International Journal of Pharmaceutics 476 (2014) 142–148
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Sustained release and enhanced bioavailability of injectable scutellarin-loaded bovine serum albumin nanoparticles Yuanfeng Wei a , Laicun Li a , Yifeng Xi a , Shuai Qian a , Yuan Gao a , Jianjun Zhang b, * a b
School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 210009, PR China Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 July 2014 Received in revised form 8 September 2014 Accepted 26 September 2014 Available online 28 September 2014
The aim of this study is to characterize the in-vitro physicochemical and in-vivo pharmacokinetic properties of the scutellarin-loaded bovine serum albumin nanoparticles (STA-BSA-NPs). STA existed as amorphous form in the nanoparticles. Reconstituted STA-BSA-NPs had an average particle size of 283.4 nm and a zeta potential of +17.95 mV. The in-vitro sustained release profile was well fitted with Weibull distribution model. In comparison to STA solution, STA-BSA-NPs exhibited a significantly higher plasma concentration from 20 min to 6 h after intravenous administration to rats. In addition, significantly higher AUC0–inf (2.8-fold), prolonged elimination half-life (4.2-fold) and lower clearance (2.7-fold) were achieved. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Scutellarin Albumin nanoparticles Sustained release Elimination half-life Bioavailability
1. Introduction Scutellarin (STA) (Fig. 1), a typical flavonoid glycoside, is the primary bioactive ingredient in breviscapine extracted from the Chinese herb Erigeron breviscapus (Vant.) Hand.-Mazz. Breviscapine has been widely used to treat cardiovascular diseases and cerebrovascular injury in clinics, such as cerebral ischemia, angina pectoris, myocardial infarction, stroke and cerebral thrombotic diseases. Various pharmacological activities of STA have been recognized, including anti-oxidation (Wang et al., 2008), angiogenesis (Gao et al., 2010), neuroprotector (Xu et al., 2007), ischemia protector (Lin et al., 2007) as well as anti-HIV (Zhang et al., 2005). Intravenous injection of STA is the most commonly used dosage form in clinical treatments. However, the fast metabolic rate, short elimination half-life, and short blood residence time limit its wide application (Li et al., 2013). A satisfactory system that exhibits a good pharmacokinetic profile has not been found in the field of colloidal particle delivery systems for STA. It is urgent to develop a drug delivery system that can prolong the elimination half-life and improve the intravenous bioavailability of STA. Albumin has been used as a versatile protein carrier to fabricate nanoparticles for drug delivery due to its nontoxic, nonimmunogenic, biocompatible and biodegradable properties (Kratz, 2008). Therapeutic drugs could be incorporated into the
* Corresponding author. Tel.: +86 25 83379418; fax: +86 25 83379418. E-mail address: [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.ijpharm.2014.09.038 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
nanoparticulate matrix due to various binding sites present in the albumin molecule (Patil, 2003). In addition, albumin nanoparticles showed great potential to prolong the half-life and bioavailability of encapsulated drugs including both small molecules (e.g., curcumin (Jithan et al., 2011)) and macromolecules such as hurudin (Jing et al., 2013). Moreover, albumin nanoparticles could also enhance the anti-tumor efficacy of some anti-cancer drugs. A successful example is the nanoparticulate albumin-bound (nabTM)-paclitaxel, which has been approved by FDA for clinical use. It can provide improving efficacy and tolerability in comparison to Cremophor based paclitaxel solution (Cortes and Saura, 2010). Various types of albumins used to prepare nanoparticles, including ovalbumin, bovine serum albumin and human serum albumin, have shown a promising prospect in the controlled delivery of nanoparticulate therapeutic agents (Elsadek and Kratz, 2012). Nevertheless, the studies on in-vivo pharmacokinetic behavior of BSA-containing nanoparticulate systems are still deficient. Generally, there are three different methods to prepare albumin nanoparticles, such as desolvation, emulsification and thermal gelation (Elzoghby et al., 2012). Desolvation process followed by cross-linking with glutaraldehyde is the commonly used technique to prepare albumin nanoparticles. In desolvation process, nanoparticles are prepared by a continuous dropwise addition of ethanol to an aqueous solution of albumin under continuous stirring until the solution becomes turbid. However, the obtained albumin nanoparticles are not sufficiently stabilized and may consequently be re-dissolved in water. Therefore, coacervates are
Y. Wei et al. / International Journal of Pharmaceutics 476 (2014) 142–148
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and the adsorption process of STA was allowed for 1 h to get the suspension containing the BSA-NPs loaded with STA (STA-BSA-NPs). After centrifugation at 8000 rpm for 10 min, the obtained precipitation was re-dispersed in 2% lactose solution and freeze-dried to get lyophilized powder.
Fig. 1. Chemical structure of scutellarin (STA).
hardened by cross-linking with glutaraldehyde to solidify the albumin nanoparticles. Previously, STA-BSA-NPs have been prepared by desolvation technique followed by cross-linking, precipitation, adsorption and freeze drying. This preparation procedure was also optimized (Xi et al., 2012). However, in-vivo behavior of STA-BSA-NPs has not been explored yet. The main objective of the present study is to employ albumin nanoparticles as the carrier for extending the half-life and bioavailability of STA. The physicochemical properties of the STA-BSA-NPs were characterized. In addition, in-vivo pharmacokinetic study of STA-BSA-NPs was performed in rats with STA solution as a control group. 2. Materials and methods 2.1. Materials Scutellarin (purity of 95.8%) was purchased from Nanjing Zelang Pharmaceutical Technology Co., Ltd. (Nanjing, China). Scutellarin reference standard (purity of 98.2%) and protocatechuic aldehyde reference standard (purity of 98%) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Bovine serum albumin was purchased from the Shanghai Hui Xing Biochemical Reagent Co., Ltd. (Shanghai, China). Lactose was obtained from the Meggle Pharma (Wasserburg, Germany). Millipore water was used throughout the study. Other chemicals were of HPLC or analytical grade. HepG2 cell lines were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science. HepG2 cells were cultured in the Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated calf serum (Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China), 100 U/mL penicillin G and 100 U/mL streptomycin (pH 7.4), in a water jacketed CO2 incubator with a humidified atmosphere of 5% CO2 at 37 C. 2.2. Methods 2.2.1. Preparation of STA-BSA-NPs STA-BSA-NPs were prepared by the optimized method developed by our research group (Xi et al., 2012). Briefly, 40 mL ethanol (95%) was added continuously into 15 mL of 10% BSA aqueous solution at a rate of 1 mL/min under stirring (600 rpm) at 25 C. 5% Glutaraldehyde solution (equivalent to 0.95 mg/mL BSA) was added and allowed for cross-linking reaction and solidification for 12 h. The solidified BSA-nanoparticles (BSA-NPs) were then collected after centrifugation at 8000 rpm for 10 min. Purification was conducted by re-dispersing the sedimentation in 20 mL of water by sonication at 5 kHz for 5 min. The purification procedure was repeated three times. The obtained purified BSA-NPs (1 g) were re-dispersed in 6 mL of water. 24 mL of ethanol was added dropwise. Under continuous stirring, 72 mL of STA ethanol solution (1.45 mg/mL) was added,
2.2.2. Particle size and zeta potential analysis The particle size analysis was performed by photon correlation spectroscopy (PCS) using a Zetasizer 3000 (Malvern Instruments, Malvern, UK). Prior to measurement, appropriate amount of lyophilized STA-BSA-NPs sample was reconstituted in 0.01 M citric acid solution and measured in triplicate. Seven-day stability test of reconstituted STA-BSA-NPs was performed by observing their particle size and zeta potential. 2.2.3. Determination of drug loading and entrapment efficiency 100 mg of STA-BSA-NPs were dispersed in 10 mL of 80% ethanol solution (to fully dissolve free STA but not disrupt the nanostructure of STA-BSA-NPs) and stirred for 10 min. The suspension was centrifuged at 15,000 rpm for 15 min. The concentration of the obtained supernatant was determined, and the amount of free unloaded STA in STA-BSA-NP suspension was calculated (m2). Another 100 mg of STA-BSA-NPs were dispersed in 1 mL of phosphate buffer (pH 6.8) and then sonicated for 10 min. 4 mL of 0.05 M phosphate buffer (pH6.8, containing pancreatic enzyme, which was used to degrade albumin for fully releasing the incorporated STA) was added and the suspension was stirred at 37 C for 3 h. After centrifugation at 15,000 rpm for 15 min, the STA concentration in the supernatant was determined by HPLC and the total amount of STA added into STA-BSA-NPs was calculated (m1). Drug loading and entrapment efficiency were calculated by: Drugloadingefficiencyð%Þ ¼
