International Journal of Pharmaceutics 473 (2014) 475–484
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
A highly stable norcantharidin loaded lipid microspheres: Preparation, biodistribution and targeting evaluation Jinlong Ma a , Huan Teng a , Juan Wang a,b , Yu Zhang a,c , Tianyang Ren a , Xing Tang a , Cuifang Cai a, * a
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China Shenyang Jinchang Pharmaceutical Co., Ltd., China c Jiangsu Kanion Pharmaceutical Co., Ltd., China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 March 2014 Received in revised form 11 July 2014 Accepted 25 July 2014 Available online 30 July 2014
The purpose of this study was to prepare norcantharidin (NCTD)-loaded lipid microspheres (LMs) with a high encapsulation efficiency (EE) and stability during sterilization. The NCTD–phospholipid complex (NPC) was produced and characterized to increase the lipophilic properties of NCTD and a novel concentrated homogenization method was applied for the preparation of LMs. The results of the UV, DSC and IR investigations confirmed the formation of NPC. The oil–water partition coefficient (log P) of NPC was significantly increased with a value of 1.34 0.06 at pH 7.4, nearly 224 times higher than that of NCTD. A concentrated emulsion was prepared based on a homogenization method and then diluted with water. After optimization of the NPC formation and emulsion preparation process, the EE was dramatically increased from 21.6% to 84.6%, and a highly sterilization stability was achieved with only a minor change in particle size from 168.2 39.4 nm to 173.4 43.5 nm. The tissue distribution of NPCLM was measured after intravenous administration to rats of a dose of 3.9 mg/kg with NCTD injection (NI) as the reference. Considerably increased concentrations of NCTD in the liver, spleen and lung were detected with NPCLM and the values were 1.67, 1.49 and 1.06 times higher than in the NI group, respectively while, in the kidney, the concentration was slightly reduced 0.96-fold. Overall, based on these techniques, this NPCLM with an improved EE and stability offers great promise in clinical applications and industrialscale production along with a potentially increased targeting effect on the liver and reduced toxicity in the kidney. ã 2014 Published by Elsevier B.V.
Keywords: Norcantharidin Phospholipid complex Encapsulation efficiency Concentrated homogenization Tissue distribution Targeting efficiency
1. Introduction Norcantharidin (NCTD), a demethylated analogue of cantharides isolated from the dried body of the blister beetle, is a potent anti-cancer drug used for the treatment of primary hepatic carcinoma, breast cancer and abdominal cancer (Wang, 1989). Compared with the other anticancer drugs, the distinguishing features of NCTD include its low degree of myelosuppression, induction of leukocytosis and minor effect on normal cells (Yi et al., 1991), (Chen et al., 2012). Therefore, NCTD is more attractive for chemotherapy because of its potent anti-hematoma activity and synergistic therapeutic effect. However, the significant side-effects of NCTD, including cardiac and renal damage, limit its use in clinical situations (Liu et al.,
* Corresponding author: Tel.: +86 24 23986343; fax: +86 24 23911736. E-mail address: [email protected] (C. Cai). http://dx.doi.org/10.1016/j.ijpharm.2014.07.047 0378-5173/ ã 2014 Published by Elsevier B.V.
1995). NCTD is usually administered by means of a high-dose injection which produces intense irritation at the injection site (Wang et al., 2006). When administered by the oral route, NCTD has to be given in a high dose to increase its antitumor efficacy due to its poor intestinal absorption, which can produce severe gastrointestinal and urinary toxic effects. Therefore, it is very important to find a new drug delivery system for NCTD to reduce these side effects. To obtain a safer and more effective NCTD treatment, many new alternative formulations have been studied to improve the targeted delivery, such as N-trimethyl chitosan nanoparticles (Guan et al., 2012), lipid microspheres (Wang et al., 2006), polycaprolactone microspheres (Wang et al., 2008), NCTD-polymer conjugates (Hong et al., 2009), and pH-sensitive liposomes (Qiaoling et al., 2012). However, most of these dosage forms have been found to be clinically unsuccessful because of lower encapsulation efficiency, the presence of an organic solvent and poor physicochemical stability during long-term storage.
