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Bovine serum albumin nanoparticles for delivery of tacrolimus to reduce its kidney uptake and functional nephrotoxicity
International Journal of Pharmaceutics 483 (2015) 180–187
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Bovine serum albumin nanoparticles for delivery of tacrolimus to reduce its kidney uptake and functional nephrotoxicity Lei Zhao, Yanxia Zhou, Yajie Gao, Shujin Ma, Chao Zhang, Jinwen Li, Dishi Wang, Xueping Li, Chengwei Li, Yan Liu * , Xinru Li * Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 November 2014 Received in revised form 31 December 2014 Accepted 10 February 2015 Available online 11 February 2015
The purpose of the present study was to develop a new nanoparticulate formulation for delivery of tacrolimus to reduce its kidney distribution and functional nephrotoxicity. Tacrolimus (TAC)-loaded bovine serum albumin (BSA) nanoparticles (TAC-BSA-NPs) were prepared by emulsification-dispersion technique. The obtained TAC-BSA-NPs, with 189.50 7.15 nm of diameter and 20.86 0.45 mV of Zeta potential determined by DLS, were spherical in shape observed by TEM. The drug loading content and encapsulation efficiency were (1.7 0.13)% and (85 3.0)%, respectively. The in vitro release of TAC-BSANPs exhibited biphasic drug release pattern with an initial burst release and subsequently sustained release. Pharmacokinetic analysis displayed that TAC-BSA-NPs could enhance the drug blood level and prolong the circulation time in comparison to Prograf1. Meanwhile, compared with Prograf1, TAC-BSANPs could deliver less TAC to kidney and simultaneously reduce the functional nephrotoxicity of TAC to kidney. In conclusion, BSA nanoparticles might be a more safe carrier for delivery of hydrophobic drug TAC. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Tacrolimus Bovine serum albumin nanoparticles Pharmacokinetics Nephrotoxicity Tissue distribution
1. Introduction Tacrolimus (TAC) (Fig. 1), a macrolide immunosuppressant, has been widely used to prevent acute rejection in organ transplants (Wallemacq and Reding, 1993). Similar to cyclosporine A, a cyclic undecapeptide immunosuppressant, the action mechanism of TAC has been demonstrated to bind to the TAC binding protein (immunophilin FKBP-12) inside the activated T-cells (Cardenas et al., 1995), which inhibits the activity of calcineurin for dephosphorylating the nuclear factor of activated T cells (NFAT), thereby reduces the generation of IL-2 and inhibits activation and proliferation of T cells, leading to suppressed immune response (Ruff and Leach, 1995; Shaw et al., 1995). TAC exhibits much greater immunosuppressive activity than cyclosporine A (Geissler and Schlitt, 2009) and provides a better side effect profile and increases long-term survival in patients (Jurewicz, 2003; Wiesner, 1998). However, its therapeutic efficacy is limited due to its poor water solubility. In addition, TAC is known to exhibit some adverse events, such as nephrotoxicity, neurotoxicity, hypertension and diabetogenic effects (Bottiger et al., 1999). Its commercial formulation Prograf1 contains high amount of polyethylene
* Corresponding authors. Tel.: +86 10 82801508. E-mail addresses: [email protected] (Y. Liu), [email protected] (X. Li). http://dx.doi.org/10.1016/j.ijpharm.2015.02.018 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
hydrogenated castor oil (HCO-60) which has been found to associate with severe side effect of anaphylaxis and still showed nephrotoxicity to some extent (Nicolai and Bunyavanich, 2012). Therefore, many efforts have been devoted to improve the solubility of TAC and reduce the side effects of TAC, including encapsulation of TAC in liposomes (Ishii et al., 2013), polymeric micelles (Wang et al., 2011), cyclodextrin inclusion (Brewster and Loftsson, 2007), nanoparticles (Xu et al., 2014), nanocapsules (Nassar et al., 2009) and self-microemulsifying drug delivery system (von Suesskind-Schwendi et al., 2013). Among these drug delivery vehicles, nanoparticles have gained much attention in recent years. Nanoparticles are made of a variety of polymers, such as polysaccharides (Fernandez-Urrusuno et al., 1999; Liu et al., 2008), proteins (Elzoghby, 2013; Harsha, 2013) and synthetic polymers (Breunig et al., 2008; Fattal et al., 1998). Among all the available materials, the versatile albumin is an ideal material to fabricate nanoparticles for drug delivery due to its nontoxic, nonimmunogenic, biocompatible and biodegradable properties (Kratz, 2008). Additionally, albumin nanoparticles exhibited high binding capacity of various drugs (Jithan et al., 2011; Kratz, 2014) and were well tolerated without any serious side effects (Ibrahim et al., 2002). Moreover, their polymeric nature controls the release of drug in a sustained and controlled manner for a longer time (Roney et al., 2005). Accordingly, albumin nanoparticles have received
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considerable attention (Elzoghby et al., 2012). Encouragingly, paclitaxel-loaded nanoparticles human serum albumin (Abraxane1) was approved by FDA for clinic use in 2005. It improved water solubility of paclitaxel with enhanced efficacy and tolerability compared with Cremophor based paclitaxel formulation (Cortes and Saura, 2010). To the best of our knowledge, albumin nanoparticles containing TAC has not been reported. Hence, this study was aimed to formulate TAC-loaded bovine serum albumin nanoparticles and evaluate their physicochemical properties, in vitro release, in vivo pharmacokinetics and biodistribution, and find out their effectiveness in reduction of functional nephrotoxicity. 2. Materials and methods 2.1. Materials Tacrolimus (TAC) was supplied by Guangzhou Yibang Company (Guangdong, China). Bovine serum albumin (BSA, purity 99%) was purchased from Beijing Xinjingke Company (Beijing, China). Cholesterol was obtained from Tianjin Bodi Company (Tianjin, China). All other reagents were of analytical grade. The kits used to assay blood urea nitrogen, serum creatinine and creatinine clearance were purchased from Nanjing Kaiji Biological Company (Jiangsu, China). SD rats weighing 250 30 g were supplied by the Experimental Animal Center of Peking University Health Science Center. All care and handling of animals were performed with approval of Institutional Authority for Laboratory Animal Care of Peking University Health Science Center.
aqueous solution (1%, w/v, 30 mL). The resultant mixture was under shearing for 3 min to form a crude emulsion, and then passed through a high-pressure homogenizer (NCJJ-0.007/200, Langfang, China) for 8 cycles at 90–100 MPa, followed by evaporation under vacuum at 30 C for about 30 min to obtain the organic solvent-free dispersion of TAC-BSA-NPs. 2.3. Characterization of TAC-loaded BSA nanoparticles The mean diameter and size distribution, and Zeta potential of TAC-BSA-NPs were determined by dynamic light scattering (DLS) using a Malvern Zeta/Sizer (Nano ZS, Malvern, UK) with a scattering angle of 90 at 25 C. Morphological examination of TAC-BSA-NPs was conducted using transmission electron microscope (TEM, JEM-1230, JEOL, Japan) following negative staining with a drop of 1 wt% phosphotungstic acid solution. To determine the encapsulation efficiency (EE) and loading content (LC) of the prepared nanoparticles, 9 mL methanol was added to 1 mL TAC-BSA-NPs suspension to precipitate protein, followed by centrifugation at 3000 rpm for 5 min. An HPLC system (HP1100, Agilent, USA) with a UV detector set at 220 nm was used to determine the concentration of TAC in the supernatant (Gao et al., 2012). The separation of TAC in 20 mL of samples was performed on a reversed phase column (ODS C18, 5 mm, 4.6 mm 250 mm; Dikma, Beijing, China) at 50 C, eluting with a mixture of acetonitrile and water at a ratio of 3:1 (v/v) at a flow rate of 1.0 mL/min. The EE and LC of TAC-BSA-NPs were calculated as follows: EEð%Þ ¼
amount of TAC loaded in nanoparticles 100 original feeding amount of TAC
LCð%Þ ¼
amount of TAC loaded in nanoparticles 100: total amount of nanoparticles
2.2. Preparation of TAC-loaded BSA nanoparticles TAC-loaded BSA nanoparticles (denoted as TAC-BSA-NPs) were prepared by the emulsification-dispersion technique as previously reported with minor modification (Zhang et al., 2011). Briefly, TAC (6 mg) and cholesterol (12 mg) were dissolved in a mixture of chloroform (0.55 mL) and ethanol (0.05 mL), and then added to BSA
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2.4. In vitro drug release profile The in vitro release behavior of TAC from TAC-BSA-NPs was monitored by using a dialysis-bag diffusion method as described previously with little modification (Li et al., 2010). Briefly, 2 mL of TAC-BSA-NPs dispersion was introduced into a dialysis bag (molecular weight cutoff of 8–14 kD). Following sealed, the dialysis bag was completely submerged into 20 mL of PBS (pH 7.4) containing 0.1% (v/v) Tween 80 at 37 C with a shaking rate of 100 rpm. Periodically, 1 mL of the release medium was taken out at various time intervals and the same volume of fresh medium was added. The concentration of released TAC in release medium was measured by HPLC method as described above. The release experiments were repeated three times and average data were reported. The release of TAC from free TAC solution in ethanol was also tested as control. 2.5. In vivo pharmacokinetics study
Fig. 1. Chemical structure of tacrolimus (TAC).