Entrapmentefficiencyð%Þ ¼
m1 m2 100 m3
m1 m2 100 m1
(1)
(2)
where m1 is the total amount of STA added into STA-BSA-NPs; m2 is the free unloaded STA in STA-BSA-NP suspension; m3 is total amount of BSA NPs. 2.2.4. Differential scanning calorimetry (DSC) DSC experiments were carried out using a DSC 204 F1 Phoenix Differential Scanning Calorimeter (Netzsch Inc., Germany) which was calibrated for temperature and cell constants using indium. Samples were placed on non-hermetic aluminum pans. The sample cell was equilibrated at 20 C and then heated at a rate of 10 C/min in a range of 20–300 C. Data analysis was performed using NETZSCH-Proteus software (version 4.2). 2.2.5. Powder X-ray diffraction analysis X-ray powder diffraction (XRPD) was used to determine the crystalline or amorphous nature of STA in the STA-BSA-NPs. XRPD analysis was recorded using a Bruker D8 Advance Powder Diffractometer (Karlsruhe Inc., Germany) with Cu-Ka radiation (1.5406 Å). The samples were gently consolidated in an aluminum holder and scanned at 40 kV and 40 mA from 5 to 40 2u using a scanning speed of 2 /min and a step size of 0.02 . 2.2.6. Surface morphological study Morphological evaluation of the nanoparticles was performed by scanning electron microscope (SEM) (Hitachi S3400, Tokyo, Japan). The sample was examined on a brass stub using carbon double-sided tape. Powder samples were glued and mounted on metal sample plates. The samples were gold coated
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(thickness 15–20 nm) with a sputter coater (Fison Instruments, UK) using an electrical potential of 2.0 kV at 25 mA for 10 min. An excitation voltage of 20 kV was used in the experiment. 2.2.7. In-vitro release In-vitro release of STA from STA-BSA-NPs was studied by dialysis method using a RC-806 dissolution tester (TDTF technology Co., Ltd., China) with the rotary basket rotating at 50 rpm. 10 mL of reconstituted STA-BSA-NPs in 0.01 M citric acid solution (equivalent to 5 mg STA) or 10 mL of STA solution (0.5 mg/mL) (n = 6) was placed in each regenerated cellulose membrane Spectra/Por dialysis bag (MWCO 8000–12,000 Da) (Spectrum Industries, Los Angeles, CA). The dialysis bags were sealed in both ends with clips and soaked in 250 mL of phosphate buffer (pH 6.8) controlled at 37 C. The pH value of this buffer is close to plasma (pH 7.4) and scutellarin can achieve sink condition in pH of above 6.8 according to preliminary studies. Therefore, it is suitable for the purpose of in-vitro release comparison. Samples (1 mL) were withdrawn and filtered at predetermined time points (0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 12, 16 and 24 h). STA concentration was analyzed by HPLC method on Shimadzu LC2010AHT system (Shimadzu Corporation, Japan). A reversed phase Shimadzu-pack VP-ODS C18 (4.6 250 mm, 5 mm) column guarded by a precolumn C18 insert was used for separation. STA was isocratically eluted with the mixture of acetonitrile and potassium dihydrogen phosphate buffer (0.05 M, pH 2.5) (22/78, v/v). The flow rate of 1.0 mL/min was maintained. The detection wavelength was set at 335 nm. The in-vitro drug release amount of STA from STA-BSA-NPs at a given time was fitted into different models, including zero-order kinetics equation, first-order kinetics equation, Higuchi equation, Peppas equation, and Weibull equation. 2.2.8. In-vivo pharmacokinetic study The animal study was approved by the Ethical Committee of China Pharmaceutical University. All animals used in this study were handled in accordance with the guidelines of the Principles of Laboratory Animal Care (State Council, revised 1988). Male Sprague-Dawley rats (200 20 g) were obtained from Nanjing Qinglongshan Laboratory Animal Center (Nanjing, China). All animals housed in standard cages on a 12 h light–dark cycles were fed with standard animal chow daily, and had free access to drinking water. One day before the pharmacokinetic study, each animal was operated with a cannula insert into the right jugular vein under anesthesia by intraperitoneal injection of pentobarbital sodium (50 mg/kg). A surgical incision was made on the ventral side of the neck of rats to expose the jugular vein. The jugular vein was then cannulated with a polyethylene tubing (0.5 mm ID, 1 mm OD, Portex Ltd., Hythe, Kent, England) that was led under the skin and exteriorized at the back of the neck for blood sampling. 50 IU/mL of heparin sodium in normal saline was filled into the catheter to prevent the blood clotting. After the exposed areas were surgically sutured, the rats were placed individually in standard cages. The animals were allowed to recover for 24 h and were fasted overnight prior to administration (Zhang et al., 2013). Before intravenous administration to rats, the lyophilized STA-BSA-NPs were reconstituted in 0.01 M citric acid solution to obtain a suspension containing 3.5 mg/mL STA. 3.5 mg/mL STA solution in 0.01 M citric acid solution was served as control. Rats were divided randomly into two groups with 6 rats in each group. The body weight of each rat was measured before the administration. Each rat received the tail vein injection at a single dose of 9.4 mg/kg STA (Lv et al., 2008). Around 400 mL of blood was collected from the jugular vein into heparinized centrifuge tube at predetermined time points (5, 10, 20, 40, 60, 90, 120, 180, 240, 360 and 480 min) post dosing. Plasma samples were immediately
separated by centrifugation of blood at 10,000 rpm for 10 min (Refrigerated Centrifuge, Sigma 1–15 K, Sigma, Germany) and stored at 20 C until analysis. Frozen plasma samples were thawed at room temperature just before sample preparation. 50 mL of protocatechuic aldehyde methanol solution (internal standard, 50.0 mg/mL) and 100 mL of phosphoric acid (1 M) were added to a 200 mL aliquot of rat plasma. After vortex-mixing for 2 min, the mixture was extracted with 3 mL of ethyl acetate and centrifuged at 10,000 rpm for 10 min. 2 mL of the obtained supernatant was transferred to a 5-mL glass tube and evaporated to dryness under nitrogen stream in the water bath (40 C). The obtained residues were reconstituted with 400 mL of mobile phase. After centrifugation for another 5 min (10 C, 10,000 rpm), an aliquot of 20 mL supernatant was injected into the HPLC system for analysis using the method as described in Section 2.2.7. Calibration standard was prepared by spiking STA standards of six known concentrations in blank rat plasma. Good linearity was obtained in the concentration range of 0.031–76.16 mg/mL (r2 = 0.996). The recoveries of STA were over 90.5%; the interand intra-day precision was below 7% RSD (relative standard deviation), and the accuracy was within 93.2–102.9%. The limit of quantification and limit of detection were 60 ng/mL and 25 ng/mL, respectively. After storage for 8 h at room temperature, 5 days at 20 C and freeze–thawing for three times, STA was found to be stable in rat plasma. Pharmacokinetic analysis was performed by means of a model independent method using the PKSolver computer program (Zhang et al., 2010). The pharmacokinetic parameters were calculated. The area under the concentration–time curve (AUC) was calculated with trapezium method. The terminal half-life (t1/2) was calculated as ln 2/lz, where lz was the first-order rate constant associated with the terminal (log-linear) portion of the curve. The area under the plasma concentration–time curve from time 0 to 480 min (AUC0–480 min) and from time 0 to infinity (AUC0–inf) were calculated by the trapezoidal rule without or with extrapolation to time infinity (AUC0–inf = AUC0–48 min + C480 min/lz). 2.2.9. MTT assay MTT assay was performed to evaluate the cytotoxicity of STA-BSA-NPs on HepG2 cells (Fotakis and Timbrell, 2006). Briefly, HepG2 cells (1 104 per well in a 96-well plate) were treated with STA-BSA-NPs suspension (50, 100, 250 and 500 mg/mL in 0.01 M citric acid solution, n = 6) for 24 h at 37 C in a 5% CO2 incubator, respectively. Then, 20 mL MTT (5 mg/mL) was added into each well and incubated for 4 h. The supernatant was discarded, and 150 mL DMSO was added. The mixture was gently shaken on a microvibrator for 10 min. The absorbance was then recorded at 570 nm using multimode microplate reader (Infinite 200 Pro Tecan, Switzerland). 2.2.10. Statistical analysis Statistical analysis was performed using a statistical software package SPSS (version 17, SPSS Inc., Chicago, IL, USA). Independentsample t-test was applied for analysis of the data from in-vitro release and in-vivo pharmacokinetic studies. A probability level of p < 0.05 was set as the criterion of significance. 3. Results and discussion 3.1. Physicochemical properties Particle size and its distribution are the most widely accepted defining characteristics of nanoparticle-based medicines since particle size can significantly influence the pharmacokinetics, biodistribution, and safety of nanoparticulate drugs (Desai, 2012).
