposomes was significantly higher than that of SMV/siRNA (P b 0.005), which indicates that the liposomes promoting the transport of SMV were through an intracellular pathway (Figure 4, D).
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R. Qi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1141–1151 Table 2 Main pharmacokinetics parameters of SMV and the two SMV formulations. Parameters
SMV
SMV/G5-NH2 complexes
SMV-liposomes
Cmax (μg/L) Tmax (h) T1/2 (h) AUC0→10 (μg/L ∙ h) AUC0→∞ (μg/L ∙ h) Fr (%)
165.46 ± 41.0 2.00 ± 0.0 1.84 ± 0.2 453.41 ± 60.1
356.66 ± 65.5⁎ 3.00 ± 0.0 1.84 ± 0.4 1153.07 ± 54.5⁎⁎
627.72 ± 119.0⁎⁎, # 2.50 ± 0.0 1.79 ± 0.6 1686.87 ± 154.7⁎⁎
536.12 ± 101.7
1229.11 ± 67.5⁎⁎
1819.33 ± 256.5⁎⁎
100
254.3
372
Figure 6. Plasma concentrations of SMV following an intragastric administration of a single dose (equivalent to 140 mg SMV/kg body weight) of free SMV, SMV/G5-NH2 complexes or SMV-liposomes in rats.
⁎ P b 0.05 ⁎⁎ P b 0.01 for SMV/G5-NH2 complexes or SMV-liposomes vs SMV. # P b 0.05 SMV-liposomes vs SMV/G5-NH2 complexes.
Supplementary Figure 5 indicates that both the liposome and the G5-NH2 could be internalized together with fluorescence Cy5 into the Caco-2 cells. It was reported that the endocytosis of the liposomes was mainly mediated by clathrin. 24 CME is associated with the assembly of “clathrin”, a coat protein on the intracellular face of the plasma membrane. CPZ can pharmacologically inhibit CME involving loss of the complexes of clathrin and AP2 adaptor. 33 The significant inhibition (60%) in the uptake of Cy5-liposomes by CPZ indicates that drug delivery mechanism of the liposomes in Caco-2 cells was mediated by clathrin, which was consistent with the literature. 23 The significantly higher (2.4-fold) intracellular Cy5 delivery by the liposomes than the G5-NH2 complexes (Figure 5) explained well the greater transepithelial transport and absorption of the SMV-liposomes than the SMV/G5-NH2 complexes. Kulhari et al found that SMV/G4-NH2 complexes improved the Cmax of SMV by a factor of 1.65. 13,14 Consistent with their results, we also found that the SMV/G5-NH2 complexes enhanced solubility and oral absorption of SMV, and resulted in a 2.2 times increase of the Cmax of SMV. The absorption enhancement provided by the G5-NH2 complexes resulted from the combined effects of an increase in solubility, inhibition of P-gp, and interruption of TJ. Moreover, it was noted in our work that the SMV-liposomes significantly improved the Cmax of SMV by a factor of 3.8, and oral bioavailability by a factor of 3.7 times (Table 2), indicating the SMV-liposomes provided greater improvement in in vivo oral absorption of SMV than the SMV/G5-NH2 complexes did. Better solubilization and encapsulation of SMV and more efficient intracellular delivery of SMV appear to be the greatest contributors to the improved absorption observed for the SMV-liposomes, as compared to the G5-NH2 complexes. These findings indicate that both SMV/G5-NH2 molecular complexes and SMV-liposomes are effective in promoting the in vitro transport and in vivo bioavailability of SMV and provide two complementary strategies for the improvement of oral administration of this drug.
References
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2015.02.011.
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