2.5.1. In vivo experiments 12 healthy male SD rats weighing 250 30 g, fasted for 12 h prior to the experiments but allowed free access to water, were randomly divided into two groups with 6 rats in each group to be intravenously given a single injection of TAC-BSA-NPs and commercial formulation Prograf1 (served as control) through the tail vein at an equivalent TAC dose of 10 mg/kg, respectively. 1 mL of blood sample was drawn from the rat’s orbit at the designated time intervals, and then stored at 20 C until analysis.
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Fig. 2. The size distribution (A), transmission electron micrograph (TEM) images (B) and Zeta potential distribution (C) of TAC-BSA-NPs. The scale bar is 1 mm.
2.5.2. Blood sample analysis Blood samples were analyzed using a slightly modified procedure (Qin et al., 2010). Briefly, in a 10 mL test tube was added 0.5 mL of whole blood, followed by 50 mL internal standard (cyclosporine A) solution with a concentration of 50 mg/mL in methanol, 2 mL of 0.1 mol/L zinc sulfate solution, and 2 mL of acetonitrile. After vortexing for 3 min, the supernatant was collected by centrifugation at 6000 rpm for 10 min and then 4 mL of diethyl ether was added. After centrifugation at 4000 rpm for 10 min, the ether layer was removed and 4 mL of diethyl ether was added followed by centrifugation. Then the same procedure was performed once. The ether phase was combined and evaporated to dryness. The residue was reconstituted with 200 mL of mobile phase (acetonitrile/water, 4/1, v/v) and then 400 mL of n-hexane was added. After vortexing and centrifugation at 4000 rpm for 3 min, the organic phase was removed and 400 mL of n-hexane was added. Then the same vortex, centrifugation and reconstitution procedure were performed as described above for 5 times. The organic layer was combined and concentrated. Finally, 20 mL of samples was taken for HPLC analysis as described above. Linearity of the calibration curves ranged from 2.00 to 50.00 mg/mL (r2 = 0.9958). The coefficient of variation of the inter-day and intra-day precision of the quality control samples ranged from 0.20% to 8.30% and accuracy ranged from 89.6% 4.47% to 95.30% 5.90% The limit of detection (LOD) and the limit of quantification (LOQ) were 0.19 mg/mL and 0.71 mg/mL, respectively. The extraction recovery ranged from 81.31% 4.7% to 89.00% 2.7%. Pharmacokinetic analysis was performed by use of a two compartment model using the DAS2.0 program. The pharmacokinetic parameters were calculated. The area under the concentration–time curve from 0 to t (AUC0t) was calculated with trapezium method. Total clearance (CL), and mean residence time from 0 to t (MRT0t) were obtained.
physiological saline to others. 14 days later, all the rats were weighed again, blood and 24-h urine were harvested and stored at 20 C until analysis. Blood urea nitrogen (BUN), serum creatinine (Scr) and creatinine clearance (CLcr) were assayed to assess renal function by using the kits according to the manufacturer's instructions. CLcr was estimated using the following equation: CLcr ¼
urine creatinine urine volume : serum creatinine 1440
2.7. In vivo biodistribution study 18 healthy male SD rats weighing 250 30 g, fasted overnight prior to the experiments but allowed free access to water, were divided randomly into two groups. TAC-BSA-NPs suspension and Prograf1 were injected intravenously to rats via tail vein at a single dose of 10 mg/kg, respectively. At predetermined time intervals (0.5 h, 10 h and 24 h) after drug administration, blood was collected from postorbital vein. Major organs and tissues of interest (heart, liver, spleen, lung and kidney) were rapidly excised after the animals were sacrificed, and then lightly rinsed with normal saline, wiped with filter paper, and weighted. Both blood and tissue samples were immediately frozen until analysis. The tissue samples were finally homogenized in ice-cold saline to prepare 0.25 g/mL homogenates. Likewise, the blank tissue samples were prepared. The same extraction procedures to blood as described above were used to dispose tissue samples. The concentration of TAC in each tissue was assayed by HPLC method mentioned above. The mean extraction recovery was 82.59% for heart, 87.02% for
2.6. Assessment of renal function 18 healthy male SD rats weighing 250 30 g were used in this study. Prior to the experiment, rats were housed for one week under standard conditions and allowed free access to standard rodent food and water. Before drug administration, rats were weighed and blood was collected, and a 24-h urine sample was collected in a metabolic cage. Then rats were randomly divided into three groups with six rats in each group. One group intravenously received 0.8 mg/kg/day TAC as Prograf1 via tail vein. Another group received the same dose of TAC as TAC-BSA-NPs formulation. Control group received the same volume of
Fig. 3. In vitro release profiles of TAC from TAC-BSA-NPs and its solution in ethanol in pH 7.4 PBS with 0.1% (v/v) Tween 80 at 37 C (n = 3).
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Table 1 The fitted model for the release kinetics of TAC from TAC-BSA-NPs. Model
Zero order
First order
Higuchi model
Fitted equation Correlation coefficient (R)
Mt/M1 = 0.7337t + 48.40 0.7128
Mt/M1 = 82.58(1e0.2544t) 0.9856
Mt/M1 = 7.941t1/2 + 32.61 0.8495
liver, 83.05% for spleen, 84.35% for lung, and 81.66% for kidney, respectively. 2.8. Statistical analysis Data are presented as the mean standard deviation. One-way analysis of variance (ANOVA) was used to determine the statistical significance of differences among multiple groups. A p-value of 0.05 or less was considered to be statistically significant. 3. Results and discussion 3.1. Preparation and characterization of TAC-BSA-NPs As known, albumin nanoparticles are fabricated by specialized nanotechnological techniques such as desolvation, emulsification, thermal gelation and recently nano-spray drying, nab-technology and self-assembly. In the present study, BSA nanoparticles were prepared by emulsification method followed by high-pressure homogenization technique. The O/W emulsion was formed by adding TAC solution (oil phase) into BSA aqueous solution (water phase) and mixing thoroughly by ultrasonication. Nanoparticles were constructed by formation of new disulfide bonds through oxidation and crosslinking of sulfhydryl groups in BSA molecules caused by the shear force of the high pressure homogenization after the crude emulsion passed through a homogenizer (Fu et al., 2009). In order to evaluate the effect of formula and process parameters on particle size and stability to obtain an optimal formula and process technology, various factors such as homogenization pressure, times of cycles as well as mass ratio of cholesterol to TAC were varied. Interestingly, we found that the addition of cholesterol in a mass ratio of 2:1 (m/m) to TAC remarkably decreased the particle size, which might be attributed to the fact that the presence of cholesterol in BSA nanoparticles provided a more lipophilic microenvironment compared to lipophilic area of BSA and thereby increased the hydrophobic interaction of TAC with nanoparticles, and improved the physical stability of the nanoparticles characterized in unremarkable increase (2%) in particle size during storage at room temperature for 24 h compared with cholesterol-free nanoparticles with pronounced increase (66%) in particle size. This indicated that the nanoparticles had considerably high thermodynamic stability
without aggregation. Moreover, our results revealed that homogenization performance under 100 MPa with 8 cycles was preferred to obtain stable nanoparticles with smaller diameter. As shown in Fig. 2A, the prepared TAC-BSA-NPs exhibited an average diameter of 189.5 7.2 nm with a polydispersity index of 0.23 0.05 determined by DLS. The morphology of TAC-BSA-NPs examined by TEM was depicted in Fig. 2B. The nanoparticles presented a nearly spherical shape. As known, Zeta potential greatly influences the stability of NPs in suspension through electrostatic repulsion between the particles. NPs with greater Zeta potential values (either positive or negative) produce greater repulsive forces which prevent nanoparticles from aggregation, and hence facilitate easy redispersion and enhance the stability of the product (Mainardes and Evangelista, 2005). Further, taking consideration of both electrostatic and steric stabilization of NPs, a zeta potential value ranged from 30 mV to +30 mV is highly desirable (Freitas and Müller, 1998). In the present study, Zeta potential of TAC-BSA-NPs was determined to be 20.9 0.5 mV (Fig. 2C), suggesting strong repellent forces among particles to prevent aggregation of the NPs, which was desirable to produce a stable NPs suspension. This was also strongly supported by the negligible change in the particle size during storage. The loading content (LC) and encapsulation efficiency (EE) of TAC-BSA-NPs was (1.7 0.13)% and (85 3.0)%, respectively, demonstrating that TAC could be effectively loaded inside the nanoparticles. 3.2. In vitro release of TAC from TAC-BSA-NPs The in vitro release of TAC from TAC-BSA-NPs was investigated by a dialysis-bag diffusion method at 37 C. Prior to conducting the release assays, a control experiment with free TAC confirmed that the dialysis membrane tubing with molecular weight cut-off of 8– 14 kD could not restrict diffusion of the released free TAC, and reached 100% release after 8 h (Fig. 3). Furthermore, the sink condition was respected by addition of 0.1% (v/v) Tween 80 in pH 7.4 PBS. It was obvious that the release of TAC from TAC-BSA-NPs was slower than the diffusion of free TAC, and exhibited a biphasic pattern characterized with relative burst drug release and sustained drug release. At the initial stage, the fast release phase, owing to burst effect, might be attributed to desorption and diffusion of TAC from the surface of BSA nanoparticles. This is favorable and should be sufficient to exhibit therapeutic effect. Subsequently, the encapsulated TAC released from the internal of BSA nanoparticles in a sustained manner due to the slow diffusion of TAC across the albumin matrix, which provided the possibility to continually take effect. The similar phenomena were observed in
Table 2 Pharmacokinetic parameters of TAC following intravenous administration of TACBSA-NPs and Prograf1 to rats at a single dose of 10 mg/kg (n = 6).