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The mean particle size and polydispersity index of prepared STABSA-NPs were determined to be 283.4 nm and 0.117, respectively (Fig. 2). The particle size in the range of 100–300 nm with narrow distribution indicates that STA-BSA-NPs will mainly distribute in the liver and spleen after intravenous administration (Gaumet et al., 2008). STA-BSA-NPs exhibited the regular spherical shape with smooth surfaces according to SEM (Fig. 3) with observed
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particle size in SEM of about 100–300 nm. This agrees well with the determined value by PCS. Zeta potential of the reconstituted STA-BSA-NPs was determined to be +17.95 mV. In seven days, no aggregation or flocculation of the reconstituted STA-BSA-NPs was observed. In addition, there was no change in their particle size and zeta potential, indicating that the reconstituted STA-BSA-NPs can be stable for at least a week. Citric acid solution used to
Fig. 2. Particle size (a) and zeta potential (b) of STA-BSA-NPs.
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(a)
onset:189.3°C onset:116.2°C
onset:227.4°C
(b)
onset:49.0°C onset:202.3°C
onset:112.0°C
(c) onset:228.5°C
onset:41.6°C
onset:227.8°C
(d)
onset:50.0°C 50
100
150
200
250
Temperature(°C)
Fig. 5. DSC thermograms of crystalline STA (a); BSA-NPs (b); physical mixture of STA and BSA-NPs (c); and STA-BSA-NPs (d). Fig. 3. Scanning electron microscope (SEM) photograph of STA-BSA-NPs.
3.2. DSC and XRPD reconstitute STA-BSA-NPs made pH value of the dispersion far away from the isoelectric point of BSA (pH 4.7) and generated a positive zeta potential. Such moderate positive zeta potential led to the moderate stability of the reconstituted nanoparticle dispersion. The entrapment efficiency and drug loading efficiency of STA-BSA-NPs are 64.46% and 6.73%, respectively. The release profiles of STA solution and STA-BSA-NPs in 0.05 M phosphate buffer (pH 6.8) are presented in Fig. 4. In comparison to STA solution, STA-BSA-NPs had an obviously slower release. Based on the model fitting results of release data, the in-vitro drug release amount (Q) of STA from STA-BSA-NPs at a given time (t) was fitted very well to Weibull distribution model (Q = 100 {1 Exp[(tb)/a]}, (a = 5.67, b = 0.81, r2 = 0.9948)), an important model for analyzing multi-mechanistic drug release from nanoparticles. The release of different compounds may be described by different models. For example, Adriamycin coupled albumin nanoparticles were reported to have the similar Weibull release pattern (Chen et al., 2010). However, the release of folateconjugated BSA nanoparticles loaded with vinorelbine tartrate corresponded with the Higuchi equation (Li et al., 2012). The in-vitro release profile of STA-BSA-NPs was characterized by the shape parameter (b < 1) to be S-shape which has steeper initial slope than the exponential (b = 1) (Barzegar-Jalali et al., 2008; Yuksel et al., 2000). In addition, the calculated release half-life (t50%) of STA-BSA-NPs was 2.5-fold longer than that of STA solution.
The DSC curves of crystalline STA, drug-free BSA-NPs, physical mixture of STA and BSA-NPs, and STA-BSA-NPs were studied to examine the status of crystallinity and potential change of STA after the cross-linking and adsorption. As shown in Fig. 5, STA had an endothermic peak at 116.2 C (melting point of STA) and two peaks at 189.3 and 196.5 C which might be due to the melting behavior of some impurities in STA. Drug-free BSA-NPs exhibited two broad endothermic peaks with onset values at 49.0 C and 227.4 C. Although different from their corresponding onset values, DSC curve of their physical mixture showed a distinct endothermic peak at 112 C, demonstrating the existence of crystalline STA in physical mixture. However, STA-BSA-NPs had significantly different thermal behavior from crystalline STA and the physical mixture of STA and BSA-NPs. No endothermic peak was observed around the melting point of STA, indicating the existence of STA as amorphous state in STA-BSA-NPs. The XRPD patterns of crystalline STA, drug-free BSA-NPs, physical mixture of STA and BSA-NPs, and STA-BSA-NPs are presented in Fig. 6. The characteristic diffraction peaks demonstrate the long-range order of crystalline STA (Fig. 6(a)). In comparison to blank BSA-NPs, physical mixture of STA and BSANPs showed two obvious diffraction peaks of STA at 25.85 and 26.75 , indicating the presence of crystalline STA in physical mixture (Fig. 6(c)). This agrees with the observation from DSC.
100 90
The cumulative release(%)
80 70 60
(a)
50
STA-BSA-NPs STA solution
40
(b)
30
(c)
20
(d)
10 0
0
5
10
15
20
25
Time (h) Fig. 4. The release profiles of STA solution and STA-BSA-NPs in 0.05 M phosphate buffer (pH 6.8).
5
10
15
20
25
30
35
40
2θ(°) Fig. 6. XRPD patterns of crystalline STA (a); BSA-NPs (b); physical mixture of STA and BSA-NPs (c); and STA-BSA-NPs (d).
Y. Wei et al. / International Journal of Pharmaceutics 476 (2014) 142–148
Plasma concentration of STA(μg/mL)
70 60
STA-BSA-NPs STA solution
50 40
*
30
*
20
* 10 0
0
*
*
100
*
* 200
* 300
400
500
Time(min) Fig. 7. Mean plasma concentration of STA versus time curves after tail vein injection of STA solution and STA-BSA-NPs to rats (n = 6).