476
J. Ma et al. / International Journal of Pharmaceutics 473 (2014) 475–484
In recent years, lipid microspheres (LMs) have attracted much attention as an ideal carrier for anticancer drugs. Compared with other drug carriers, LMs are physically stable, biodegradable, biocompatible, and easy to prepare (Lundberg et al., 1996; Fukui et al., 2003). Moreover, sustained drug release can be obtained because the drug is contained in the inner phase and does not come into direct contact with body tissues and fluids (Lu et al., 2005). In addition, it also improves the stability of hydrolyzable materials and reduces the effect of the drug on tissues. Consequently, LM is an ideal carrier for NCTD. LMs are usually used as parenteral delivery carriers for lipophilic drugs, such as prostaglandin E1, diazepam, and nonsteroidal anti-inflammatory drugs (Constantinides, 1995; Singh and Ravin, 1986; Yamaguchi and Mizushima, 1994). To ensure an encapsulation efficiency of more than 80%, the oil–water partition coefficient (log P) of the drug must be greater than 3. However, the problem is that all drugs of commercial interest to be formulated as i.v. emulsions do not have a sufficiently high solubility and oil– water partition coefficient in standard LCT and MCT oils, while the development of a new oil phase is too expensive (Akkar and Müller , 2003). The oil–water partition coefficient (log P) of NCTD is approximately 3.69 (pH 7.4) so during the process of preparing a coarse emulsion, NCTD dissolved in the oil phase would diffuse into the water, which would reduce the encapsulation efficiency of the formulation. In order to improve the lipophilicity of NCTD, phospholipid complexes of NCTD were developed. Phospholipid complexes are currently used for two groups of drugs for different purposes. One is to reduce the gastrointestinal toxicity caused by non-steroidal anti-inflammatory drugs (NSAIDs), while the other is to increase the solubility and the bioavailability of highly lipophilic and poorly absorbed drugs. (Hüsch et al., 2011). In the case of phytopharmaceuticals, Indena S.p.A. introduced a new product called Phytosom1 (Semalty et al., 2010). According to the structure of NCTD, it is prone to hydrolysis and forms a dicarboxylic acid structure which can interact with the amino end of phospholipids, forming hydrogen bonds or exhibiting charge effects. Also, NCTD is a low molecular weight drug and, therefore, the formation of NPC is a suitable way of increasing the lipophilicity of NCTD. In addition, to further increase the EE and reduce the exposure of the drug to water, a concentrated homogenization method was used (initial preparation of a 20% o/w emulsion which was then diluted to 10%). The resulting emulsion had a high encapsulation efficiency, excellent stability, and efficient tissue targeting. This paper describes the preparation of a new kind of NCTD o/w lipid microspheres loaded with an NCTD–phospholipid complex (NPCLM). The preparation process was systemically investigated and optimized. As a result, we developed a novel method which is suitable for the drug NCTD (log P 3.69, pH 7.4) and provides an encapsulation efficiency greater than 80%. All the experimental results prove that NPCLM can withstand thermal sterilization and it has good physical stability and high encapsulation efficiency. Moreover, it is suitable for industrial-scale production. 2. Material and methods
from BASF AG (Ludwigshafen, Germany), EDTA (Toshihito Pharmaceutical Co., Ltd. in Hangzhou, China) and glycerol (Zhejiang Suichang Glycerol Plant, Zhejiang, China), All other chemicals and reagents were obtained from Tianjin Concord Technology Ltd. Co., Tianjin, China, and were of analytical or chromatographic grade. All the animals used in this study were obtained from the Experimental Animal Center (Shenyang Pharmaceutical University, Shenyang, China). The experimental protocol were evaluated and approved by the University Ethics Committee for the use of experimental animals and conformed to the Guide for Care and Use of Laboratory Animals. 