Fig. 4. Blood concentration–time curve of TAC after intravenous administration of Prograf1 and TAC-BSA-NPs to rats at a single dose of 10 mg/kg (n = 6).
Parameter
Prograf1
TAC-BSA-NPs
AUC024 h (min mg/L) CL (L/h/kg) MRT024 h (h)
84.4 10.98 0.072 0.018 8.90 0.67
151.5 46.69* 0.038 0.016* 10.58 0.57*
AUC0–24 h, area under whole blood concentration–time curve from 0 to 24 h; CL, total clearance; MRT0–24 h, mean residence time from 0 to 24 h. * p < 0.05 TAC-BSA-NPs vs Prograf1.
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previous BSA-containing nanoparticulate systems (Wilson et al., 2012; Wilson et al., 2014). The release mechanism of TAC from TAC-BSA-NPs was determined by fitting drug release data to various kinetic models. Comparison of the correlation coefficient R values from the applied models (Table 1) suggested that first order kinetics was the best fit kinetic model with R value of 0.9856 to describe the release kinetics of TAC-BSA-NPs. 3.3. In vivo pharmacokinetics of TAC-BSA-NPs SD rats were treated with the developed TAC-BSA-NPs at a single dose of 10 mg/kg TAC to rats to evaluate their in vivo kinetic performance, and commercial formulation Prograf1 was also studied as control. The whole concentration–time curves of TAC for two formulations were shown in Fig. 4 after intravenous administration to rats. Compared with Prograf1, TAC-BSA-NPs showed similar initial phase of rapid decrease in whole blood concentration of TAC in the first 1 h, while much slower decline after 1.5 h post dose. From 1.5 h to 12 h, TAC-BSA-NPs showed a remarkably delayed blood clearance with higher TAC concentration at each time point, which could be explained by the sustained release characteristic of TAC-BSA-NPs mentioned above.
Fig. 6. Concentration of TAC in the tissues and blood at 0.5 h, 10 h and 24 h after intravenous administration of Prograf1 (A) and TAC-BSA-NPs (B) to rats at a single dose of 10 mg/kg (n = 3). (C) Kidney/blood levels ratios at 0.5 h, 10 h and 24 h after administration of TAC-BSA-NPs and Prograf1 at a single dose of 10 mg/kg of TAC.
Pharmacokinetic profiles of TAC for the two formulations were found to be well fitted to two-compartment model. The calculated pharmacokinetic parameters were listed in Table 2. Results revealed a difference in the pharmacokinetics for the two formulations. Compared with Prograf1, which is a solution of TAC in mixture of ethanol with polyethylene hydrogenated castor oil (HCO-60) (40:60, v/v), TAC-BSA-NPs had significantly higher (1.80-fold higher) area under the whole blood concentration–time curve from 0 to 24 h (AUC024 h) (p < 0.05), which might be attributed to the fact that encapsulated TAC could be protected from metabolizing enzyme in the liver (Li and Huang, 2008), and TAC-BSA-NPs were big enough to avoid or reduce the clearance by renal filtration (Duan and Li, 2013). Consequently, Prograf1 exhibited a fast clearance (CL) (1.90-fold higher), whereas TACBSA-NPs showed a significantly slower clearance on account of the combined effect of protected liver metabolism and reduced renal clearance. This effect was strongly supported by the significantly increased mean residence time (MRT) of TAC-BSA-NPs, which indicated that TACI-BSA-NPs prolonged the systemic circulation time. These results were similar to the previous report (Wei et al., 2014). 3.4. Renal function The immunosuppressive effect of TAC is 50–100 times higher than that of ciclosporin, however, they have the same side effects associated with nephrotoxic, diabetogenic, neurological, and cardiovascular effects (Scott et al., 2003). Due to the marked nephrotoxicity, presumably related with inhibition of the activation of calcineurin resulting from the increase in the Ca2+ influx into tubular cells and thereby inhibition of protein synthesis and cell proliferation in tubular cells (Moutabarrik et al., 1991), the use of TAC is always limited in clinic. The renal function study in the present paper was aimed to evaluate TAC-induced functional nephrotoxicity in rats for TAC-BSA-NPs. Serum creatinine (Scr), creatinine clearance (CLcr) and blood urea nitrogen (BUN) were selected as biochemical markers to manifest the TAC-induced functional nephrotoxicity in previous document (Chen et al., 2007). In addition, BUN, Scr and CLcr are commonly used as assessment indicators of renal function in clinic (Chen et al., 2009). The concentration of Scr and BUN depends on the glomerular filtration rate (GFR), and CLcr is usually used to calculate GFR. As known, renal dysfunction results in reduction of glomerular filtration of Scr and BUN, thus Scr and BUN increase. Hence, in the present study, creatinine level in urine samples, and creatinine and urea nitrogen levels in blood samples were determined before (day 0, initial) and after (day 14) administration of TAC-BSA-NPs. Saline and Prograf1 were tested as negative and positive control, respectively. It was found that there were no significant changes in Scr BUN and CLcr from baseline for control animals given saline after 14 days of treatment. In contrast, repeated dose of Prograf1 caused highly significant increases in BUN (91.80%) (Fig. 5A) and Scr (410.80%) (Fig. 5B) (p < 0.01), whereas TAC-BSA-NPs caused significant increase in BUN (42.50%) (Fig. 5A) (p < 0.05) and highly significant increase in Scr (175.00%) (Fig. 5B) (p < 0.01). These treatments also resulted in highly significant decreases in CLcr (86.40% for TAC-BSA-NPs and 70.20% for Prograf1, respectively) (Fig. 5C) (p < 0.01). Overall, the degree of increase in BUN for different treatment groups was ranked as saline < TAC-BSANPs < Prograf1. The degree of increase in Scr followed the same order. The level of decrease in mean CLcr for different treatment groups was ranked as saline < TAC-BSA-NPs < Prograf1. These indicated that both TAC-BSA-NPs and Prograf1 had effect on renal function to some extent. Further, compared with Prograf1, TACBSANPs exhibited highly significantly lower increase in Scr and BUN (p < 0.01), and significantly lower reduction in CLcr (p < 0.05).
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Fig. 5. Changes in (A) blood urea nitrogen (BUN), (B) serum creatinine (Scr), (C) creatinine clearance (CLcr) and (D) body weight of rats after 14 days treatments with saline, 0.8 mg/kg/day TAC as TAC-BSA-NPs and Prograf1 expressed as a percent of variable measured in the same treatment group on day zero (n = 6). *p < 0.05, **p < 0.01, nsp > 0.05.