After the formation of nanoparticles, STA-BSA-NPs presented a XRPD pattern without the characteristic diffraction peaks of STA, suggesting the amorphous state of STA in nanoparticles (Fig. 6(d)). This transformation might be attributed the interaction between STA and BSA. The similar phenomena have been observed in previous BSA-containing nanoparticulate systems (Foldbjerg et al., 2013; Zhao et al., 2010). 3.3. In-vivo pharmacokinetic study To evaluate the in-vivo performance of the developed STA-BSA-NPs, the systemic pharmacokinetics of STA was studied with STA solution as the control. The plasma concentration-versustime profiles of STA in two formulations are given in Fig. 7. The calculated pharmacokinetic parameters are summarized in Table 1. In comparison to STA solution, intravenous STA-BSA-NPs showed similar initial phase of rapid decrease in plasma concentration in 10 min, while much slower decline after 20 min post dose. From 20 min to 6 h, the plasma concentrations of STA after intravenous administration of STA-BSA-NPs were significantly higher than those of STA solution (p < 0.05). Pharmacokinetic profiles of STA after intravenous administration of both STA solution and STA-BSA-NPs were verified to be well fitted to open two-compartment model. Such well fitted model agrees with other studies of the in-vivo behavior of STA (Huang Table 1 Pharmacokinetic parameters of STA after tail vein injection of STA solution and STABSA-NPs in rats.
k10/(min1) k12/(min1) k21/(min1) t1/2a/(min) t1/2b/(min) C0/(mg mL1) CL/(mL min1) AUC0–480 min/(mg min mL1) AUC0–inf/(mg min mL1) AUMC/(mg min2 mL1) MRT/(min)
STA solution
STA-BSA-NPs
0.064 0.014 0.016 0.011 0.022 0.003 8.67 2.32 42.24 3.25 70.79 14.48 2.69 0.13 1099 56.82 1116 56.78 29,910 3648 26.75 2.28
0.017 0.004 0.011 0.007 0.008 0.003 23.90 7.98** 176.8 22.98** 51.45 13.78* 0.98 0.05** 2836 166.4** 3070 161.0** 45,1614 21,168** 147.4 11.49**
k10, elimination rate constant; k12,k21, rate constant between the two compartments; t1/2a, distribution half-life; t1/2b, elimination half-life; CL, total clearance; C0, the instantaneous concentration after intravenous injection; AUC0–t, area under plasma concentration versus time curve (total area from 0 to t); AUC0–inf, area under plasma concentration versus time curve (total area from 0 to infinity); AUMC, partial area under the moment curve; MRT, mean residence time. * p < 0.05, STA-BSA-NPs versus STA solution. ** p < 0.01, STA-BSA-NPs versus STA solution.
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et al., 2005; Lv et al., 2005). In comparison to STA solution, STA-BSA-NPs achieved the significantly higher AUC0–inf (p < 0.01) with a relative bioavailability of 275%. Several factors are involved in the enhancement of STA bioavailability. When drugs are encapsulated in nanoparticles, they are protected from metabolizing enzyme in the liver before their release in intravenous vein (Li and Huang, 2008). For instance, albumin coated 5-fluorouridine nanoparticles can even circumvent its pre-systemic degradation (Arbos et al., 2004). In addition, since the cut-off size of renal clearance is lower than 15 nm, the nanoparticulate drugs in systemic circulation will be blocked by glomerulus and the renal clearance of encapsulated drugs will be reduced regardless of their partition coefficients (Kadam et al., 2012). As a lipophilic drug, STA will undergo rapid and extensive biotransformation in the liver to hydrophilic aglycone conjugated metabolites and then the rapid biliary or renal excretion starts (Huang et al., 2005). Therefore, the intravenous STA solution has a fast clearance in-vivo (CL = 2.69 mL min1). However, STA-BSA-NPs achieved a 2.7-fold lower clearance as the result of combined effect of protected liver metabolism and reduced renal clearance. Such effect has also been reflected on the prolonged elimination half-life (t1/2b) (4.2-fold) and increased mean residence time (MRT) (5.5-fold) after intravenous administration of STA-BSA-NPs. 3.4. MTT assay The cell viability of HepG2 from MTT assay was more than 98% at all tested STA-BSA-NPs concentrations (50–500 mg/mL). There was no statistical difference among the four tested concentrations (p > 0.05). The MTT assay indicates that the STA-BSA-NPs are rather safe even at a relative high concentration (500 mg/mL) toward liver cells. 4. Conclusion In the present investigation, the potential of BSA nanoparticles as a novel drug delivery system for STA was evaluated based on their physicochemical and pharmacokinetic characterization. STA-BSA-NPs appeared to be the regular spherical shape with smooth surfaces in morphology and exhibited an in-vitro sustained release profile. In comparison to STA solution, STA-BSA-NPs achieved a significantly enhanced bioavailability and lower elimination half-life as well as longer MRT after intravenous administration. This study provides a great potential to utilize albumin nanoparticles as the effective intravenous formulation strategy for drugs with fast clearance and low bioavailability. Acknowledgments This research was supported by the National Natural Science Fund (No. 81202988), Natural Science Foundation of Jiangsu Province (BK20141351), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and Qing Lan Project of Jiangsu Province. References Arbos, P., Campanero, M.A., Arangoa, M.A., Irache, J.M., 2004. Nanoparticles with specific bioadhesive properties to circumvent the pre-systemic degradation of fluorinated pyrimidines. J. Control. Release 96, 55–65. Barzegar-Jalali, M., Adibkia, K., Valizadeh, H., Shadbad, M.R.S., Nokhodchi, A., Omidi, Y., Mohammadi, G., Nezhadi, S.H., Hasan, M., 2008. Kinetic analysis of drug release from nanoparticles. J. Pharm. Pharm. Sci. 11, 167–177. Chen, D., Tang, Q., Xue, W., Xiang, J., Zhang, L., Wang, X., 2010. The preparation and characterization of folate-conjugated human serum albumin magnetic cisplatin nanoparticles. J. Biomed. Res. 24, 26–32. Cortes, J., Saura, C., 2010. Nanoparticle albumin-bound (nabTM)-paclitaxel: improving efficacy and tolerability by targeted drug delivery in metastatic breast cancer. EJC Suppl. 8, 1–10.