2.2. Preparation of the NCTD–phospholipid complex (NPC) The NPC was prepared with phospholipids (E80), cholesterol and NCTD in a suitable ratio. The required amounts of the above materials were dissolved in 10 ml anhydrous alcohol then the mixture was allowed to react at a temperature of 40 C for 4 h with constant stirring at 100 rpm. Then, the ethanol was completely evaporated in a rotary evaporator and the dried NPC was collected for further processing. 2.3. Characterization of NPC 2.3.1. UV absorption spectra Appropriate amounts of NCTD and NPC were dissolved in methanol and their UV absorption was scanned from 200–400 nm. Differences in UV absorption characteristics between the test samples were compared. 2.3.2. Fourier transforms infrared spectroscopy (FT-IR) Fourier transform infrared spectrophotometry (FT-IR Spectrometer, BRUKER IF S-55, Switzerland) was used to study the interaction between NCTD and phospholipid. The IR spectra of NCTD, phospholipid, a physical mixture and NPC were obtained by the KBr method. 2.3.3. Differential scanning calorimetry (DSC) Samples were sealed in aluminum crimped cells and heated at a rate of 10 C min1 from 30 C to 150 C in a nitrogen atmosphere (DSC-60, Shimadzu, Japan). The peak transition maximum temperatures of NCTD, phospholipid, physical mixture and NPC were determined and compared using a Thermal Analyzer (TA-60 WS, Shimadzu, Japan). 2.4. Solubility studies 2.4.1. Water and oil solubility In brief, an excess of NCTD was placed in vials, then phosphate buffer solution (PBS) with different pH values was added followed by distilled water, MCT and LCT. The oversaturated solutions were placed in a shaking air bath (HZQ-C, Dongming Medical Instrument Co., Harbin, China) operated at 100 rpm and 25 C for 72 h to achieve a solubility equilibrium. The resultant suspensions were passed through a 0.45 mm microporous filter then the filtrate was diluted for HPLC analysis. All solubility samples were processed in triplicate.
2.1. Materials The following materials were purchased from the sources in brackets: NCTD (Surui Medicine and Chemical Industry Ltd. Co., Suzhou, China), PL-100 M (Advanced Vehicle Technology Ltd. Co., Shanghai China), Lipoid E801, oleic acid, cholesterol and mediumchain triglyceride (MCT) (Lipoid KG, Ludwigshafen, Germany), long-chain triglyceride (LCT) (Tieling Beiya Pharmaceutical Co., Tieling, China), Poloxamer 188 (Pluronic F681) was purchased
2.4.2. Oil/water apparent partition coefficient (log P) of NCTD and NPC Briefly, an excess of NCTD and NPC were added to a solution containing 5 ml double -distilled water (pre-saturated with noctanol) and 5 ml n-octanol (pre-saturated with double -distilled water) in sealed glass containers at 25 C. Each experiment was performed in triplicate. The liquids were agitated at 100 rpm for 72 h, and then centrifuged to remove excess NCTD or NPC (10 min, 8000 rpm). The supernatants were then passed through a 0.45 mm
J. Ma et al. / International Journal of Pharmaceutics 473 (2014) 475–484
membrane filter and then examined using the methods described in Section 2.9. The oil–water partition coefficient was calculated based on the following equation: Co lgP ¼ lg Cw
(1)
Where Co and Cw are the concentration of NCTD in n-octanol and water, respectively. 2.5. Preparation of the NPCLM/NLM The NPCLM was prepared by a concentrated homogenization method. Briefly, NPC was dissolved in an oil phase consisting of MCT 10% (w/v) and oleic acid 0.06% (w/v), at 40 C in a water bath. The aqueous phase consisting of 2.5% (w/v) glycerol, 0.4% (w/v) F68, 0.04% (w/v) EDTA, and 0.8% (w/v) PL-100 M was also heated to 40 C in the water bath. Then, the water phase was rapidly added to the oil phase followed by high speed shear mixing (ULTRA RURRAX1IKA1T18 basic, Germany) at 10,000 rpm for 1.5 min. The coarse emulsion was then subjected to high-pressure microfluidizer homogenization (Microfluidizer Processor M-110EH, USA). The homogenization conditions were 10,000 psi, one cycle; after adding the rest of the double-distilled water, the pressure was increased to 15,000 psi for two cycles to obtain the final emulsion. The whole process was carried out at 20 C with cooling being achieved using a cold trap. The final emulsion was adjusted to pH 8.2 with 1.0 mol/l NaOH and then nitrogen was passed through the emulsion before it was sealed in vials and sterilized by autoclaving at 121 C for 8 min. The NLM was prepared according to the method reported previously (Lin et al., 2012).