Based on these results, it appeared that the damage of TAC by intravenous administration of TAC-BSA-NPs to renal function was significantly lower than that of Prograf1. Further, after 14 days of treatments, changes in body weight of rats given saline, TAC-BSA-NPs and Prograf1 were depicted in Fig. 5D. An interesting phenomenon observed was that rats given saline gained body weight 40.6% of initial, whereas rats receiving Prograf1 gained body weight only 28.0% of initial. In contrast, the increase (30.9% of initial) in body weight of rats given TAC-BSA-NPs was more than that of rats receiving Prograf1, although the difference was not significant and the weight increase for TAC-BSANPs group was not as high as the saline group. In conclusion, TACBSA-NPs could reduce the functional nephrotoxicity caused by TAC compared with Prograf1. 3.5. Tissue distribution Previous document reported that polymeric micelles had a potential in restricting the nephrotoxicity of cyclosporine A by reducing kidney uptake of cyclosporine A by 2.6-fold, and increased cyclosporine A levels in blood by 2.1-fold (Aliabadi et al., 2008). This promoted us to explore the plausible difference in in vivo distribution of TAC for TAC-BSA-NPs and Prograf1. Therefore, biodistribution of TAC in SD rats was evaluated at 0.5 h, 10 h and 24 h after intravenous administration of TAC-BSANPs and commercial formulation Prograf1 at a single dose of 10 mg/kg, respectively. The concentration of TAC found in sampled tissues and blood at each sampling point was expressed in the content of TAC per gram tissue or per milliliter blood (Fig. 6A and B). The results showed that TAC was widely distributed to the rat tissues when treated with the two formulations, which was in good agreement with the previous document (Yura et al., 1999), and the concentration of TAC reduced with time in all tissue samples. For the formulation of Prograf1 (Fig. 6A), the lung and heart had higher TAC concentration among all the detected tissues at each time point. The TAC level in tissues were found to decrease
in the order of lung > heart > spleen > kidney > liver at 0.5 h, lung > heart > spleen > liver > kidney at 10 h, and lung > heart > liver > spleen > kidney at 24 h, respectively, which was similar to previous report (Venkataramanan et al., 1990). In comparison, TAC-BSA-NPs changed the distribution of TAC in tissues (Fig. 6B). 0.5 h after dosing, the level of TAC in the lung was the highest, followed by spleen, as previously reported (Yokogawa et al., 1999). The TAC concentration in tissues were found to decrease in the order of lung > spleen > liver heart > kidney at 0.5 h, lung > liver heart > spleen > kidney at 10 h, and liver > lung > heart > spleen > kidney at 24 h, respectively. Specifically, TAC-BSA-NPs had significantly lower AUC024 h (94.54 12.12 min mg/g) of kidney compared with Prograf1 (123.57 13.82 min mg/g) (p < 0.05). Further, the TAC concentration in heart, spleen and lung for TAC-BSA-NPs was significantly lower than that for Prograf1. However, TAC level in liver for TAC-BSA-NPs was significantly higher than that for Prograf1, which might be attributed to the fact that the size of BSA nanoparticles made TAC-BSA-NPs more easily be extracted by liver (Kapoor et al., 2008). More importantly, TAC-BSA-NPs conspicuously enhanced TAC level in blood and reduced TAC accumulation in kidney, which is desirable to decrease kidney toxicity. Specifically, in comparison to Prograf1, analysis of the results revealed a reduced delivery of TAC by TAC-BSA-NPs to kidneys by 1.8-fold, 1.2-fold and 1.2-fold, and increased TAC levels in blood by 1.6-fold, 1.7-fold and 1.6-fold at 0.5 h, 10 h and 24 h, respectively, which was similar to cyclosporine A-loaded polymeric micelles (Aliabadi et al., 2008). To further validate the effect of distribution of TAC on renal function, the ratio of TAC level in kidney to that in blood was compared between TACBSA-NPs and Prograf1. As was seen in Fig. 6C, the concentration ratios of TAC in kidney to those in blood over the course of the study for TAC-BSA-NPs were significantly lower than those of Prograf1 (p < 0.05). These could explain the improvement of renal function by TAC-BSA-NPs compared with Prograf1. All together, these results demonstrated that with the aid of TAC-BSA-NPs, more TAC could be accumulated in blood whereas a subsequent reduced
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distribution in other organs, especially in kidney, which was beneficial to reduction of kidney toxicity. 4. Conclusions In the present work, TAC-BSA-NPs with uniform particle size below 190 nm were successfully prepared by the emulsificationdispersion method and exhibited a biphasic release pattern characterized with an initial burst release and sustained drug release. TAC-BSA-NPs had more favorable pharmacokinetics and biodistribution characteristics compared with Prograf1, therefore showing a potential to reduce TAC delivery to kidney and thereby functional nephrotoxicity while maintaining high blood levels. In summary, the findings of this study further provided important convincing evidence of the effectiveness of appropriately designed drug delivery systems to enhance the therapeutic index of drugs with serious side effects. Acknowledgements This research was financially supported by the National Natural Science Foundation of China (No. 81172990), the National Key Science Research Program of China (973 Program, 2015CB932100) and the Innovation Team of Ministry of Education (No. BMU20110263). References Aliabadi, H.M., Elhasi, S., Brocks, D.R., Lavasanifar, A., 2008. Polymeric micellar delivery reduces kidney distribution and nephrotoxic effects of cyclosporine A after multiple dosing. J. Pharm. Sci. 97, 1916–1926. Bottiger, Y., Brattstrom, C., Tyden, G., Sawe, J., Groth, C.G., 1999. Tacrolimus whole blood concentrations correlate closely to side-effects in renal transplant recipients. Br. J. Clin. Pharmacol. 48, 445–448. Breunig, M., Bauer, S., Goepferich, A., 2008. Polymers and nanoparticles: intelligent tools for intracellular targeting? Eur. J. Pharm. Biopharm. 68, 112–128. Brewster, M.E., Loftsson, T., 2007. Cyclodextrins as pharmaceutical solubilizers. Adv. Drug Deliv. Rev. 59, 645–666. Cardenas, M.E., Zhu, D., Heitman, J., 1995. Molecular mechanisms of immunosuppression by cyclosporine FK506, and rapamycin. Curr. Opin. Nephrol. Hypertens. 4, 472–477. Chen, Y.H., Liang, Y.X., Chen, L.Q., Liang, J.J., Zhang, J., Qiu, J., Li, W.L., Hu, J.B., Xie, K.J., Zhong, W.D., Chen, L.Z., Zheng, K.L., 2007. Prevention of diltiazem in tacrolimusinduced nephrotoxicity: experiment with rats. Zhonghua Yi Xue Za Zhi 87, 2235–2237. Chen, Y.H., Liang, Y.X., Chen, L.Q., Lu, Y.M., Liang, J.J., Zhang, J., Qiu, J., Xie, K.J., Hu, J.B., Zhong, W.D., Wang, B., Chen, L.Z., Zheng, K.L., 2009. Nephrotoxicity of tacrolimus and preventive effect of diltiazem: experiment with rats. Zhonghua Yi Xue Za Zhi 89, 704–708. Cortes, J., Saura, C., 2010. Nanoparticle albumin-bound (nabTM)-paclitaxel: improving efficacy and tolerability by targeted drug delivery in metastatic breast cancer. Eur. J. Cancer Suppl. 8, 1–10. Duan, X., Li, Y., 2013. Physicochemical characteristics of nanoparticles affect circulation, biodistribution, cellular internalization, and trafficking. Small 9, 1521–1532. Elzoghby, A.O., 2013. Gelatin-based nanoparticles as drug and gene delivery systems: reviewing three decades of research. J. Control. Release 172, 1075– 1091. Elzoghby, A.O., Samy, W.M., Elgindy, N.A., 2012. Albumin-based nanoparticles as potential controlled release drug delivery systems. J. Control. Release 157, 168– 182. Fattal, E., Vauthier, C., Aynie, I., Nakada, Y., Lambert, G., Malvy, C., Couvreur, P., 1998. Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides. J. Control. Release 53, 137–143. Fernandez-Urrusuno, R., Calvo, P., Remunan-Lopez, C., Vila-Jato, J.L., Alonso, M.J., 1999. Enhancement of nasal absorption of insulin using chitosan nanoparticles. Pharm. Res. 16, 1576–1581. Freitas, C., Müller, R.H., 1998. Effect of light and temperature on zeta potential and physical stability in solid lipid nanoparticle (SLNTM) dispersions. Int. J. Pharm. 168, 221–229. Fu, Q., Sun, J., Zhang, W., Sui, X., Yan, Z., He, Z., 2009. Nanoparticle albumin-bound (NAB) technology is a promising method for anti-cancer drug delivery. Recent Pat. Anti-Cancer Drug Discov. 4, 262–272. Gao, S., Sun, J., Fu, D., Zhao, H., Lan, M., Gao, F., 2012. Preparation, characterization and pharmacokinetic studies of tacrolimus-dimethyl-beta-cyclodextrin inclusion complex-loaded albumin nanoparticles. Int. J. Pharm. 427, 410–416.