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Li, Y., Zhao, X., Zu, Y., Han, X., Ge, Y., Wang, W., Yu, X., 2012. A novel active targeting preparation, vinorelbine tartrate (vlbt) encapsulated by folate-conjugated bovine serum albumin (BSA) nanoparticles: preparation, characterization and in vitro release study. Materials 5, 2403–2422. Lin, L.L., Liu, A.J., Liu, J.G., Yu, X.H., Qin, L.P., Su, D.F., 2007. Protective effects of scutellarin and breviscapine on brain and heart ischemia in rats. J. Cardiovasc. Pharmacol. 50, 327–332. Lv, W., Guo, J.X., Li, J., Huang, L.S., Ping, Q.N., 2005. Distribution of liposomal breviscapine in brain following intravenous injection in rats. Int. J. Pharm. 306, 99–106. Lv, W.L., Guo, J.X., Ping, Q.N., Song, Y.M., Li, J., 2008. Comparative pharmacokinetics of breviscapine liposomes in dogs, rabbits and rats. Int. J. Pharm. 359, 118–122. Patil, G.V., 2003. Biopolymer albumin for diagnosis and in drug delivery. Drug Dev. Res. 58, 219–247. Wang, M., Xie, C., Cai, R.L., Li, X.H., Luo, X.Z., Qi, Y., 2008. Studies on antioxidant activities of breviscapine in the cell-free system. Am. J. Chin. Med. 36, 1199–1207. Xi, Y.F., Zhou, H., Li, L.C., Gao, Y., 2012. Central composite design–response surface methodology for the preparation optimization of adsorbed breviscapine by bovine serum albumin nanoparticles. J. Chin. Pharm. Univ. 43, 216–221. Xu, W., Zha, R.P., Wang, W.Y., Wang, Y.P., 2007. Effects of scutellarin on PKCgamma in PC12 cell injury induced by oxygen and glucose deprivation. Acta Pharmacol. Sinica 28, 1573–1579. Yuksel, N., Kanik, A.E., Baykara, T., 2000. Comparison of in vitro dissolution profiles by ANOVA-based: model-dependent and -independent methods. Int. J. Pharm. 209, 57–67. Zhang, G.H., Wang, Q., Chen, J.J., Zhang, X.M., Tam, S.C., Zheng, Y.T., 2005. The antiHIV-1 effect of scutellarin. Biochem. Biophys. Res. Commun. 334, 812–816. Zhang, J.J., Huang, Y.T., Liu, D.P., Gao, Y., Qian, S., 2013. Preparation of apigenin nanocrystals using supercritical antisolvent process for dissolution and bioavailability enhancement. Eur. J. Pharm. Sci. 48, 740–747. Zhang, Y., Huo, M.R., Zhou, J.P., Xie, S.F., 2010. PKSolver: an add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput. Methods Programs Biomed. 99, 306–314. Zhao, D.M., Zhao, X.H., Zu, Y.G., Li, J.L., Zhang, Y., Jiang, R., Zhang, Z.H., 2010. Preparation, characterization, and in vitro targeted delivery of folate-decorated paclitaxel-loaded bovine serum albumin nanoparticles. Int. J. Nanomed. 5, 669–677.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Sustained release and enhanced bioavailability of injectable scutellarin-loaded bovine serum albumin nanoparticles Yuanfeng Wei a , Laicun Li a , Yifeng Xi a , Shuai Qian a , Yuan Gao a , Jianjun Zhang b, * a b
School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 210009, PR China Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 July 2014 Received in revised form 8 September 2014 Accepted 26 September 2014 Available online 28 September 2014
The aim of this study is to characterize the in-vitro physicochemical and in-vivo pharmacokinetic properties of the scutellarin-loaded bovine serum albumin nanoparticles (STA-BSA-NPs). STA existed as amorphous form in the nanoparticles. Reconstituted STA-BSA-NPs had an average particle size of 283.4 nm and a zeta potential of +17.95 mV. The in-vitro sustained release profile was well fitted with Weibull distribution model. In comparison to STA solution, STA-BSA-NPs exhibited a significantly higher plasma concentration from 20 min to 6 h after intravenous administration to rats. In addition, significantly higher AUC0–inf (2.8-fold), prolonged elimination half-life (4.2-fold) and lower clearance (2.7-fold) were achieved. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Scutellarin Albumin nanoparticles Sustained release Elimination half-life Bioavailability
1. Introduction Scutellarin (STA) (Fig. 1), a typical flavonoid glycoside, is the primary bioactive ingredient in breviscapine extracted from the Chinese herb Erigeron breviscapus (Vant.) Hand.-Mazz. Breviscapine has been widely used to treat cardiovascular diseases and cerebrovascular injury in clinics, such as cerebral ischemia, angina pectoris, myocardial infarction, stroke and cerebral thrombotic diseases. Various pharmacological activities of STA have been recognized, including anti-oxidation (Wang et al., 2008), angiogenesis (Gao et al., 2010), neuroprotector (Xu et al., 2007), ischemia protector (Lin et al., 2007) as well as anti-HIV (Zhang et al., 2005). Intravenous injection of STA is the most commonly used dosage form in clinical treatments. However, the fast metabolic rate, short elimination half-life, and short blood residence time limit its wide application (Li et al., 2013). A satisfactory system that exhibits a good pharmacokinetic profile has not been found in the field of colloidal particle delivery systems for STA. It is urgent to develop a drug delivery system that can prolong the elimination half-life and improve the intravenous bioavailability of STA. Albumin has been used as a versatile protein carrier to fabricate nanoparticles for drug delivery due to its nontoxic, nonimmunogenic, biocompatible and biodegradable properties (Kratz, 2008). Therapeutic drugs could be incorporated into the
* Corresponding author. Tel.: +86 25 83379418; fax: +86 25 83379418. E-mail address: [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.ijpharm.2014.09.038 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
nanoparticulate matrix due to various binding sites present in the albumin molecule (Patil, 2003). In addition, albumin nanoparticles showed great potential to prolong the half-life and bioavailability of encapsulated drugs including both small molecules (e.g., curcumin (Jithan et al., 2011)) and macromolecules such as hurudin (Jing et al., 2013). Moreover, albumin nanoparticles could also enhance the anti-tumor efficacy of some anti-cancer drugs. A successful example is the nanoparticulate albumin-bound (nabTM)-paclitaxel, which has been approved by FDA for clinical use. It can provide improving efficacy and tolerability in comparison to Cremophor based paclitaxel solution (Cortes and Saura, 2010). Various types of albumins used to prepare nanoparticles, including ovalbumin, bovine serum albumin and human serum albumin, have shown a promising prospect in the controlled delivery of nanoparticulate therapeutic agents (Elsadek and Kratz, 2012). Nevertheless, the studies on in-vivo pharmacokinetic behavior of BSA-containing nanoparticulate systems are still deficient. Generally, there are three different methods to prepare albumin nanoparticles, such as desolvation, emulsification and thermal gelation (Elzoghby et al., 2012). Desolvation process followed by cross-linking with glutaraldehyde is the commonly used technique to prepare albumin nanoparticles. In desolvation process, nanoparticles are prepared by a continuous dropwise addition of ethanol to an aqueous solution of albumin under continuous stirring until the solution becomes turbid. However, the obtained albumin nanoparticles are not sufficiently stabilized and may consequently be re-dissolved in water. Therefore, coacervates are
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and the adsorption process of STA was allowed for 1 h to get the suspension containing the BSA-NPs loaded with STA (STA-BSA-NPs). After centrifugation at 8000 rpm for 10 min, the obtained precipitation was re-dispersed in 2% lactose solution and freeze-dried to get lyophilized powder.
Fig. 1. Chemical structure of scutellarin (STA).