477
2.8. Drug release in vitro Samples equivalent to about 4 mg NCTD were directly added to dialysis tubes (MWCO 3500, Shanghai Green Bird Science and Technology Development Co., Ltd. Shanghai, China), which were then immersed in dissolution medium (pH 7.4 PBS phosphate buffer saline, 150 ml) in a flask with continuous stirring at 100 rpm. The temperature was maintained at 37 C in a water bath. At predetermined intervals (5, 15, 30, 45, 60, 120 and 240 min), 1 ml aliquots were withdrawn and replaced with fresh medium. The sample solutions were passed through a 0.22 mm polyamides filter membrane and then analyzed by HPLC. Each sample was analyzed in triplicate. 2.9. Drug content determination in vitro HPLC analysis was performed on a Hitachi-Chromaster (Tokyo, Japan) equipped with a 5110 Pump, a 5210 Autosampler, a 5310Column Oven, and a 5410 UV detector. A HiQ sil C18 column (5 mm, 4.6 mm 250 mm, Kromasil, SE-445 80Bohus, Sweden) was used. The mobile phase consisted of PBS (pH 2.5)-acetonitrile at a ratio of 92:8, the flow rate was 1.0 ml min1; the detection wavelength was 210 nm; the column temperature was maintained at 30 C and the injection volume was 20 mL. 2.10. Long term and accelerated stability test A new batch of optimum NPCLM was prepared and stored at 4 C for 24 months, 25 C for 6 months. At pre-determined time intervals, samples were removed and their physical appearance, particle size distribution, drug content and EE were monitored to evaluate the NPCLM stability.
2.6. Characterization of NPCLM 2.11. Tissue distribution The particle size and zeta potential were measured using a Nicomp TM 380 particle sizing system (Santa Barbara, USA) which was based on the principle of photon correlation spectroscopy (DLS) and electrophoretic light scattering (ELS). The emulsion sample was diluted 1:5000 with double distilled water immediately before measurement at 25 C. The pH of the emulsion was measured using a Sartorius PB-10 standard pH-meter (Sartorius Scientific Instruments Ltd. Beijing, China) with a microelectrode at 25 C. The entrapment efficiency (EE) of NPCLM was determined by measuring free NCTD in the aqueous phase. Ultrafiltration centrifuge tubes (Millipore, 10000D)were used to isolate the aqueous phase from LM at 3000 rpm for 20 min. The drug in the aqueous phase was determined by HPLC as described in Section 2.9 and the EE (%) was calculated using the following equation (Férézou et al., 1994; Groves et al., 1985), EE ¼ 1
Ww Ww þ o
(2)
in which Ww is the drug content in the aqueous phase, and Ww + o is the total amount of drug.
2.11.1. Study design and procedures Animals were randomly divided into two groups (Kunming strain mice weighing 18–22 g). Then, NPCLM and NCTD injections (NI) were given to the two groups at a dose of 3.9 mg/kg via the tail vein after a 5-fold dilution with 5% (w/w) glucose solution. After injection, the mice were exsanguinated at 0.5, 1, 3, 6, 12 and 24 h. Blood was collected and heart, liver, spleen, lung, kidney, brain, stomach and intestine were excised, washed in normal saline and blotted dry with filter paper. The blood samples were centrifuged at 4000 rpm for 10 min to obtain plasma. All the biological samples were stored frozen at 80 C until analysis. 2.11.2. Bioanalytical methods NCTD in serum and tissue was determined by HPLC-MS/MS using an Agilent 1100HPLC system (Agilent Corp., Santa Clara, CA) which was connected to an API 2000 triple-quadrupole tandem mass spectrometer (Waters Corp., Manchester, UK) via an electrospray ionization source (Turbo Ionspray, Waters Corp., Manchester, UK). The analytical method used for the tissue sample preparation and sample determination was that previously reported (Lin et al., 2012) and found to be suitable for the analysis of biological samples.
2.7. Transmission electron microscopy (TEM) Five microliters of NPCLM was dropped onto a holey carbon copper grid, and the excess liquid was removed using filter paper after 5 min. The grids were then incubated in phosphotungstic acid (1.5% w/v; pH 7.0) for 5 min and airdried at room temperature. Finally, the samples were observed using a G220 microscope (FEI, Hillsboro, Oregon, USA).