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Bovine serum albumin nanoparticles for delivery of tacrolimus to reduce its kidney uptake and functional nephrotoxicity Lei Zhao, Yanxia Zhou, Yajie Gao, Shujin Ma, Chao Zhang, Jinwen Li, Dishi Wang, Xueping Li, Chengwei Li, Yan Liu * , Xinru Li * Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 November 2014 Received in revised form 31 December 2014 Accepted 10 February 2015 Available online 11 February 2015
The purpose of the present study was to develop a new nanoparticulate formulation for delivery of tacrolimus to reduce its kidney distribution and functional nephrotoxicity. Tacrolimus (TAC)-loaded bovine serum albumin (BSA) nanoparticles (TAC-BSA-NPs) were prepared by emulsification-dispersion technique. The obtained TAC-BSA-NPs, with 189.50 7.15 nm of diameter and 20.86 0.45 mV of Zeta potential determined by DLS, were spherical in shape observed by TEM. The drug loading content and encapsulation efficiency were (1.7 0.13)% and (85 3.0)%, respectively. The in vitro release of TAC-BSANPs exhibited biphasic drug release pattern with an initial burst release and subsequently sustained release. Pharmacokinetic analysis displayed that TAC-BSA-NPs could enhance the drug blood level and prolong the circulation time in comparison to Prograf1. Meanwhile, compared with Prograf1, TAC-BSANPs could deliver less TAC to kidney and simultaneously reduce the functional nephrotoxicity of TAC to kidney. In conclusion, BSA nanoparticles might be a more safe carrier for delivery of hydrophobic drug TAC. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Tacrolimus Bovine serum albumin nanoparticles Pharmacokinetics Nephrotoxicity Tissue distribution
1. Introduction Tacrolimus (TAC) (Fig. 1), a macrolide immunosuppressant, has been widely used to prevent acute rejection in organ transplants (Wallemacq and Reding, 1993). Similar to cyclosporine A, a cyclic undecapeptide immunosuppressant, the action mechanism of TAC has been demonstrated to bind to the TAC binding protein (immunophilin FKBP-12) inside the activated T-cells (Cardenas et al., 1995), which inhibits the activity of calcineurin for dephosphorylating the nuclear factor of activated T cells (NFAT), thereby reduces the generation of IL-2 and inhibits activation and proliferation of T cells, leading to suppressed immune response (Ruff and Leach, 1995; Shaw et al., 1995). TAC exhibits much greater immunosuppressive activity than cyclosporine A (Geissler and Schlitt, 2009) and provides a better side effect profile and increases long-term survival in patients (Jurewicz, 2003; Wiesner, 1998). However, its therapeutic efficacy is limited due to its poor water solubility. In addition, TAC is known to exhibit some adverse events, such as nephrotoxicity, neurotoxicity, hypertension and diabetogenic effects (Bottiger et al., 1999). Its commercial formulation Prograf1 contains high amount of polyethylene
* Corresponding authors. Tel.: +86 10 82801508. E-mail addresses: [email protected] (Y. Liu), [email protected] (X. Li). http://dx.doi.org/10.1016/j.ijpharm.2015.02.018 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
hydrogenated castor oil (HCO-60) which has been found to associate with severe side effect of anaphylaxis and still showed nephrotoxicity to some extent (Nicolai and Bunyavanich, 2012). Therefore, many efforts have been devoted to improve the solubility of TAC and reduce the side effects of TAC, including encapsulation of TAC in liposomes (Ishii et al., 2013), polymeric micelles (Wang et al., 2011), cyclodextrin inclusion (Brewster and Loftsson, 2007), nanoparticles (Xu et al., 2014), nanocapsules (Nassar et al., 2009) and self-microemulsifying drug delivery system (von Suesskind-Schwendi et al., 2013). Among these drug delivery vehicles, nanoparticles have gained much attention in recent years. Nanoparticles are made of a variety of polymers, such as polysaccharides (Fernandez-Urrusuno et al., 1999; Liu et al., 2008), proteins (Elzoghby, 2013; Harsha, 2013) and synthetic polymers (Breunig et al., 2008; Fattal et al., 1998). Among all the available materials, the versatile albumin is an ideal material to fabricate nanoparticles for drug delivery due to its nontoxic, nonimmunogenic, biocompatible and biodegradable properties (Kratz, 2008). Additionally, albumin nanoparticles exhibited high binding capacity of various drugs (Jithan et al., 2011; Kratz, 2014) and were well tolerated without any serious side effects (Ibrahim et al., 2002). Moreover, their polymeric nature controls the release of drug in a sustained and controlled manner for a longer time (Roney et al., 2005). Accordingly, albumin nanoparticles have received
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considerable attention (Elzoghby et al., 2012). Encouragingly, paclitaxel-loaded nanoparticles human serum albumin (Abraxane1) was approved by FDA for clinic use in 2005. It improved water solubility of paclitaxel with enhanced efficacy and tolerability compared with Cremophor based paclitaxel formulation (Cortes and Saura, 2010). To the best of our knowledge, albumin nanoparticles containing TAC has not been reported. Hence, this study was aimed to formulate TAC-loaded bovine serum albumin nanoparticles and evaluate their physicochemical properties, in vitro release, in vivo pharmacokinetics and biodistribution, and find out their effectiveness in reduction of functional nephrotoxicity. 2. Materials and methods 2.1. Materials Tacrolimus (TAC) was supplied by Guangzhou Yibang Company (Guangdong, China). Bovine serum albumin (BSA, purity 99%) was purchased from Beijing Xinjingke Company (Beijing, China). Cholesterol was obtained from Tianjin Bodi Company (Tianjin, China). All other reagents were of analytical grade. The kits used to assay blood urea nitrogen, serum creatinine and creatinine clearance were purchased from Nanjing Kaiji Biological Company (Jiangsu, China). SD rats weighing 250 30 g were supplied by the Experimental Animal Center of Peking University Health Science Center. All care and handling of animals were performed with approval of Institutional Authority for Laboratory Animal Care of Peking University Health Science Center.
aqueous solution (1%, w/v, 30 mL). The resultant mixture was under shearing for 3 min to form a crude emulsion, and then passed through a high-pressure homogenizer (NCJJ-0.007/200, Langfang, China) for 8 cycles at 90–100 MPa, followed by evaporation under vacuum at 30 C for about 30 min to obtain the organic solvent-free dispersion of TAC-BSA-NPs. 2.3. Characterization of TAC-loaded BSA nanoparticles The mean diameter and size distribution, and Zeta potential of TAC-BSA-NPs were determined by dynamic light scattering (DLS) using a Malvern Zeta/Sizer (Nano ZS, Malvern, UK) with a scattering angle of 90 at 25 C. Morphological examination of TAC-BSA-NPs was conducted using transmission electron microscope (TEM, JEM-1230, JEOL, Japan) following negative staining with a drop of 1 wt% phosphotungstic acid solution. To determine the encapsulation efficiency (EE) and loading content (LC) of the prepared nanoparticles, 9 mL methanol was added to 1 mL TAC-BSA-NPs suspension to precipitate protein, followed by centrifugation at 3000 rpm for 5 min. An HPLC system (HP1100, Agilent, USA) with a UV detector set at 220 nm was used to determine the concentration of TAC in the supernatant (Gao et al., 2012). The separation of TAC in 20 mL of samples was performed on a reversed phase column (ODS C18, 5 mm, 4.6 mm 250 mm; Dikma, Beijing, China) at 50 C, eluting with a mixture of acetonitrile and water at a ratio of 3:1 (v/v) at a flow rate of 1.0 mL/min. The EE and LC of TAC-BSA-NPs were calculated as follows: EEð%Þ ¼
amount of TAC loaded in nanoparticles 100 original feeding amount of TAC
LCð%Þ ¼
amount of TAC loaded in nanoparticles 100: total amount of nanoparticles
2.2. Preparation of TAC-loaded BSA nanoparticles TAC-loaded BSA nanoparticles (denoted as TAC-BSA-NPs) were prepared by the emulsification-dispersion technique as previously reported with minor modification (Zhang et al., 2011). Briefly, TAC (6 mg) and cholesterol (12 mg) were dissolved in a mixture of chloroform (0.55 mL) and ethanol (0.05 mL), and then added to BSA
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2.4. In vitro drug release profile The in vitro release behavior of TAC from TAC-BSA-NPs was monitored by using a dialysis-bag diffusion method as described previously with little modification (Li et al., 2010). Briefly, 2 mL of TAC-BSA-NPs dispersion was introduced into a dialysis bag (molecular weight cutoff of 8–14 kD). Following sealed, the dialysis bag was completely submerged into 20 mL of PBS (pH 7.4) containing 0.1% (v/v) Tween 80 at 37 C with a shaking rate of 100 rpm. Periodically, 1 mL of the release medium was taken out at various time intervals and the same volume of fresh medium was added. The concentration of released TAC in release medium was measured by HPLC method as described above. The release experiments were repeated three times and average data were reported. The release of TAC from free TAC solution in ethanol was also tested as control. 2.5. In vivo pharmacokinetics study
Fig. 1. Chemical structure of tacrolimus (TAC).