hardened by cross-linking with glutaraldehyde to solidify the albumin nanoparticles. Previously, STA-BSA-NPs have been prepared by desolvation technique followed by cross-linking, precipitation, adsorption and freeze drying. This preparation procedure was also optimized (Xi et al., 2012). However, in-vivo behavior of STA-BSA-NPs has not been explored yet. The main objective of the present study is to employ albumin nanoparticles as the carrier for extending the half-life and bioavailability of STA. The physicochemical properties of the STA-BSA-NPs were characterized. In addition, in-vivo pharmacokinetic study of STA-BSA-NPs was performed in rats with STA solution as a control group. 2. Materials and methods 2.1. Materials Scutellarin (purity of 95.8%) was purchased from Nanjing Zelang Pharmaceutical Technology Co., Ltd. (Nanjing, China). Scutellarin reference standard (purity of 98.2%) and protocatechuic aldehyde reference standard (purity of 98%) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Bovine serum albumin was purchased from the Shanghai Hui Xing Biochemical Reagent Co., Ltd. (Shanghai, China). Lactose was obtained from the Meggle Pharma (Wasserburg, Germany). Millipore water was used throughout the study. Other chemicals were of HPLC or analytical grade. HepG2 cell lines were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science. HepG2 cells were cultured in the Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated calf serum (Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China), 100 U/mL penicillin G and 100 U/mL streptomycin (pH 7.4), in a water jacketed CO2 incubator with a humidified atmosphere of 5% CO2 at 37 C. 2.2. Methods 2.2.1. Preparation of STA-BSA-NPs STA-BSA-NPs were prepared by the optimized method developed by our research group (Xi et al., 2012). Briefly, 40 mL ethanol (95%) was added continuously into 15 mL of 10% BSA aqueous solution at a rate of 1 mL/min under stirring (600 rpm) at 25 C. 5% Glutaraldehyde solution (equivalent to 0.95 mg/mL BSA) was added and allowed for cross-linking reaction and solidification for 12 h. The solidified BSA-nanoparticles (BSA-NPs) were then collected after centrifugation at 8000 rpm for 10 min. Purification was conducted by re-dispersing the sedimentation in 20 mL of water by sonication at 5 kHz for 5 min. The purification procedure was repeated three times. The obtained purified BSA-NPs (1 g) were re-dispersed in 6 mL of water. 24 mL of ethanol was added dropwise. Under continuous stirring, 72 mL of STA ethanol solution (1.45 mg/mL) was added,
2.2.2. Particle size and zeta potential analysis The particle size analysis was performed by photon correlation spectroscopy (PCS) using a Zetasizer 3000 (Malvern Instruments, Malvern, UK). Prior to measurement, appropriate amount of lyophilized STA-BSA-NPs sample was reconstituted in 0.01 M citric acid solution and measured in triplicate. Seven-day stability test of reconstituted STA-BSA-NPs was performed by observing their particle size and zeta potential. 2.2.3. Determination of drug loading and entrapment efficiency 100 mg of STA-BSA-NPs were dispersed in 10 mL of 80% ethanol solution (to fully dissolve free STA but not disrupt the nanostructure of STA-BSA-NPs) and stirred for 10 min. The suspension was centrifuged at 15,000 rpm for 15 min. The concentration of the obtained supernatant was determined, and the amount of free unloaded STA in STA-BSA-NP suspension was calculated (m2). Another 100 mg of STA-BSA-NPs were dispersed in 1 mL of phosphate buffer (pH 6.8) and then sonicated for 10 min. 4 mL of 0.05 M phosphate buffer (pH6.8, containing pancreatic enzyme, which was used to degrade albumin for fully releasing the incorporated STA) was added and the suspension was stirred at 37 C for 3 h. After centrifugation at 15,000 rpm for 15 min, the STA concentration in the supernatant was determined by HPLC and the total amount of STA added into STA-BSA-NPs was calculated (m1). Drug loading and entrapment efficiency were calculated by: Drugloadingefficiencyð%Þ ¼
Entrapmentefficiencyð%Þ ¼
m1 m2 100 m3
m1 m2 100 m1
(1)
(2)
where m1 is the total amount of STA added into STA-BSA-NPs; m2 is the free unloaded STA in STA-BSA-NP suspension; m3 is total amount of BSA NPs. 2.2.4. Differential scanning calorimetry (DSC) DSC experiments were carried out using a DSC 204 F1 Phoenix Differential Scanning Calorimeter (Netzsch Inc., Germany) which was calibrated for temperature and cell constants using indium. Samples were placed on non-hermetic aluminum pans. The sample cell was equilibrated at 20 C and then heated at a rate of 10 C/min in a range of 20–300 C. Data analysis was performed using NETZSCH-Proteus software (version 4.2). 2.2.5. Powder X-ray diffraction analysis X-ray powder diffraction (XRPD) was used to determine the crystalline or amorphous nature of STA in the STA-BSA-NPs. XRPD analysis was recorded using a Bruker D8 Advance Powder Diffractometer (Karlsruhe Inc., Germany) with Cu-Ka radiation (1.5406 Å). The samples were gently consolidated in an aluminum holder and scanned at 40 kV and 40 mA from 5 to 40 2u using a scanning speed of 2 /min and a step size of 0.02 . 2.2.6. Surface morphological study Morphological evaluation of the nanoparticles was performed by scanning electron microscope (SEM) (Hitachi S3400, Tokyo, Japan). The sample was examined on a brass stub using carbon double-sided tape. Powder samples were glued and mounted on metal sample plates. The samples were gold coated
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(thickness 15–20 nm) with a sputter coater (Fison Instruments, UK) using an electrical potential of 2.0 kV at 25 mA for 10 min. An excitation voltage of 20 kV was used in the experiment. 2.2.7. In-vitro release In-vitro release of STA from STA-BSA-NPs was studied by dialysis method using a RC-806 dissolution tester (TDTF technology Co., Ltd., China) with the rotary basket rotating at 50 rpm. 10 mL of reconstituted STA-BSA-NPs in 0.01 M citric acid solution (equivalent to 5 mg STA) or 10 mL of STA solution (0.5 mg/mL) (n = 6) was placed in each regenerated cellulose membrane Spectra/Por dialysis bag (MWCO 8000–12,000 Da) (Spectrum Industries, Los Angeles, CA). The dialysis bags were sealed in both ends with clips and soaked in 250 mL of phosphate buffer (pH 6.8) controlled at 37 C. The pH value of this buffer is close to plasma (pH 7.4) and scutellarin can achieve sink condition in pH of above 6.8 according to preliminary studies. Therefore, it is suitable for the purpose of in-vitro release comparison. Samples (1 mL) were withdrawn and filtered at predetermined time points (0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 12, 16 and 24 h). STA concentration was analyzed by HPLC method on Shimadzu LC2010AHT system (Shimadzu Corporation, Japan). A reversed phase Shimadzu-pack VP-ODS C18 (4.6 250 mm, 5 mm) column guarded by a precolumn C18 insert was used for separation. STA was isocratically eluted with the mixture of acetonitrile and potassium dihydrogen phosphate buffer (0.05 M, pH 2.5) (22/78, v/v). The flow rate of 1.0 mL/min was maintained. The detection wavelength was set at 335 nm. The in-vitro drug release amount of STA from STA-BSA-NPs at a given time was fitted into different models, including zero-order kinetics equation, first-order kinetics equation, Higuchi equation, Peppas equation, and Weibull equation. 2.2.8. In-vivo pharmacokinetic study The animal study was approved by the Ethical Committee of China Pharmaceutical University. All animals used in this study were handled in accordance with the guidelines of the Principles of Laboratory Animal Care (State Council, revised 1988). Male Sprague-Dawley rats (200 20 g) were obtained from Nanjing Qinglongshan Laboratory Animal Center (Nanjing, China). All animals housed in standard cages on a 12 h light–dark cycles were fed with standard animal chow daily, and had free access to drinking water. One day before the pharmacokinetic study, each animal was operated with a cannula insert into the right jugular vein under anesthesia by intraperitoneal injection of pentobarbital sodium (50 mg/kg). A surgical incision was made on the ventral side of the neck of rats to expose the jugular vein. The jugular vein was then cannulated with a polyethylene tubing (0.5 mm ID, 1 mm OD, Portex Ltd., Hythe, Kent, England) that was led under the skin and exteriorized at the back of the neck for blood sampling. 50 IU/mL of heparin sodium in normal saline was filled into the catheter to prevent the blood clotting. After the exposed areas were surgically sutured, the rats were placed individually in standard cages. The animals were allowed to recover for 24 h and were fasted overnight prior to administration (Zhang et al., 2013). Before intravenous administration to rats, the lyophilized STA-BSA-NPs were reconstituted in 0.01 M citric acid solution to obtain a suspension containing 3.5 mg/mL STA. 3.5 mg/mL STA solution in 0.01 M citric acid solution was served as control. Rats were divided randomly into two groups with 6 rats in each group. The body weight of each rat was measured before the administration. Each rat received the tail vein injection at a single dose of 9.4 mg/kg STA (Lv et al., 2008). Around 400 mL of blood was collected from the jugular vein into heparinized centrifuge tube at predetermined time points (5, 10, 20, 40, 60, 90, 120, 180, 240, 360 and 480 min) post dosing. Plasma samples were immediately