2.11.3. Pharmacokinetics and statistical analysis The data were analyzed by drug and statistics (DAS) version 2.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China). The tissue distribution results were analyzed statistically using Student's independent sample t-test and expressed as one-way p-values. When comparisons between groups yielded a p value
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
A highly stable norcantharidin loaded lipid microspheres: Preparation, biodistribution and targeting evaluation Jinlong Ma a , Huan Teng a , Juan Wang a,b , Yu Zhang a,c , Tianyang Ren a , Xing Tang a , Cuifang Cai a, * a
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China Shenyang Jinchang Pharmaceutical Co., Ltd., China c Jiangsu Kanion Pharmaceutical Co., Ltd., China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 March 2014 Received in revised form 11 July 2014 Accepted 25 July 2014 Available online 30 July 2014
The purpose of this study was to prepare norcantharidin (NCTD)-loaded lipid microspheres (LMs) with a high encapsulation efficiency (EE) and stability during sterilization. The NCTD–phospholipid complex (NPC) was produced and characterized to increase the lipophilic properties of NCTD and a novel concentrated homogenization method was applied for the preparation of LMs. The results of the UV, DSC and IR investigations confirmed the formation of NPC. The oil–water partition coefficient (log P) of NPC was significantly increased with a value of 1.34 0.06 at pH 7.4, nearly 224 times higher than that of NCTD. A concentrated emulsion was prepared based on a homogenization method and then diluted with water. After optimization of the NPC formation and emulsion preparation process, the EE was dramatically increased from 21.6% to 84.6%, and a highly sterilization stability was achieved with only a minor change in particle size from 168.2 39.4 nm to 173.4 43.5 nm. The tissue distribution of NPCLM was measured after intravenous administration to rats of a dose of 3.9 mg/kg with NCTD injection (NI) as the reference. Considerably increased concentrations of NCTD in the liver, spleen and lung were detected with NPCLM and the values were 1.67, 1.49 and 1.06 times higher than in the NI group, respectively while, in the kidney, the concentration was slightly reduced 0.96-fold. Overall, based on these techniques, this NPCLM with an improved EE and stability offers great promise in clinical applications and industrialscale production along with a potentially increased targeting effect on the liver and reduced toxicity in the kidney. ã 2014 Published by Elsevier B.V.
Keywords: Norcantharidin Phospholipid complex Encapsulation efficiency Concentrated homogenization Tissue distribution Targeting efficiency
1. Introduction Norcantharidin (NCTD), a demethylated analogue of cantharides isolated from the dried body of the blister beetle, is a potent anti-cancer drug used for the treatment of primary hepatic carcinoma, breast cancer and abdominal cancer (Wang, 1989). Compared with the other anticancer drugs, the distinguishing features of NCTD include its low degree of myelosuppression, induction of leukocytosis and minor effect on normal cells (Yi et al., 1991), (Chen et al., 2012). Therefore, NCTD is more attractive for chemotherapy because of its potent anti-hematoma activity and synergistic therapeutic effect. However, the significant side-effects of NCTD, including cardiac and renal damage, limit its use in clinical situations (Liu et al.,
* Corresponding author: Tel.: +86 24 23986343; fax: +86 24 23911736. E-mail address: [email protected] (C. Cai). http://dx.doi.org/10.1016/j.ijpharm.2014.07.047 0378-5173/ ã 2014 Published by Elsevier B.V.
1995). NCTD is usually administered by means of a high-dose injection which produces intense irritation at the injection site (Wang et al., 2006). When administered by the oral route, NCTD has to be given in a high dose to increase its antitumor efficacy due to its poor intestinal absorption, which can produce severe gastrointestinal and urinary toxic effects. Therefore, it is very important to find a new drug delivery system for NCTD to reduce these side effects. To obtain a safer and more effective NCTD treatment, many new alternative formulations have been studied to improve the targeted delivery, such as N-trimethyl chitosan nanoparticles (Guan et al., 2012), lipid microspheres (Wang et al., 2006), polycaprolactone microspheres (Wang et al., 2008), NCTD-polymer conjugates (Hong et al., 2009), and pH-sensitive liposomes (Qiaoling et al., 2012). However, most of these dosage forms have been found to be clinically unsuccessful because of lower encapsulation efficiency, the presence of an organic solvent and poor physicochemical stability during long-term storage.