2.5.1. In vivo experiments 12 healthy male SD rats weighing 250 30 g, fasted for 12 h prior to the experiments but allowed free access to water, were randomly divided into two groups with 6 rats in each group to be intravenously given a single injection of TAC-BSA-NPs and commercial formulation Prograf1 (served as control) through the tail vein at an equivalent TAC dose of 10 mg/kg, respectively. 1 mL of blood sample was drawn from the rat’s orbit at the designated time intervals, and then stored at 20 C until analysis.
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Fig. 2. The size distribution (A), transmission electron micrograph (TEM) images (B) and Zeta potential distribution (C) of TAC-BSA-NPs. The scale bar is 1 mm.
2.5.2. Blood sample analysis Blood samples were analyzed using a slightly modified procedure (Qin et al., 2010). Briefly, in a 10 mL test tube was added 0.5 mL of whole blood, followed by 50 mL internal standard (cyclosporine A) solution with a concentration of 50 mg/mL in methanol, 2 mL of 0.1 mol/L zinc sulfate solution, and 2 mL of acetonitrile. After vortexing for 3 min, the supernatant was collected by centrifugation at 6000 rpm for 10 min and then 4 mL of diethyl ether was added. After centrifugation at 4000 rpm for 10 min, the ether layer was removed and 4 mL of diethyl ether was added followed by centrifugation. Then the same procedure was performed once. The ether phase was combined and evaporated to dryness. The residue was reconstituted with 200 mL of mobile phase (acetonitrile/water, 4/1, v/v) and then 400 mL of n-hexane was added. After vortexing and centrifugation at 4000 rpm for 3 min, the organic phase was removed and 400 mL of n-hexane was added. Then the same vortex, centrifugation and reconstitution procedure were performed as described above for 5 times. The organic layer was combined and concentrated. Finally, 20 mL of samples was taken for HPLC analysis as described above. Linearity of the calibration curves ranged from 2.00 to 50.00 mg/mL (r2 = 0.9958). The coefficient of variation of the inter-day and intra-day precision of the quality control samples ranged from 0.20% to 8.30% and accuracy ranged from 89.6% 4.47% to 95.30% 5.90% The limit of detection (LOD) and the limit of quantification (LOQ) were 0.19 mg/mL and 0.71 mg/mL, respectively. The extraction recovery ranged from 81.31% 4.7% to 89.00% 2.7%. Pharmacokinetic analysis was performed by use of a two compartment model using the DAS2.0 program. The pharmacokinetic parameters were calculated. The area under the concentration–time curve from 0 to t (AUC0t) was calculated with trapezium method. Total clearance (CL), and mean residence time from 0 to t (MRT0t) were obtained.
physiological saline to others. 14 days later, all the rats were weighed again, blood and 24-h urine were harvested and stored at 20 C until analysis. Blood urea nitrogen (BUN), serum creatinine (Scr) and creatinine clearance (CLcr) were assayed to assess renal function by using the kits according to the manufacturer's instructions. CLcr was estimated using the following equation: CLcr ¼
urine creatinine urine volume : serum creatinine 1440
2.7. In vivo biodistribution study 18 healthy male SD rats weighing 250 30 g, fasted overnight prior to the experiments but allowed free access to water, were divided randomly into two groups. TAC-BSA-NPs suspension and Prograf1 were injected intravenously to rats via tail vein at a single dose of 10 mg/kg, respectively. At predetermined time intervals (0.5 h, 10 h and 24 h) after drug administration, blood was collected from postorbital vein. Major organs and tissues of interest (heart, liver, spleen, lung and kidney) were rapidly excised after the animals were sacrificed, and then lightly rinsed with normal saline, wiped with filter paper, and weighted. Both blood and tissue samples were immediately frozen until analysis. The tissue samples were finally homogenized in ice-cold saline to prepare 0.25 g/mL homogenates. Likewise, the blank tissue samples were prepared. The same extraction procedures to blood as described above were used to dispose tissue samples. The concentration of TAC in each tissue was assayed by HPLC method mentioned above. The mean extraction recovery was 82.59% for heart, 87.02% for
2.6. Assessment of renal function 18 healthy male SD rats weighing 250 30 g were used in this study. Prior to the experiment, rats were housed for one week under standard conditions and allowed free access to standard rodent food and water. Before drug administration, rats were weighed and blood was collected, and a 24-h urine sample was collected in a metabolic cage. Then rats were randomly divided into three groups with six rats in each group. One group intravenously received 0.8 mg/kg/day TAC as Prograf1 via tail vein. Another group received the same dose of TAC as TAC-BSA-NPs formulation. Control group received the same volume of
Fig. 3. In vitro release profiles of TAC from TAC-BSA-NPs and its solution in ethanol in pH 7.4 PBS with 0.1% (v/v) Tween 80 at 37 C (n = 3).
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Table 1 The fitted model for the release kinetics of TAC from TAC-BSA-NPs. Model
Zero order
First order
Higuchi model
Fitted equation Correlation coefficient (R)
Mt/M1 = 0.7337t + 48.40 0.7128
Mt/M1 = 82.58(1e0.2544t) 0.9856
Mt/M1 = 7.941t1/2 + 32.61 0.8495
liver, 83.05% for spleen, 84.35% for lung, and 81.66% for kidney, respectively. 2.8. Statistical analysis Data are presented as the mean standard deviation. One-way analysis of variance (ANOVA) was used to determine the statistical significance of differences among multiple groups. A p-value of 0.05 or less was considered to be statistically significant. 3. Results and discussion 3.1. Preparation and characterization of TAC-BSA-NPs As known, albumin nanoparticles are fabricated by specialized nanotechnological techniques such as desolvation, emulsification, thermal gelation and recently nano-spray drying, nab-technology and self-assembly. In the present study, BSA nanoparticles were prepared by emulsification method followed by high-pressure homogenization technique. The O/W emulsion was formed by adding TAC solution (oil phase) into BSA aqueous solution (water phase) and mixing thoroughly by ultrasonication. Nanoparticles were constructed by formation of new disulfide bonds through oxidation and crosslinking of sulfhydryl groups in BSA molecules caused by the shear force of the high pressure homogenization after the crude emulsion passed through a homogenizer (Fu et al., 2009). In order to evaluate the effect of formula and process parameters on particle size and stability to obtain an optimal formula and process technology, various factors such as homogenization pressure, times of cycles as well as mass ratio of cholesterol to TAC were varied. Interestingly, we found that the addition of cholesterol in a mass ratio of 2:1 (m/m) to TAC remarkably decreased the particle size, which might be attributed to the fact that the presence of cholesterol in BSA nanoparticles provided a more lipophilic microenvironment compared to lipophilic area of BSA and thereby increased the hydrophobic interaction of TAC with nanoparticles, and improved the physical stability of the nanoparticles characterized in unremarkable increase (2%) in particle size during storage at room temperature for 24 h compared with cholesterol-free nanoparticles with pronounced increase (66%) in particle size. This indicated that the nanoparticles had considerably high thermodynamic stability
without aggregation. Moreover, our results revealed that homogenization performance under 100 MPa with 8 cycles was preferred to obtain stable nanoparticles with smaller diameter. As shown in Fig. 2A, the prepared TAC-BSA-NPs exhibited an average diameter of 189.5 7.2 nm with a polydispersity index of 0.23 0.05 determined by DLS. The morphology of TAC-BSA-NPs examined by TEM was depicted in Fig. 2B. The nanoparticles presented a nearly spherical shape. As known, Zeta potential greatly influences the stability of NPs in suspension through electrostatic repulsion between the particles. NPs with greater Zeta potential values (either positive or negative) produce greater repulsive forces which prevent nanoparticles from aggregation, and hence facilitate easy redispersion and enhance the stability of the product (Mainardes and Evangelista, 2005). Further, taking consideration of both electrostatic and steric stabilization of NPs, a zeta potential value ranged from 30 mV to +30 mV is highly desirable (Freitas and Müller, 1998). In the present study, Zeta potential of TAC-BSA-NPs was determined to be 20.9 0.5 mV (Fig. 2C), suggesting strong repellent forces among particles to prevent aggregation of the NPs, which was desirable to produce a stable NPs suspension. This was also strongly supported by the negligible change in the particle size during storage. The loading content (LC) and encapsulation efficiency (EE) of TAC-BSA-NPs was (1.7 0.13)% and (85 3.0)%, respectively, demonstrating that TAC could be effectively loaded inside the nanoparticles. 3.2. In vitro release of TAC from TAC-BSA-NPs The in vitro release of TAC from TAC-BSA-NPs was investigated by a dialysis-bag diffusion method at 37 C. Prior to conducting the release assays, a control experiment with free TAC confirmed that the dialysis membrane tubing with molecular weight cut-off of 8– 14 kD could not restrict diffusion of the released free TAC, and reached 100% release after 8 h (Fig. 3). Furthermore, the sink condition was respected by addition of 0.1% (v/v) Tween 80 in pH 7.4 PBS. It was obvious that the release of TAC from TAC-BSA-NPs was slower than the diffusion of free TAC, and exhibited a biphasic pattern characterized with relative burst drug release and sustained drug release. At the initial stage, the fast release phase, owing to burst effect, might be attributed to desorption and diffusion of TAC from the surface of BSA nanoparticles. This is favorable and should be sufficient to exhibit therapeutic effect. Subsequently, the encapsulated TAC released from the internal of BSA nanoparticles in a sustained manner due to the slow diffusion of TAC across the albumin matrix, which provided the possibility to continually take effect. The similar phenomena were observed in
Table 2 Pharmacokinetic parameters of TAC following intravenous administration of TACBSA-NPs and Prograf1 to rats at a single dose of 10 mg/kg (n = 6).