separated by centrifugation of blood at 10,000 rpm for 10 min (Refrigerated Centrifuge, Sigma 1–15 K, Sigma, Germany) and stored at 20 C until analysis. Frozen plasma samples were thawed at room temperature just before sample preparation. 50 mL of protocatechuic aldehyde methanol solution (internal standard, 50.0 mg/mL) and 100 mL of phosphoric acid (1 M) were added to a 200 mL aliquot of rat plasma. After vortex-mixing for 2 min, the mixture was extracted with 3 mL of ethyl acetate and centrifuged at 10,000 rpm for 10 min. 2 mL of the obtained supernatant was transferred to a 5-mL glass tube and evaporated to dryness under nitrogen stream in the water bath (40 C). The obtained residues were reconstituted with 400 mL of mobile phase. After centrifugation for another 5 min (10 C, 10,000 rpm), an aliquot of 20 mL supernatant was injected into the HPLC system for analysis using the method as described in Section 2.2.7. Calibration standard was prepared by spiking STA standards of six known concentrations in blank rat plasma. Good linearity was obtained in the concentration range of 0.031–76.16 mg/mL (r2 = 0.996). The recoveries of STA were over 90.5%; the interand intra-day precision was below 7% RSD (relative standard deviation), and the accuracy was within 93.2–102.9%. The limit of quantification and limit of detection were 60 ng/mL and 25 ng/mL, respectively. After storage for 8 h at room temperature, 5 days at 20 C and freeze–thawing for three times, STA was found to be stable in rat plasma. Pharmacokinetic analysis was performed by means of a model independent method using the PKSolver computer program (Zhang et al., 2010). The pharmacokinetic parameters were calculated. The area under the concentration–time curve (AUC) was calculated with trapezium method. The terminal half-life (t1/2) was calculated as ln 2/lz, where lz was the first-order rate constant associated with the terminal (log-linear) portion of the curve. The area under the plasma concentration–time curve from time 0 to 480 min (AUC0–480 min) and from time 0 to infinity (AUC0–inf) were calculated by the trapezoidal rule without or with extrapolation to time infinity (AUC0–inf = AUC0–48 min + C480 min/lz). 2.2.9. MTT assay MTT assay was performed to evaluate the cytotoxicity of STA-BSA-NPs on HepG2 cells (Fotakis and Timbrell, 2006). Briefly, HepG2 cells (1 104 per well in a 96-well plate) were treated with STA-BSA-NPs suspension (50, 100, 250 and 500 mg/mL in 0.01 M citric acid solution, n = 6) for 24 h at 37 C in a 5% CO2 incubator, respectively. Then, 20 mL MTT (5 mg/mL) was added into each well and incubated for 4 h. The supernatant was discarded, and 150 mL DMSO was added. The mixture was gently shaken on a microvibrator for 10 min. The absorbance was then recorded at 570 nm using multimode microplate reader (Infinite 200 Pro Tecan, Switzerland). 2.2.10. Statistical analysis Statistical analysis was performed using a statistical software package SPSS (version 17, SPSS Inc., Chicago, IL, USA). Independentsample t-test was applied for analysis of the data from in-vitro release and in-vivo pharmacokinetic studies. A probability level of p < 0.05 was set as the criterion of significance. 3. Results and discussion 3.1. Physicochemical properties Particle size and its distribution are the most widely accepted defining characteristics of nanoparticle-based medicines since particle size can significantly influence the pharmacokinetics, biodistribution, and safety of nanoparticulate drugs (Desai, 2012).
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The mean particle size and polydispersity index of prepared STABSA-NPs were determined to be 283.4 nm and 0.117, respectively (Fig. 2). The particle size in the range of 100–300 nm with narrow distribution indicates that STA-BSA-NPs will mainly distribute in the liver and spleen after intravenous administration (Gaumet et al., 2008). STA-BSA-NPs exhibited the regular spherical shape with smooth surfaces according to SEM (Fig. 3) with observed
145
particle size in SEM of about 100–300 nm. This agrees well with the determined value by PCS. Zeta potential of the reconstituted STA-BSA-NPs was determined to be +17.95 mV. In seven days, no aggregation or flocculation of the reconstituted STA-BSA-NPs was observed. In addition, there was no change in their particle size and zeta potential, indicating that the reconstituted STA-BSA-NPs can be stable for at least a week. Citric acid solution used to
Fig. 2. Particle size (a) and zeta potential (b) of STA-BSA-NPs.
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(a)
onset:189.3°C onset:116.2°C
onset:227.4°C
(b)
onset:49.0°C onset:202.3°C
onset:112.0°C
(c) onset:228.5°C
onset:41.6°C
onset:227.8°C
(d)
onset:50.0°C 50
100
150
200
250
Temperature(°C)
Fig. 5. DSC thermograms of crystalline STA (a); BSA-NPs (b); physical mixture of STA and BSA-NPs (c); and STA-BSA-NPs (d). Fig. 3. Scanning electron microscope (SEM) photograph of STA-BSA-NPs.
3.2. DSC and XRPD reconstitute STA-BSA-NPs made pH value of the dispersion far away from the isoelectric point of BSA (pH 4.7) and generated a positive zeta potential. Such moderate positive zeta potential led to the moderate stability of the reconstituted nanoparticle dispersion. The entrapment efficiency and drug loading efficiency of STA-BSA-NPs are 64.46% and 6.73%, respectively. The release profiles of STA solution and STA-BSA-NPs in 0.05 M phosphate buffer (pH 6.8) are presented in Fig. 4. In comparison to STA solution, STA-BSA-NPs had an obviously slower release. Based on the model fitting results of release data, the in-vitro drug release amount (Q) of STA from STA-BSA-NPs at a given time (t) was fitted very well to Weibull distribution model (Q = 100 {1 Exp[(tb)/a]}, (a = 5.67, b = 0.81, r2 = 0.9948)), an important model for analyzing multi-mechanistic drug release from nanoparticles. The release of different compounds may be described by different models. For example, Adriamycin coupled albumin nanoparticles were reported to have the similar Weibull release pattern (Chen et al., 2010). However, the release of folateconjugated BSA nanoparticles loaded with vinorelbine tartrate corresponded with the Higuchi equation (Li et al., 2012). The in-vitro release profile of STA-BSA-NPs was characterized by the shape parameter (b < 1) to be S-shape which has steeper initial slope than the exponential (b = 1) (Barzegar-Jalali et al., 2008; Yuksel et al., 2000). In addition, the calculated release half-life (t50%) of STA-BSA-NPs was 2.5-fold longer than that of STA solution.
The DSC curves of crystalline STA, drug-free BSA-NPs, physical mixture of STA and BSA-NPs, and STA-BSA-NPs were studied to examine the status of crystallinity and potential change of STA after the cross-linking and adsorption. As shown in Fig. 5, STA had an endothermic peak at 116.2 C (melting point of STA) and two peaks at 189.3 and 196.5 C which might be due to the melting behavior of some impurities in STA. Drug-free BSA-NPs exhibited two broad endothermic peaks with onset values at 49.0 C and 227.4 C. Although different from their corresponding onset values, DSC curve of their physical mixture showed a distinct endothermic peak at 112 C, demonstrating the existence of crystalline STA in physical mixture. However, STA-BSA-NPs had significantly different thermal behavior from crystalline STA and the physical mixture of STA and BSA-NPs. No endothermic peak was observed around the melting point of STA, indicating the existence of STA as amorphous state in STA-BSA-NPs. The XRPD patterns of crystalline STA, drug-free BSA-NPs, physical mixture of STA and BSA-NPs, and STA-BSA-NPs are presented in Fig. 6. The characteristic diffraction peaks demonstrate the long-range order of crystalline STA (Fig. 6(a)). In comparison to blank BSA-NPs, physical mixture of STA and BSANPs showed two obvious diffraction peaks of STA at 25.85 and 26.75 , indicating the presence of crystalline STA in physical mixture (Fig. 6(c)). This agrees with the observation from DSC.