476
J. Ma et al. / International Journal of Pharmaceutics 473 (2014) 475–484
In recent years, lipid microspheres (LMs) have attracted much attention as an ideal carrier for anticancer drugs. Compared with other drug carriers, LMs are physically stable, biodegradable, biocompatible, and easy to prepare (Lundberg et al., 1996; Fukui et al., 2003). Moreover, sustained drug release can be obtained because the drug is contained in the inner phase and does not come into direct contact with body tissues and fluids (Lu et al., 2005). In addition, it also improves the stability of hydrolyzable materials and reduces the effect of the drug on tissues. Consequently, LM is an ideal carrier for NCTD. LMs are usually used as parenteral delivery carriers for lipophilic drugs, such as prostaglandin E1, diazepam, and nonsteroidal anti-inflammatory drugs (Constantinides, 1995; Singh and Ravin, 1986; Yamaguchi and Mizushima, 1994). To ensure an encapsulation efficiency of more than 80%, the oil–water partition coefficient (log P) of the drug must be greater than 3. However, the problem is that all drugs of commercial interest to be formulated as i.v. emulsions do not have a sufficiently high solubility and oil– water partition coefficient in standard LCT and MCT oils, while the development of a new oil phase is too expensive (Akkar and Müller , 2003). The oil–water partition coefficient (log P) of NCTD is approximately 3.69 (pH 7.4) so during the process of preparing a coarse emulsion, NCTD dissolved in the oil phase would diffuse into the water, which would reduce the encapsulation efficiency of the formulation. In order to improve the lipophilicity of NCTD, phospholipid complexes of NCTD were developed. Phospholipid complexes are currently used for two groups of drugs for different purposes. One is to reduce the gastrointestinal toxicity caused by non-steroidal anti-inflammatory drugs (NSAIDs), while the other is to increase the solubility and the bioavailability of highly lipophilic and poorly absorbed drugs. (Hüsch et al., 2011). In the case of phytopharmaceuticals, Indena S.p.A. introduced a new product called Phytosom1 (Semalty et al., 2010). According to the structure of NCTD, it is prone to hydrolysis and forms a dicarboxylic acid structure which can interact with the amino end of phospholipids, forming hydrogen bonds or exhibiting charge effects. Also, NCTD is a low molecular weight drug and, therefore, the formation of NPC is a suitable way of increasing the lipophilicity of NCTD. In addition, to further increase the EE and reduce the exposure of the drug to water, a concentrated homogenization method was used (initial preparation of a 20% o/w emulsion which was then diluted to 10%). The resulting emulsion had a high encapsulation efficiency, excellent stability, and efficient tissue targeting. This paper describes the preparation of a new kind of NCTD o/w lipid microspheres loaded with an NCTD–phospholipid complex (NPCLM). The preparation process was systemically investigated and optimized. As a result, we developed a novel method which is suitable for the drug NCTD (log P 3.69, pH 7.4) and provides an encapsulation efficiency greater than 80%. All the experimental results prove that NPCLM can withstand thermal sterilization and it has good physical stability and high encapsulation efficiency. Moreover, it is suitable for industrial-scale production. 2. Material and methods
from BASF AG (Ludwigshafen, Germany), EDTA (Toshihito Pharmaceutical Co., Ltd. in Hangzhou, China) and glycerol (Zhejiang Suichang Glycerol Plant, Zhejiang, China), All other chemicals and reagents were obtained from Tianjin Concord Technology Ltd. Co., Tianjin, China, and were of analytical or chromatographic grade. All the animals used in this study were obtained from the Experimental Animal Center (Shenyang Pharmaceutical University, Shenyang, China). The experimental protocol were evaluated and approved by the University Ethics Committee for the use of experimental animals and conformed to the Guide for Care and Use of Laboratory Animals. 