Fig. 4. Blood concentration–time curve of TAC after intravenous administration of Prograf1 and TAC-BSA-NPs to rats at a single dose of 10 mg/kg (n = 6).
Parameter
Prograf1
TAC-BSA-NPs
AUC024 h (min mg/L) CL (L/h/kg) MRT024 h (h)
84.4 10.98 0.072 0.018 8.90 0.67
151.5 46.69* 0.038 0.016* 10.58 0.57*
AUC0–24 h, area under whole blood concentration–time curve from 0 to 24 h; CL, total clearance; MRT0–24 h, mean residence time from 0 to 24 h. * p < 0.05 TAC-BSA-NPs vs Prograf1.
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previous BSA-containing nanoparticulate systems (Wilson et al., 2012; Wilson et al., 2014). The release mechanism of TAC from TAC-BSA-NPs was determined by fitting drug release data to various kinetic models. Comparison of the correlation coefficient R values from the applied models (Table 1) suggested that first order kinetics was the best fit kinetic model with R value of 0.9856 to describe the release kinetics of TAC-BSA-NPs. 3.3. In vivo pharmacokinetics of TAC-BSA-NPs SD rats were treated with the developed TAC-BSA-NPs at a single dose of 10 mg/kg TAC to rats to evaluate their in vivo kinetic performance, and commercial formulation Prograf1 was also studied as control. The whole concentration–time curves of TAC for two formulations were shown in Fig. 4 after intravenous administration to rats. Compared with Prograf1, TAC-BSA-NPs showed similar initial phase of rapid decrease in whole blood concentration of TAC in the first 1 h, while much slower decline after 1.5 h post dose. From 1.5 h to 12 h, TAC-BSA-NPs showed a remarkably delayed blood clearance with higher TAC concentration at each time point, which could be explained by the sustained release characteristic of TAC-BSA-NPs mentioned above.
Fig. 6. Concentration of TAC in the tissues and blood at 0.5 h, 10 h and 24 h after intravenous administration of Prograf1 (A) and TAC-BSA-NPs (B) to rats at a single dose of 10 mg/kg (n = 3). (C) Kidney/blood levels ratios at 0.5 h, 10 h and 24 h after administration of TAC-BSA-NPs and Prograf1 at a single dose of 10 mg/kg of TAC.
Pharmacokinetic profiles of TAC for the two formulations were found to be well fitted to two-compartment model. The calculated pharmacokinetic parameters were listed in Table 2. Results revealed a difference in the pharmacokinetics for the two formulations. Compared with Prograf1, which is a solution of TAC in mixture of ethanol with polyethylene hydrogenated castor oil (HCO-60) (40:60, v/v), TAC-BSA-NPs had significantly higher (1.80-fold higher) area under the whole blood concentration–time curve from 0 to 24 h (AUC024 h) (p < 0.05), which might be attributed to the fact that encapsulated TAC could be protected from metabolizing enzyme in the liver (Li and Huang, 2008), and TAC-BSA-NPs were big enough to avoid or reduce the clearance by renal filtration (Duan and Li, 2013). Consequently, Prograf1 exhibited a fast clearance (CL) (1.90-fold higher), whereas TACBSA-NPs showed a significantly slower clearance on account of the combined effect of protected liver metabolism and reduced renal clearance. This effect was strongly supported by the significantly increased mean residence time (MRT) of TAC-BSA-NPs, which indicated that TACI-BSA-NPs prolonged the systemic circulation time. These results were similar to the previous report (Wei et al., 2014). 3.4. Renal function The immunosuppressive effect of TAC is 50–100 times higher than that of ciclosporin, however, they have the same side effects associated with nephrotoxic, diabetogenic, neurological, and cardiovascular effects (Scott et al., 2003). Due to the marked nephrotoxicity, presumably related with inhibition of the activation of calcineurin resulting from the increase in the Ca2+ influx into tubular cells and thereby inhibition of protein synthesis and cell proliferation in tubular cells (Moutabarrik et al., 1991), the use of TAC is always limited in clinic. The renal function study in the present paper was aimed to evaluate TAC-induced functional nephrotoxicity in rats for TAC-BSA-NPs. Serum creatinine (Scr), creatinine clearance (CLcr) and blood urea nitrogen (BUN) were selected as biochemical markers to manifest the TAC-induced functional nephrotoxicity in previous document (Chen et al., 2007). In addition, BUN, Scr and CLcr are commonly used as assessment indicators of renal function in clinic (Chen et al., 2009). The concentration of Scr and BUN depends on the glomerular filtration rate (GFR), and CLcr is usually used to calculate GFR. As known, renal dysfunction results in reduction of glomerular filtration of Scr and BUN, thus Scr and BUN increase. Hence, in the present study, creatinine level in urine samples, and creatinine and urea nitrogen levels in blood samples were determined before (day 0, initial) and after (day 14) administration of TAC-BSA-NPs. Saline and Prograf1 were tested as negative and positive control, respectively. It was found that there were no significant changes in Scr BUN and CLcr from baseline for control animals given saline after 14 days of treatment. In contrast, repeated dose of Prograf1 caused highly significant increases in BUN (91.80%) (Fig. 5A) and Scr (410.80%) (Fig. 5B) (p < 0.01), whereas TAC-BSA-NPs caused significant increase in BUN (42.50%) (Fig. 5A) (p < 0.05) and highly significant increase in Scr (175.00%) (Fig. 5B) (p < 0.01). These treatments also resulted in highly significant decreases in CLcr (86.40% for TAC-BSA-NPs and 70.20% for Prograf1, respectively) (Fig. 5C) (p < 0.01). Overall, the degree of increase in BUN for different treatment groups was ranked as saline < TAC-BSANPs < Prograf1. The degree of increase in Scr followed the same order. The level of decrease in mean CLcr for different treatment groups was ranked as saline < TAC-BSA-NPs < Prograf1. These indicated that both TAC-BSA-NPs and Prograf1 had effect on renal function to some extent. Further, compared with Prograf1, TACBSANPs exhibited highly significantly lower increase in Scr and BUN (p < 0.01), and significantly lower reduction in CLcr (p < 0.05).
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Fig. 5. Changes in (A) blood urea nitrogen (BUN), (B) serum creatinine (Scr), (C) creatinine clearance (CLcr) and (D) body weight of rats after 14 days treatments with saline, 0.8 mg/kg/day TAC as TAC-BSA-NPs and Prograf1 expressed as a percent of variable measured in the same treatment group on day zero (n = 6). *p < 0.05, **p < 0.01, nsp > 0.05.