100 90
The cumulative release(%)
80 70 60
(a)
50
STA-BSA-NPs STA solution
40
(b)
30
(c)
20
(d)
10 0
0
5
10
15
20
25
Time (h) Fig. 4. The release profiles of STA solution and STA-BSA-NPs in 0.05 M phosphate buffer (pH 6.8).
5
10
15
20
25
30
35
40
2θ(°) Fig. 6. XRPD patterns of crystalline STA (a); BSA-NPs (b); physical mixture of STA and BSA-NPs (c); and STA-BSA-NPs (d).
Y. Wei et al. / International Journal of Pharmaceutics 476 (2014) 142–148
Plasma concentration of STA(μg/mL)
70 60
STA-BSA-NPs STA solution
50 40
*
30
*
20
* 10 0
0
*
*
100
*
* 200
* 300
400
500
Time(min) Fig. 7. Mean plasma concentration of STA versus time curves after tail vein injection of STA solution and STA-BSA-NPs to rats (n = 6).
After the formation of nanoparticles, STA-BSA-NPs presented a XRPD pattern without the characteristic diffraction peaks of STA, suggesting the amorphous state of STA in nanoparticles (Fig. 6(d)). This transformation might be attributed the interaction between STA and BSA. The similar phenomena have been observed in previous BSA-containing nanoparticulate systems (Foldbjerg et al., 2013; Zhao et al., 2010). 3.3. In-vivo pharmacokinetic study To evaluate the in-vivo performance of the developed STA-BSA-NPs, the systemic pharmacokinetics of STA was studied with STA solution as the control. The plasma concentration-versustime profiles of STA in two formulations are given in Fig. 7. The calculated pharmacokinetic parameters are summarized in Table 1. In comparison to STA solution, intravenous STA-BSA-NPs showed similar initial phase of rapid decrease in plasma concentration in 10 min, while much slower decline after 20 min post dose. From 20 min to 6 h, the plasma concentrations of STA after intravenous administration of STA-BSA-NPs were significantly higher than those of STA solution (p < 0.05). Pharmacokinetic profiles of STA after intravenous administration of both STA solution and STA-BSA-NPs were verified to be well fitted to open two-compartment model. Such well fitted model agrees with other studies of the in-vivo behavior of STA (Huang Table 1 Pharmacokinetic parameters of STA after tail vein injection of STA solution and STABSA-NPs in rats.
k10/(min1) k12/(min1) k21/(min1) t1/2a/(min) t1/2b/(min) C0/(mg mL1) CL/(mL min1) AUC0–480 min/(mg min mL1) AUC0–inf/(mg min mL1) AUMC/(mg min2 mL1) MRT/(min)
STA solution
STA-BSA-NPs
0.064 0.014 0.016 0.011 0.022 0.003 8.67 2.32 42.24 3.25 70.79 14.48 2.69 0.13 1099 56.82 1116 56.78 29,910 3648 26.75 2.28
0.017 0.004 0.011 0.007 0.008 0.003 23.90 7.98** 176.8 22.98** 51.45 13.78* 0.98 0.05** 2836 166.4** 3070 161.0** 45,1614 21,168** 147.4 11.49**
k10, elimination rate constant; k12,k21, rate constant between the two compartments; t1/2a, distribution half-life; t1/2b, elimination half-life; CL, total clearance; C0, the instantaneous concentration after intravenous injection; AUC0–t, area under plasma concentration versus time curve (total area from 0 to t); AUC0–inf, area under plasma concentration versus time curve (total area from 0 to infinity); AUMC, partial area under the moment curve; MRT, mean residence time. * p < 0.05, STA-BSA-NPs versus STA solution. ** p < 0.01, STA-BSA-NPs versus STA solution.
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et al., 2005; Lv et al., 2005). In comparison to STA solution, STA-BSA-NPs achieved the significantly higher AUC0–inf (p < 0.01) with a relative bioavailability of 275%. Several factors are involved in the enhancement of STA bioavailability. When drugs are encapsulated in nanoparticles, they are protected from metabolizing enzyme in the liver before their release in intravenous vein (Li and Huang, 2008). For instance, albumin coated 5-fluorouridine nanoparticles can even circumvent its pre-systemic degradation (Arbos et al., 2004). In addition, since the cut-off size of renal clearance is lower than 15 nm, the nanoparticulate drugs in systemic circulation will be blocked by glomerulus and the renal clearance of encapsulated drugs will be reduced regardless of their partition coefficients (Kadam et al., 2012). As a lipophilic drug, STA will undergo rapid and extensive biotransformation in the liver to hydrophilic aglycone conjugated metabolites and then the rapid biliary or renal excretion starts (Huang et al., 2005). Therefore, the intravenous STA solution has a fast clearance in-vivo (CL = 2.69 mL min1). However, STA-BSA-NPs achieved a 2.7-fold lower clearance as the result of combined effect of protected liver metabolism and reduced renal clearance. Such effect has also been reflected on the prolonged elimination half-life (t1/2b) (4.2-fold) and increased mean residence time (MRT) (5.5-fold) after intravenous administration of STA-BSA-NPs. 3.4. MTT assay The cell viability of HepG2 from MTT assay was more than 98% at all tested STA-BSA-NPs concentrations (50–500 mg/mL). There was no statistical difference among the four tested concentrations (p > 0.05). The MTT assay indicates that the STA-BSA-NPs are rather safe even at a relative high concentration (500 mg/mL) toward liver cells. 4. Conclusion In the present investigation, the potential of BSA nanoparticles as a novel drug delivery system for STA was evaluated based on their physicochemical and pharmacokinetic characterization. STA-BSA-NPs appeared to be the regular spherical shape with smooth surfaces in morphology and exhibited an in-vitro sustained release profile. In comparison to STA solution, STA-BSA-NPs achieved a significantly enhanced bioavailability and lower elimination half-life as well as longer MRT after intravenous administration. This study provides a great potential to utilize albumin nanoparticles as the effective intravenous formulation strategy for drugs with fast clearance and low bioavailability. Acknowledgments This research was supported by the National Natural Science Fund (No. 81202988), Natural Science Foundation of Jiangsu Province (BK20141351), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and Qing Lan Project of Jiangsu Province. References Arbos, P., Campanero, M.A., Arangoa, M.A., Irache, J.M., 2004. Nanoparticles with specific bioadhesive properties to circumvent the pre-systemic degradation of fluorinated pyrimidines. J. Control. Release 96, 55–65. Barzegar-Jalali, M., Adibkia, K., Valizadeh, H., Shadbad, M.R.S., Nokhodchi, A., Omidi, Y., Mohammadi, G., Nezhadi, S.H., Hasan, M., 2008. Kinetic analysis of drug release from nanoparticles. J. Pharm. Pharm. Sci. 11, 167–177. Chen, D., Tang, Q., Xue, W., Xiang, J., Zhang, L., Wang, X., 2010. The preparation and characterization of folate-conjugated human serum albumin magnetic cisplatin nanoparticles. J. Biomed. Res. 24, 26–32. Cortes, J., Saura, C., 2010. Nanoparticle albumin-bound (nabTM)-paclitaxel: improving efficacy and tolerability by targeted drug delivery in metastatic breast cancer. EJC Suppl. 8, 1–10.
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