2.2. Preparation of the NCTD–phospholipid complex (NPC) The NPC was prepared with phospholipids (E80), cholesterol and NCTD in a suitable ratio. The required amounts of the above materials were dissolved in 10 ml anhydrous alcohol then the mixture was allowed to react at a temperature of 40 C for 4 h with constant stirring at 100 rpm. Then, the ethanol was completely evaporated in a rotary evaporator and the dried NPC was collected for further processing. 2.3. Characterization of NPC 2.3.1. UV absorption spectra Appropriate amounts of NCTD and NPC were dissolved in methanol and their UV absorption was scanned from 200–400 nm. Differences in UV absorption characteristics between the test samples were compared. 2.3.2. Fourier transforms infrared spectroscopy (FT-IR) Fourier transform infrared spectrophotometry (FT-IR Spectrometer, BRUKER IF S-55, Switzerland) was used to study the interaction between NCTD and phospholipid. The IR spectra of NCTD, phospholipid, a physical mixture and NPC were obtained by the KBr method. 2.3.3. Differential scanning calorimetry (DSC) Samples were sealed in aluminum crimped cells and heated at a rate of 10 C min1 from 30 C to 150 C in a nitrogen atmosphere (DSC-60, Shimadzu, Japan). The peak transition maximum temperatures of NCTD, phospholipid, physical mixture and NPC were determined and compared using a Thermal Analyzer (TA-60 WS, Shimadzu, Japan). 2.4. Solubility studies 2.4.1. Water and oil solubility In brief, an excess of NCTD was placed in vials, then phosphate buffer solution (PBS) with different pH values was added followed by distilled water, MCT and LCT. The oversaturated solutions were placed in a shaking air bath (HZQ-C, Dongming Medical Instrument Co., Harbin, China) operated at 100 rpm and 25 C for 72 h to achieve a solubility equilibrium. The resultant suspensions were passed through a 0.45 mm microporous filter then the filtrate was diluted for HPLC analysis. All solubility samples were processed in triplicate.
2.1. Materials The following materials were purchased from the sources in brackets: NCTD (Surui Medicine and Chemical Industry Ltd. Co., Suzhou, China), PL-100 M (Advanced Vehicle Technology Ltd. Co., Shanghai China), Lipoid E801, oleic acid, cholesterol and mediumchain triglyceride (MCT) (Lipoid KG, Ludwigshafen, Germany), long-chain triglyceride (LCT) (Tieling Beiya Pharmaceutical Co., Tieling, China), Poloxamer 188 (Pluronic F681) was purchased
2.4.2. Oil/water apparent partition coefficient (log P) of NCTD and NPC Briefly, an excess of NCTD and NPC were added to a solution containing 5 ml double -distilled water (pre-saturated with noctanol) and 5 ml n-octanol (pre-saturated with double -distilled water) in sealed glass containers at 25 C. Each experiment was performed in triplicate. The liquids were agitated at 100 rpm for 72 h, and then centrifuged to remove excess NCTD or NPC (10 min, 8000 rpm). The supernatants were then passed through a 0.45 mm
J. Ma et al. / International Journal of Pharmaceutics 473 (2014) 475–484
membrane filter and then examined using the methods described in Section 2.9. The oil–water partition coefficient was calculated based on the following equation: Co lgP ¼ lg Cw
(1)
Where Co and Cw are the concentration of NCTD in n-octanol and water, respectively. 2.5. Preparation of the NPCLM/NLM The NPCLM was prepared by a concentrated homogenization method. Briefly, NPC was dissolved in an oil phase consisting of MCT 10% (w/v) and oleic acid 0.06% (w/v), at 40 C in a water bath. The aqueous phase consisting of 2.5% (w/v) glycerol, 0.4% (w/v) F68, 0.04% (w/v) EDTA, and 0.8% (w/v) PL-100 M was also heated to 40 C in the water bath. Then, the water phase was rapidly added to the oil phase followed by high speed shear mixing (ULTRA RURRAX1IKA1T18 basic, Germany) at 10,000 rpm for 1.5 min. The coarse emulsion was then subjected to high-pressure microfluidizer homogenization (Microfluidizer Processor M-110EH, USA). The homogenization conditions were 10,000 psi, one cycle; after adding the rest of the double-distilled water, the pressure was increased to 15,000 psi for two cycles to obtain the final emulsion. The whole process was carried out at 20 C with cooling being achieved using a cold trap. The final emulsion was adjusted to pH 8.2 with 1.0 mol/l NaOH and then nitrogen was passed through the emulsion before it was sealed in vials and sterilized by autoclaving at 121 C for 8 min. The NLM was prepared according to the method reported previously (Lin et al., 2012).