Based on these results, it appeared that the damage of TAC by intravenous administration of TAC-BSA-NPs to renal function was significantly lower than that of Prograf1. Further, after 14 days of treatments, changes in body weight of rats given saline, TAC-BSA-NPs and Prograf1 were depicted in Fig. 5D. An interesting phenomenon observed was that rats given saline gained body weight 40.6% of initial, whereas rats receiving Prograf1 gained body weight only 28.0% of initial. In contrast, the increase (30.9% of initial) in body weight of rats given TAC-BSA-NPs was more than that of rats receiving Prograf1, although the difference was not significant and the weight increase for TAC-BSANPs group was not as high as the saline group. In conclusion, TACBSA-NPs could reduce the functional nephrotoxicity caused by TAC compared with Prograf1. 3.5. Tissue distribution Previous document reported that polymeric micelles had a potential in restricting the nephrotoxicity of cyclosporine A by reducing kidney uptake of cyclosporine A by 2.6-fold, and increased cyclosporine A levels in blood by 2.1-fold (Aliabadi et al., 2008). This promoted us to explore the plausible difference in in vivo distribution of TAC for TAC-BSA-NPs and Prograf1. Therefore, biodistribution of TAC in SD rats was evaluated at 0.5 h, 10 h and 24 h after intravenous administration of TAC-BSANPs and commercial formulation Prograf1 at a single dose of 10 mg/kg, respectively. The concentration of TAC found in sampled tissues and blood at each sampling point was expressed in the content of TAC per gram tissue or per milliliter blood (Fig. 6A and B). The results showed that TAC was widely distributed to the rat tissues when treated with the two formulations, which was in good agreement with the previous document (Yura et al., 1999), and the concentration of TAC reduced with time in all tissue samples. For the formulation of Prograf1 (Fig. 6A), the lung and heart had higher TAC concentration among all the detected tissues at each time point. The TAC level in tissues were found to decrease
in the order of lung > heart > spleen > kidney > liver at 0.5 h, lung > heart > spleen > liver > kidney at 10 h, and lung > heart > liver > spleen > kidney at 24 h, respectively, which was similar to previous report (Venkataramanan et al., 1990). In comparison, TAC-BSA-NPs changed the distribution of TAC in tissues (Fig. 6B). 0.5 h after dosing, the level of TAC in the lung was the highest, followed by spleen, as previously reported (Yokogawa et al., 1999). The TAC concentration in tissues were found to decrease in the order of lung > spleen > liver heart > kidney at 0.5 h, lung > liver heart > spleen > kidney at 10 h, and liver > lung > heart > spleen > kidney at 24 h, respectively. Specifically, TAC-BSA-NPs had significantly lower AUC024 h (94.54 12.12 min mg/g) of kidney compared with Prograf1 (123.57 13.82 min mg/g) (p < 0.05). Further, the TAC concentration in heart, spleen and lung for TAC-BSA-NPs was significantly lower than that for Prograf1. However, TAC level in liver for TAC-BSA-NPs was significantly higher than that for Prograf1, which might be attributed to the fact that the size of BSA nanoparticles made TAC-BSA-NPs more easily be extracted by liver (Kapoor et al., 2008). More importantly, TAC-BSA-NPs conspicuously enhanced TAC level in blood and reduced TAC accumulation in kidney, which is desirable to decrease kidney toxicity. Specifically, in comparison to Prograf1, analysis of the results revealed a reduced delivery of TAC by TAC-BSA-NPs to kidneys by 1.8-fold, 1.2-fold and 1.2-fold, and increased TAC levels in blood by 1.6-fold, 1.7-fold and 1.6-fold at 0.5 h, 10 h and 24 h, respectively, which was similar to cyclosporine A-loaded polymeric micelles (Aliabadi et al., 2008). To further validate the effect of distribution of TAC on renal function, the ratio of TAC level in kidney to that in blood was compared between TACBSA-NPs and Prograf1. As was seen in Fig. 6C, the concentration ratios of TAC in kidney to those in blood over the course of the study for TAC-BSA-NPs were significantly lower than those of Prograf1 (p < 0.05). These could explain the improvement of renal function by TAC-BSA-NPs compared with Prograf1. All together, these results demonstrated that with the aid of TAC-BSA-NPs, more TAC could be accumulated in blood whereas a subsequent reduced
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distribution in other organs, especially in kidney, which was beneficial to reduction of kidney toxicity. 4. Conclusions In the present work, TAC-BSA-NPs with uniform particle size below 190 nm were successfully prepared by the emulsificationdispersion method and exhibited a biphasic release pattern characterized with an initial burst release and sustained drug release. TAC-BSA-NPs had more favorable pharmacokinetics and biodistribution characteristics compared with Prograf1, therefore showing a potential to reduce TAC delivery to kidney and thereby functional nephrotoxicity while maintaining high blood levels. In summary, the findings of this study further provided important convincing evidence of the effectiveness of appropriately designed drug delivery systems to enhance the therapeutic index of drugs with serious side effects. Acknowledgements This research was financially supported by the National Natural Science Foundation of China (No. 81172990), the National Key Science Research Program of China (973 Program, 2015CB932100) and the Innovation Team of Ministry of Education (No. BMU20110263). References Aliabadi, H.M., Elhasi, S., Brocks, D.R., Lavasanifar, A., 2008. Polymeric micellar delivery reduces kidney distribution and nephrotoxic effects of cyclosporine A after multiple dosing. J. Pharm. Sci. 97, 1916–1926. Bottiger, Y., Brattstrom, C., Tyden, G., Sawe, J., Groth, C.G., 1999. Tacrolimus whole blood concentrations correlate closely to side-effects in renal transplant recipients. Br. J. Clin. Pharmacol. 48, 445–448. Breunig, M., Bauer, S., Goepferich, A., 2008. Polymers and nanoparticles: intelligent tools for intracellular targeting? Eur. J. Pharm. Biopharm. 68, 112–128. Brewster, M.E., Loftsson, T., 2007. Cyclodextrins as pharmaceutical solubilizers. Adv. Drug Deliv. Rev. 59, 645–666. Cardenas, M.E., Zhu, D., Heitman, J., 1995. Molecular mechanisms of immunosuppression by cyclosporine FK506, and rapamycin. Curr. Opin. Nephrol. Hypertens. 4, 472–477. Chen, Y.H., Liang, Y.X., Chen, L.Q., Liang, J.J., Zhang, J., Qiu, J., Li, W.L., Hu, J.B., Xie, K.J., Zhong, W.D., Chen, L.Z., Zheng, K.L., 2007. Prevention of diltiazem in tacrolimusinduced nephrotoxicity: experiment with rats. Zhonghua Yi Xue Za Zhi 87, 2235–2237. Chen, Y.H., Liang, Y.X., Chen, L.Q., Lu, Y.M., Liang, J.J., Zhang, J., Qiu, J., Xie, K.J., Hu, J.B., Zhong, W.D., Wang, B., Chen, L.Z., Zheng, K.L., 2009. Nephrotoxicity of tacrolimus and preventive effect of diltiazem: experiment with rats. Zhonghua Yi Xue Za Zhi 89, 704–708. Cortes, J., Saura, C., 2010. Nanoparticle albumin-bound (nabTM)-paclitaxel: improving efficacy and tolerability by targeted drug delivery in metastatic breast cancer. Eur. J. Cancer Suppl. 8, 1–10. Duan, X., Li, Y., 2013. Physicochemical characteristics of nanoparticles affect circulation, biodistribution, cellular internalization, and trafficking. Small 9, 1521–1532. Elzoghby, A.O., 2013. Gelatin-based nanoparticles as drug and gene delivery systems: reviewing three decades of research. J. Control. Release 172, 1075– 1091. Elzoghby, A.O., Samy, W.M., Elgindy, N.A., 2012. Albumin-based nanoparticles as potential controlled release drug delivery systems. J. Control. Release 157, 168– 182. Fattal, E., Vauthier, C., Aynie, I., Nakada, Y., Lambert, G., Malvy, C., Couvreur, P., 1998. Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides. J. Control. Release 53, 137–143. Fernandez-Urrusuno, R., Calvo, P., Remunan-Lopez, C., Vila-Jato, J.L., Alonso, M.J., 1999. Enhancement of nasal absorption of insulin using chitosan nanoparticles. Pharm. Res. 16, 1576–1581. Freitas, C., Müller, R.H., 1998. Effect of light and temperature on zeta potential and physical stability in solid lipid nanoparticle (SLNTM) dispersions. Int. J. Pharm. 168, 221–229. Fu, Q., Sun, J., Zhang, W., Sui, X., Yan, Z., He, Z., 2009. Nanoparticle albumin-bound (NAB) technology is a promising method for anti-cancer drug delivery. Recent Pat. Anti-Cancer Drug Discov. 4, 262–272. Gao, S., Sun, J., Fu, D., Zhao, H., Lan, M., Gao, F., 2012. Preparation, characterization and pharmacokinetic studies of tacrolimus-dimethyl-beta-cyclodextrin inclusion complex-loaded albumin nanoparticles. Int. J. Pharm. 427, 410–416.
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