477
2.8. Drug release in vitro Samples equivalent to about 4 mg NCTD were directly added to dialysis tubes (MWCO 3500, Shanghai Green Bird Science and Technology Development Co., Ltd. Shanghai, China), which were then immersed in dissolution medium (pH 7.4 PBS phosphate buffer saline, 150 ml) in a flask with continuous stirring at 100 rpm. The temperature was maintained at 37 C in a water bath. At predetermined intervals (5, 15, 30, 45, 60, 120 and 240 min), 1 ml aliquots were withdrawn and replaced with fresh medium. The sample solutions were passed through a 0.22 mm polyamides filter membrane and then analyzed by HPLC. Each sample was analyzed in triplicate. 2.9. Drug content determination in vitro HPLC analysis was performed on a Hitachi-Chromaster (Tokyo, Japan) equipped with a 5110 Pump, a 5210 Autosampler, a 5310Column Oven, and a 5410 UV detector. A HiQ sil C18 column (5 mm, 4.6 mm 250 mm, Kromasil, SE-445 80Bohus, Sweden) was used. The mobile phase consisted of PBS (pH 2.5)-acetonitrile at a ratio of 92:8, the flow rate was 1.0 ml min1; the detection wavelength was 210 nm; the column temperature was maintained at 30 C and the injection volume was 20 mL. 2.10. Long term and accelerated stability test A new batch of optimum NPCLM was prepared and stored at 4 C for 24 months, 25 C for 6 months. At pre-determined time intervals, samples were removed and their physical appearance, particle size distribution, drug content and EE were monitored to evaluate the NPCLM stability.
2.6. Characterization of NPCLM 2.11. Tissue distribution The particle size and zeta potential were measured using a Nicomp TM 380 particle sizing system (Santa Barbara, USA) which was based on the principle of photon correlation spectroscopy (DLS) and electrophoretic light scattering (ELS). The emulsion sample was diluted 1:5000 with double distilled water immediately before measurement at 25 C. The pH of the emulsion was measured using a Sartorius PB-10 standard pH-meter (Sartorius Scientific Instruments Ltd. Beijing, China) with a microelectrode at 25 C. The entrapment efficiency (EE) of NPCLM was determined by measuring free NCTD in the aqueous phase. Ultrafiltration centrifuge tubes (Millipore, 10000D)were used to isolate the aqueous phase from LM at 3000 rpm for 20 min. The drug in the aqueous phase was determined by HPLC as described in Section 2.9 and the EE (%) was calculated using the following equation (Férézou et al., 1994; Groves et al., 1985), EE ¼ 1
Ww Ww þ o
(2)
in which Ww is the drug content in the aqueous phase, and Ww + o is the total amount of drug.
2.11.1. Study design and procedures Animals were randomly divided into two groups (Kunming strain mice weighing 18–22 g). Then, NPCLM and NCTD injections (NI) were given to the two groups at a dose of 3.9 mg/kg via the tail vein after a 5-fold dilution with 5% (w/w) glucose solution. After injection, the mice were exsanguinated at 0.5, 1, 3, 6, 12 and 24 h. Blood was collected and heart, liver, spleen, lung, kidney, brain, stomach and intestine were excised, washed in normal saline and blotted dry with filter paper. The blood samples were centrifuged at 4000 rpm for 10 min to obtain plasma. All the biological samples were stored frozen at 80 C until analysis. 2.11.2. Bioanalytical methods NCTD in serum and tissue was determined by HPLC-MS/MS using an Agilent 1100HPLC system (Agilent Corp., Santa Clara, CA) which was connected to an API 2000 triple-quadrupole tandem mass spectrometer (Waters Corp., Manchester, UK) via an electrospray ionization source (Turbo Ionspray, Waters Corp., Manchester, UK). The analytical method used for the tissue sample preparation and sample determination was that previously reported (Lin et al., 2012) and found to be suitable for the analysis of biological samples.
2.7. Transmission electron microscopy (TEM) Five microliters of NPCLM was dropped onto a holey carbon copper grid, and the excess liquid was removed using filter paper after 5 min. The grids were then incubated in phosphotungstic acid (1.5% w/v; pH 7.0) for 5 min and airdried at room temperature. Finally, the samples were observed using a G220 microscope (FEI, Hillsboro, Oregon, USA).
2.11.3. Pharmacokinetics and statistical analysis The data were analyzed by drug and statistics (DAS) version 2.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China). The tissue distribution results were analyzed statistically using Student's independent sample t-test and expressed as one-way p-values. When comparisons between groups yielded a p value
